ascorbic-acid and beta-glycerophosphoric-acid

The standard procedure for the osteogenic differentiation of multipotent stem cells is treatment of a confluent monolayer with a cocktail of dexamethasone (Dex), ascorbic acid (Asc) and β-glycerophosphate (β-Gly). This review describes the effects of these substances on intracellular signaling cascades that lead to osteogenic differentiation of bone marrow stroma-derived stem cells. We conclude that Dex induces Runx2 expression by FHL2/β-catenin-mediated transcriptional activation and that Dex enhances Runx2 activity by upregulation of TAZ and MKP1. Asc leads to the increased secretion of collagen type I (Col1), which in turn leads to increased Col1/α2β1 integrin-mediated intracellular signaling. The phosphate from β-Gly serves as a source for the phosphate in hydroxylapatite and in addition influences intracellular signaling molecules. In this context we give special attention to the differences between dystrophic and bone-specific mineralization.

Topics: Ascorbic Acid; Bone Marrow Cells; Dexamethasone; Glycerophosphates; Humans; Mesenchymal Stem Cells; Osteogenesis; Signal Transduction

The purpose of this study was to generate tissue-engineered bone using human periosteal-derived osteoblasts (PO) and polydioxanone/pluronic F127 (PDO/pluronic F127) scaffold with preseeded human periosteal-derived CD146 positive endothelial-like cells (PE). PE were purified from the periosteal cell population by cell sorting. One of the important factors to consider in generating tissue-engineered bone using osteoprecursor and endothelial cells and a specific scaffold is whether the function of osteoprecursor and endothelial cells can be maintained in originally different culture medium conditions. After human PE were preseeded into PDO/pluronic F127 scaffold and cultured in endothelial cell basal medium-2 for 7 days, human PO were seeded into the PDO/pluronic F127 scaffold with PE, and then, this cell-scaffold construct was cultured in endothelial cell basal medium-2 with osteogenic induction factors, including ascorbic acid, dexamethasone, and β-glycerophosphate, for a further 7 days. Then, this 2-week cultured construct was grafted into the mandibular defect of miniature pig. Twelve weeks after implantation, the animal was sacrificed. Clinical examination revealed that newly formed bone was seen more clearly in the defect with human PO and PDO/pluronic F127 scaffold with preseeded human PE. The experimental results suggest that tissue-engineered bone formation using human PO and PDO/pluronic F127 scaffold with preseeded human PE can be used to restore skeletal integrity to various bony defects when used in clinics.

Topics: Adolescent; Antioxidants; Ascorbic Acid; CD146 Antigen; Cells, Cultured; Dexamethasone; Endothelial Cells; Female; Glucocorticoids; Glycerophosphates; Humans; Male; Osteoblasts; Osteogenesis; Periosteum; Poloxamer; Polydioxanone; Time Factors; Tissue Scaffolds

In in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1-SHED + Dulbecco's Modified Eagles' Medium (DMEM) + fetal bovine serum (FBS); G2-SHED + DMEM + FBS + DEX; G3-SHED + DMEM + FBS + ASC + βGLY; G4-SHED + DMEM + FBS + ASC + βGLY + DEX; G5-MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

Topics: 3T3 Cells; Animals; Ascorbic Acid; Calcium; Cell Differentiation; Culture Media; Dexamethasone; DNA; Extracellular Matrix; Glycerophosphates; Humans; In Vitro Techniques; Mice; Osteogenesis; Stem Cells; Tooth, Deciduous

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Aorta; Ascorbic Acid; Atherosclerosis; Cell Transdifferentiation; Chondrocytes; Diphosphates; Glycerophosphates; Humans; Magnetic Resonance Spectroscopy; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphates; Vascular Calcification

We studied the expression of transcription factors RUNX2 and Osterix after addition of a concentrate of osteogenic-conditioned medium to the culture medium for osteogenic differentiation of mesenchymal stem cells (MSC). The obtained concentrate of osteogenic-conditioned medium containing a complex of bioactive substances with a molecular weight >10 kDa provided MSC differentiation into osteoblasts, which was confirmed by high level of expression of transcription factors RUNX2 and Osterix in comparison with the negative control. The highest expression of transcription factor Osterix was revealed on day 14 of MSC culturing in the presence of osteogenic supplement StemPro (positive control) and the studied concentrate of osteogenic-conditioned medium.

Topics: Animals; Ascorbic Acid; Biomarkers; Bone Marrow Cells; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Culture Media, Conditioned; Dexamethasone; Gene Expression; Glycerophosphates; Mesenchymal Stem Cells; Osteoblasts; Osteogenesis; Primary Cell Culture; Rats; Rats, Wistar; Transcription Factors

The periodontal ligament displays a reservoir of mesenchymal stem cells which can account for periodontal regeneration. Despite the numerous studies directed at the definition of optimal culture conditions for long-term expansion of periodontal ligament stem cells (PDLSCs), no consensus has been reached as to what is the ideal protocol. The aim of the present study was to determine the optimal medium formulation for long-term expansion and stemness maintenance of PDLSCs, in order to obtain a sufficient number of cells for therapeutic approaches. For this purpose, the effects of three different culture medium formulations were evaluated on PDLSCs obtained from three periodontal ligament samples of the same patient: minimum essential medium Eagle, alpha modification (α-MEM), Dulbecco's modified Eagle's medium (DMEM), both supplemented with 10% fetal bovine serum (FBS), and a new medium formulation, Ham's F12 medium, supplemented with 10% FBS, heparin 0.5 U/ml, epidermal growth factor (EGF) 50 ng/ml, fibroblast growth factor (FGF) 25 ng/ml, and bovine serum albumin (BSA) 1% (enriched Ham's F12 medium; EHFM). PDLSCs grown in EHFM displayed a higher PE-CD73 mean fluorescence intensity compared with cells maintained in α-MEM and DMEM, even at later passages. Cells maintained in EHFM displayed an increased population doubling and a reduced population doubling time compared with cells grown in DMEM or α-MEM. α-MEM, DMEM and EHFM with added dexamethasone, 2-phospho-L-ascorbic acid, and β-glycerophosphate were all able to promote alkaline phosphatase activity; however, no calcium deposition was detected in PDLSCs cultured in EHFM-differentiation medium. When EHFM-, α-MEM- and DMEM-expanded PDLSCs were transferred to a commercial culture medium for the osteogenesis, mineralization became much more evident in confluent monolayers of EHFM-expanded PDLSCs compared with DMEM and α-MEM. The results suggest EHFM is the optimal medium formulation for growth and stemness maintenance of primary PDLSCs. Moreover, EHFM confers higher osteogenic potential to PDLSCs compared with cells maintained in the other culture media. Overall, the results of the present work confirmed the advantages of using EHFM for long-term expansion of mesenchymal cells in vitro and the preservation of high osteogenic potential.

Topics: Adult; Ascorbic Acid; Cell Differentiation; Cell Proliferation; Dexamethasone; Flow Cytometry; Glycerophosphates; Humans; Osteoblasts; Osteogenesis; Periodontal Ligament; Real-Time Polymerase Chain Reaction; Serum Albumin, Bovine; Young Adult

Streptococcus gordonii is commonly found in the periapical endodontic lesions of patients with apical periodontitis, a condition characterized by inflammation and periapical bone loss. Since bone metabolism is controlled by osteoclastic bone resorption and osteoblastic bone formation, we investigated the effects of S. gordonii on the differentiation and function of osteoclasts and osteoblasts. For the determination of bone resorption activity in vivo, collagen sheets soaked with heat-killed S. gordonii were implanted on mouse calvaria, and the calvarial bones were scanned by micro-computed tomography. Mouse bone marrow-derived macrophages (BMMs) were stimulated with M-CSF and RANKL for 2 days and then differentiated into osteoclasts in the presence or absence of heat-killed S. gordonii. Tartrate-resistant acid phosphatase staining was performed to determine osteoclast differentiation. Primary osteoblast precursors were differentiated into osteoblasts with ascorbic acid and β-glycerophosphate in the presence or absence of heat-killed S. gordonii. Alkaline phosphatase staining and alizarin red S staining were conducted to determine osteoblast differentiation. Western blotting was performed to examine the expression of transcription factors including c-Fos, NFATc1, and Runx2. Heat-killed S. gordonii induced bone destruction in a mouse calvarial implantation model. The differentiation of RANKL-primed BMMs into osteoclasts was enhanced in the presence of heat-killed S. gordonii. Heat-killed S. gordonii increased the expression of c-Fos and NFATc1, which are essential transcription factors for osteoclast differentiation. On the other hand, heat-killed S. gordonii inhibited osteoblast differentiation and reduced the expression of Runx2, an essential transcription factor for osteoblast differentiation. S. gordonii exerts bone resorptive activity by increasing osteoclast differentiation and reducing osteoblast differentiation, which may be involved in periapical bone resorption.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Resorption; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Cytokines; Disease Models, Animal; Glycerophosphates; Macrophage Colony-Stimulating Factor; Macrophages; Male; Mice; Mice, Inbred C57BL; NFATC Transcription Factors; Osteoblasts; Osteoclasts; Osteogenesis; Periapical Periodontitis; Proto-Oncogene Proteins c-fos; RANK Ligand; Streptococcus gordonii; Transcription Factors; Up-Regulation; X-Ray Microtomography

Comparative in vitro study examined the osteogenic potential of interstitial cells of aortic valve obtained from the patients with aortic stenosis and from control recipients of orthotopic heart transplantation with intact aortic valve. The osteogenic inductors augmented mineralization of aortic valve interstitial cells (AVIC) in patients with aortic stenosis in comparison with the control level. Native AVIC culture of aortic stenosis patients demonstrated overexpression of osteopontin gene (OPN) and underexpression of osteoprotegerin gene (OPG) in comparison with control levels. In both groups, AVIC differentiation was associated with overexpression of RUNX2 and SPRY1 genes. In AVIC of aortic stenosis patients, expression of BMP2 gene was significantly greater than the control level. The study revealed an enhanced sensitivity of AVIC to osteogenic inductors in aortic stenosis patients, which indicates probable implication of OPN, OPG, and BMP2 genes in pathogenesis of aortic valve calcification.

Topics: Aged; Aortic Valve; Aortic Valve Stenosis; Ascorbic Acid; Bone Morphogenetic Protein 2; Calcinosis; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Dexamethasone; Female; Gene Expression Regulation; Glycerophosphates; Heart Transplantation; Humans; Male; Membrane Proteins; Middle Aged; Osteoblasts; Osteogenesis; Osteopontin; Osteoprotegerin; Phosphoproteins; Primary Cell Culture; Stromal Cells; Tricuspid Valve

Ascorbic acid, β-glycerophosphate, and dexamethasone have been used in osteogenesis differentiation medium for in vitro cell culture, nothing is known for delivering these three bioactive compounds in vivo. In this study, we synthesized a novel bioactive scaffold by combining these three compounds with a lysine diisocyanate-based polyurethane. These bioactive compounds were released from the scaffold during the degradation process. The cell culture showed that the sponge-like structure in the scaffold was critical in providing a large surface area to support cell growth and all degradation products of the polymer were non-toxic. This bioactive scaffold enhanced the bone regeneration as evidenced by increasing the expression of three bone-related genes including collagen type I, Runx-2 and osteocalcin in rabbit bone marrow stem cells (BMSCs) in vitro and in vivo. The osteogenesis differentiation of BMSCs cultured in this bioactive scaffold was similar to that in osteogenesis differentiation medium and more extensive in this bioactive scaffold compared to the scaffold without these three bioactive compounds. These results indicated that the scaffold containing three bioactive compounds was a good osteogenesis differentiation promoter to enhance the osteogenesis differentiation and new bone formation in vivo.

Topics: Animals; Ascorbic Acid; Biocompatible Materials; Bone Regeneration; Cell Differentiation; Cells, Cultured; Dexamethasone; Glycerophosphates; Mesenchymal Stem Cells; Osteogenesis; Polymers; Rabbits; Rats; Tissue Engineering; Tissue Scaffolds

To investigate the effects of PRP on odontoblastic differentiation using dental pulp progenitor cells derived from the dental papilla of rat incisors.. Monolayer cultures of odontoblastic lineage KN-3 cells were incubated with PRP for various time periods. The expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein-1 (DMP-1) was determined using real-time reverse transcription-polymerase chain reaction and Western blot analyses. To further clarify the role of PRP in odontogenesis, KN-3 cells were stimulated with PRP in the presence of ascorbic acid and β-glycerophosphate. The cells were stained for alkaline phosphatase (ALP), and ALP activity was quantified in cell lysates. The formation of mineralized nodules was assessed by alizarin red staining. Statistical analysis was performed by one-way analysis of variance.. PRP increased the mRNA and protein expressions of odontoblastic markers, such as DSPP and DMP-1. Furthermore, PRP stimulated the ALP activity and mineralized nodule formation induced by ascorbic acid and β-glycerophosphate in a time-dependent manner.. PRP enhances odontoblastic differentiation of KN-3 cells. These results indicate that PRP could be a potential candidate for use in the regeneration of dentine-pulp complex.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Blotting, Western; Calcium; Cell Differentiation; Cells, Cultured; Dental Pulp; Extracellular Matrix Proteins; Glycerophosphates; Humans; Odontoblasts; Phosphoproteins; Platelet-Rich Plasma; Rats; Real-Time Polymerase Chain Reaction; Sialoglycoproteins

Vitamin D deficiency (hypovitaminosis D) causes osteomalacia and poor long bone mineralization. In apparent contrast, hypovitaminosis D has been reported in patients with primary brain calcifications ("Fahr's disease"). We evaluated the expression of two phosphate transporters which we have found to be associated with primary brain calcification (SLC20A2, whose promoter has a predicted vitamin D receptor binding site, and XPR1), and one unassociated (SLC20A1), in an in vitro model of calcification. Expression of all three genes was significantly decreased in calcifying human bone osteosarcoma (SaOs-2) cells. Further, we confirmed that vitamin D (calcitriol) reduced calcification as measured by Alizarin Red staining. Cells incubated with calcitriol under calcifying conditions specifically maintained expression of the phosphate transporter SLC20A2 at higher levels relative to controls, by RT-qPCR. Neither SLC20A1 nor XPR1 were affected by calcitriol treatment and remained suppressed. Critically, knockdown of SLC20A2 gene and protein with CRISPR technology in SaOs2 cells significantly ablated vitamin D mediated inhibition of calcification. This study elucidates the mechanistic importance of SLC20A2 in suppressing the calcification process. It also suggests that vitamin D might be used to regulate SLC20A2 gene expression, as well as reduce brain calcification which occurs in Fahr's disease and normal aging.

Topics: Ascorbic Acid; Calcinosis; Calcitriol; Cell Differentiation; Cell Line, Tumor; CRISPR-Cas Systems; Gene Knockdown Techniques; Glycerophosphates; Humans; Models, Biological; Phosphate Transport Proteins; Receptors, Calcitriol; Receptors, G-Protein-Coupled; Receptors, Virus; RNA, Messenger; Sodium-Phosphate Cotransporter Proteins, Type III; Up-Regulation; Xenotropic and Polytropic Retrovirus Receptor

Activin A is a growth factor released by mature osteoblasts that has a critical effect on bone formation. We investigated the effect of bone morphogenetic protein (BMP)-4 on activin A gene expression during in vitro osteogenic differentiation of mouse embryonic stem (ES) cells. Embryoid bodies were cultured in retinoic acid (RA) for three days and then without RA for two days. Seeded cells received osteogenic medium with β-glycerophosphate, L-ascorbic acid 2-phosphate and dexamethasone during 19 days, with or without BMP-4. Six independent experiments were carried out. Real-time PCR was used to detect gene expression of activin A, Oct-4, Nanog, osteocalcin, RUNX2 and bone alkaline phosphatase. Immunofluorescence was used to co-localize activin A with the undifferentiation marker stage-specific embryonic antigen 1. Cells treated with BMP-4 had an increased gene expression of activin A, osteocalcin and bone alkaline phosphatase (p < 0.05). In conclusion, BMP-4 increases activin A gene expression during mouse ES cell differentiation into bone precursors.

Topics: Activins; Animals; Ascorbic Acid; Bone Morphogenetic Protein 4; Cell Differentiation; Culture Media; Dexamethasone; DNA Primers; Fibroblasts; Gene Expression Regulation, Developmental; Glycerophosphates; Mice; Microscopy, Fluorescence; Mouse Embryonic Stem Cells; Osteogenesis; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tretinoin

During mammalian embryogenesis, sclerotome-derived chondrocytes in the limb bud are arranged into a complicated bone shape with specific areas undergoing hypertrophy and calcification, creating a region-specific mineralized pattern in the cartilage. To follow chondrogenesis progression in vitro, we isolated limb cartilage from mice on embryonic day 13 (E13) and cultured it at the air-liquid interface after microsurgical removal of the ectoderm/epidermis. Explants underwent proper morphogenesis, giving rise to complete templates for limb bones in vitro. We found that region-specific calcification patterns resembling limbs of prepartum mature embryos could be induced in explants using culture medium containing high concentrations of CaCl2 (Ca), ascorbic acid (AA), and β-glycerophosphoric acid (BGP). In this culture system, excess amounts of all-trans retinoic acid (RA) severely disrupted morphogenesis and calcification patterns in limb cartilage. These effects were more pronounced in forearms than in phalanges. Although dissociated, the nascent chondrocytes in culture did not give rise to cartilage units even though augmented calcification was induced in these cell aggregates in the presence of RA. Taken together, our newly established culture system revealed that RA independently regulates three-dimensional morphogenesis and calcification.

Topics: Animals; Ascorbic Acid; Calcification, Physiologic; Cartilage; Cell Differentiation; Cell Line; Extremities; Glycerophosphates; Mice; Morphogenesis; Tissue Culture Techniques; Tretinoin

An osteogenic inducer (OI) consisting of dexamethasone, vitamin C, and β-sodium glycerophosphate has the capacity to induce bone formation in vitro. The aim of this study was to assess the efficacy of the application of this OI on extraction socket healing. The bilateral first mandibular premolars were extracted from 75 New Zealand rabbits. Gelatin sponges carrying OI were implanted into the sockets. Sockets undergoing implantation of gelatin sponges alone were also evaluated, as well as non-implantation sockets. Specimens from each group were evaluated radiographically, histologically, and histomorphometrically using haematoxylin-eosin staining. Results showed earlier new bone formation and higher bone quality and quantity in the OI group compared to the other groups, and the differences were significant at 2, 4, 8, and 12 weeks postoperative. The OI significantly reduced the absorption of alveolar bone in terms of height; however, changes in the width were not significantly different between the three groups (P>0.05). The OI was shown to have a positive effect on healing of the tooth extraction sockets, was inexpensive, and was convenient to use during the operational procedure; therefore this could represent a promising implant material for human clinical application.

Topics: Administration, Topical; Animals; Ascorbic Acid; Cone-Beam Computed Tomography; Dexamethasone; Drug Therapy, Combination; Glycerophosphates; Rabbits; Tooth Extraction; Tooth Socket; Wound Healing

To establish human heart valve interstitial cells calcification culture model in vitro, and observe the effect of bone morphogenetic protein-2 (BMP-2) on calcification of human heart valve interstitial cells.. Human heart valve interstitial cells were cultured in vitro, and divided into control group: cells were cultured in conventional media plus recombinant human BMP-2 treatment and experimental group: besides above treaments, calcification inducers ( recombinant human BMP-2, β-glycerophosphate, L-ascorbic acid, dexamethasone) were added to the culture media. The two group of cells were cultured for 14 days and were stained by Von Kossa, then the cell calcification was observed in this valvular interstitial cells calcification culture model in vitro. Protein expression of intercellular adhesion molecule 1 (ICAM-1), interleukin 8, BMP-2 and BMP-4 was determined by Western blot and BMP-2 secretion was measured by ELISA.. In the control group, the structure of human heart valve interstitial cells was clear, and the spindle and radial growth shaped cellular morphology was visible, and Von Kossa staining was negative. In the experimental group, the nuclei become darker in color, and granular sediment distribution was seen surrounding cells, and Von Kossa staining was positive, the cells were forming nodules of calcification. The protein expression of ICAM-1, interleukin 8, BMP-2 and BMP-4 in the experimental was significantly higher than that of the control group (all P < 0.05). The expression of BMP-2 in the experimental group was also significantly higher than that in control group ((92.5 ± 4.9) pg/ml vs. (22.2 ± 1.9) pg/ml, P < 0.05).. Human BMP-2, β-glycerophosphate, L-ascorbic acid, and dexamethasone can induce human heart valve interstitial cells calcification and enhance inflammation in vitro by stimulating the secretion of BMP-2.

Topics: Ascorbic Acid; Bone Morphogenetic Protein 2; Calcinosis; Cells, Cultured; Glycerophosphates; Heart Valve Diseases; Humans; Recombinant Proteins; Transforming Growth Factor beta

The hypothesis of this work was that human bone marrow-derived mesenchymal stem cells (MSCs) are regulated by pulsed electromagnetic fields (PEMFs) and by intracrine conversion of an adrenal prohormone to dihydrotestosterone. The effect of PEMF and dehydroepiandrosterone (DHEA) on viability and osteogenic differentiation of human MSCs and on the viability of osteoblastic SaOS-2 cells was evaluated. It was found that PEMF promoted the viability rate of both cell types, whereas DHEA decreased the viability rate in a concentration-dependent manner. PEMF did not have major effects on osteo-induction at this low seeding density level (3000 cells/cm(2) ). Instead, DHEA, after MSC-mediated and 5α-reductase-dependent conversion to dihydrotestosterone, clearly promoted the osteo-induction of MSCs induced with β-glyserophosphate, ascorbate and dexamethasone. Alkaline phosphatase (ALP), SMAD1, RUNX2, osteopontin (OP) and osteocalcin (OC) RNA levels were increased and alizarin red S- and hydroxyapatite-specific OsteoImage(TM) stainings disclosed a promoted mineralization process. In addition, DHEA increased OP and OC mRNA levels of non-induced MSCs. A sequential use of mitogenic PEMF early during the fracture healing, followed by later administration of DHEA with osteogenic differentiating effect, might be worth subjecting to a randomized clinical trial.

Topics: Ascorbic Acid; Bone and Bones; Bone Marrow Cells; Cell Differentiation; Cell Line; Cell Survival; Cholestenone 5 alpha-Reductase; Dehydroepiandrosterone; Dexamethasone; Durapatite; Electromagnetic Fields; Glycerophosphates; Humans; Mesenchymal Stem Cells; Osteoblasts; Osteocalcin; Osteogenesis; Osteopontin; Regeneration

The in vitro culture of calvarial osteoblasts from neonatal rodents remains an important method for studying the regulation of bone formation. The widespread use of transgenic mice has created a particular need for a reliable, simple method that allows the differentiation and bone‑forming activity of murine osteoblasts to be studied. In the present study, we established such a method and identified key differences in optimal culture conditions between mouse and rat osteoblasts. Cells isolated from neonatal rodent calvariae by collagenase digestion were cultured for 14‑28 days before staining for tissue non-specific alkaline phosphatase (TNAP) and bone mineralisation (alizarin red). The reliable differentiation of mouse osteoblasts, resulting in abundant TNAP expression and the formation of mineralised 'trabecular‑shaped' bone nodules, occurred only following culture in α minimum essential medium (αMEM) and took 21‑28 days. Dexamethasone (10 nM) inhibited bone mineralisation in the mouse osteoblasts. By contrast, TNAP expression and bone formation by rat osteoblasts were observed following culture in both αMEM and Dulbecco's modified Eagle's medium (DMEM) after approximately 14 days (although ~3‑fold more effectively in αMEM) and was strongly dependent on dexamethasone. Both the mouse and rat osteoblasts required ascorbate (50 µg/ml) for osteogenic differentiation and β‑glycerophosphate (2 mM) for mineralisation. The rat and mouse osteoblasts showed similar sensitivity to the well‑established inhibitors of mineralisation, inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP; 1‑100 µM). The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former. These findings offer a reliable technique for inducing mouse osteoblasts to form bone in vitro and a more effective method for culturing bone‑forming rat osteoblasts.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Calcification, Physiologic; Cell Differentiation; Culture Media; Dexamethasone; Glycerophosphates; Mice; Osteoblasts; Osteogenesis; Primary Cell Culture; Rats; Skull

Osteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis).. In this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization.. Our study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma.

Topics: Ascorbic Acid; Bone Neoplasms; Calcification, Physiologic; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Surface Extensions; Cell Survival; Collagen Type I; Glycerophosphates; Humans; Neoplasm Invasiveness; Osteosarcoma; Phenotype; Proteolysis

A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p < 0.01) and resulted in a consistent matrix mineralization. The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, β-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro.

Topics: Alkaline Phosphatase; Ascorbic Acid; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 6; Calcification, Physiologic; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Collagen Type I; Collagen Type V; Core Binding Factor Alpha 1 Subunit; Culture Media; Dexamethasone; Glycerophosphates; Growth Differentiation Factor 2; Growth Differentiation Factors; Homeodomain Proteins; Humans; Insulin-Like Growth Factor II; Intercellular Signaling Peptides and Proteins; Mesenchymal Stem Cells; Osteoblasts; Osteocalcin; Osteogenesis; Osteonectin; Phenotype; Transcription Factors

Ca-phosphate/hydroxyapatite crystals constitute the mineralic matrix of vertebrate bones, while Ca-carbonate dominates the inorganic matrix of otoliths. In addition, Ca-carbonate has been identified in lower percentage in apatite crystals. By using the human osteogenic SaOS-2 cells it could be shown that after exposure of the cells to Ca-bicarbonate in vitro, at concentrations between 1 and 10 mm, a significant increase of Ca-deposit formation results. The crystallite nodules formed on the surfaces of SaOS-2 cells become denser and larger in the presence of bicarbonate if simultaneously added together with the mineralization activation cocktail (β-glycerophosphate/ascorbic acid/dexamethasone). In parallel, with the increase of Ca-deposit formation, the expression of the carbonic anhydrase-II (CA-II) gene becomes upregulated. This effect, measured on transcriptional level is also substantiated by immunohistological studies. The stimulatory effect of bicarbonate on Ca-deposit formation is prevented if the carbonic anhydrase inhibitor acetazolamide is added to the cultures. Mapping the surface of the Ca-deposit producing SaOS-2 cells by scanning electron microscopy coupled with energy-dispersive X-ray analysis revealed an accumulation of the signals for the element carbon and, as expected, also for phosphorus. Finally, it is shown that ortho-phosphate and hydrolysis products of polyphosphate inhibit CA-II activity, suggesting a feedback regulatory system between the CA-driven Ca-carbonate deposition and a subsequent inactivation of this process by ortho-phosphate. Based on the presented data we suggest that Ca-carbonate deposits act as bioseeds for a downstream Ca-phosphate deposition process. We propose that activators for CA, especially for CA-II, might be beneficial for the treatment of bone deficiency diseases.

Topics: Acetazolamide; Ascorbic Acid; Bicarbonates; Bone Resorption; Calcium Phosphates; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Cell Line; Dexamethasone; Escherichia coli; Gene Expression Regulation, Developmental; Glycerophosphates; Humans; Microscopy, Electron; Up-Regulation; X-Rays

Frontline research progresses the applicability of bone marrow and adipose tissue in regenerative medicine, but fails to account for the functional improvement of the diseased. The justification for the failure in terms of stem cell survival, proliferation and regeneration is unclear. However, hyperglycemia rising during pathological conditions might be one such stumbling block. The prevailing literature accounts for both detrimental and beneficial effect of high glucose on mesenchymal stem cells (MSCs) leading to perplexity. Thus, this study focuses on the effect of high glucose on mesenchymal stem cells derived from subcutaneous fat, omentum fat and bone marrow in extensive cultures. We provide evidence for the retention of MSC characteristics of all sources with regards to surface marker profiling, proliferation, differentiation and karyotyping when cultured extensively under DMEM-HG containing glucose concentration of 25 mmol.l(-1) . Thus, it can be concluded that hyperglycemia in vivo (11 mmol.l(-1) ) might not be a barrier for the ineffective functional improvement of transplanted stem cells. Furthermore, we elucidated subcutaneous and omentum fat as better sources of MSCs when compared with bone marrow, thereby making these sources optimal for therapies during hyperglycemic conditions. However, further research is needed to clear the path for efficient stem cell transplantation.

Topics: 1-Methyl-3-isobutylxanthine; Adipogenesis; Adult; Ascorbic Acid; Bone Marrow Cells; Cell Culture Techniques; Cell Proliferation; Cell Separation; Cells, Cultured; Culture Media; Dexamethasone; Female; Glucose; Glycerophosphates; Humans; Immunophenotyping; Indomethacin; Insulin; Karyotyping; Male; Mesenchymal Stem Cells; Middle Aged; Omentum; Osteogenesis; Subcutaneous Fat

Ascorbic acid (AA), β-glycerophosphate (GP), and dexamethasone (DEX) are the compounds known to favor the expression of the osteoblastic phenotype in several bone cell systems.. In this report, the combination effects of differentiation agents on osteoprecursor cells were evaluated. The effect on cell proliferation was determined by a cell viability test with morphologic analysis. Differentiation and mineralization were evaluated using an alkaline phosphatase activity test and alizarin red-S staining. Protein expressions related to bone formation, such as transforming growth factor-beta (TGF-β), estrogen receptor-alpha (ER-α), and osteopontin (OPN) were evaluated by using a Western blot analysis.. AA and GP provided an inductive effect for differentiation of osteoprecusor cells, while short-term application of DEX seemed to lead to a dose-dependent increase of cellular differentiation. Long-term use of DEX seemed to reduce mineralization. These effects may seem to be regulated by the expression of ER-α, OPN, and TGF-β. Further studies related to this mechanism within the in vivo model may be necessary to ascertain greater detail.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Calcification, Physiologic; Cell Differentiation; Cell Line; Cell Proliferation; Cell Survival; Cells, Cultured; Dexamethasone; Dose-Response Relationship, Drug; Glycerophosphates; Mice; Models, Animal; Osteoblasts; Osteopontin; Phenotype; Receptors, Estrogen; Transforming Growth Factor beta

The amniotic membrane (AM) has been widely used in the field of tissue engineering because of the favorable biological properties for scaffolding material. However, little is known about the effects of an acellular AM matrix on the osteogenic differentiation of mesenchymal stem cells. In this study, it was found that both basement membrane side and collagenous stroma side of the acellular AM matrix were capable of providing a preferential environment for driving the osteogenic differentiation of human dental apical papilla cells (APCs) with proven stem cell characteristics. Acellular AM matrix potentiated the induction effect of osteogenic supplements (OS) such as ascorbic acid, β-glycerophosphate, and dexamethasone and enhanced the osteogenic differentiation of APCs, as seen by increased core-binding factor alpha 1 (Cbfa-1) phosphorylation, alkaline phosphatase activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Even in the absence of soluble OS, acellular AM matrix also could exert the substrate-induced effect on initiating APCs' differentiation. Especially, the collagenous stroma side was more effective than the basement membrane side. Moreover, the AM-induced effect was significantly inhibited by U0126, an inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) signaling. Taken together, the osteogenic differentiation promoting effect on APCs is AM-specific, which provides potential applications of acellular AM matrix in bone/tooth tissue engineering.

Topics: Alkaline Phosphatase; Amnion; Ascorbic Acid; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Dental Papilla; Dexamethasone; Gene Expression Regulation; Genetic Markers; Glycerophosphates; Humans; MAP Kinase Signaling System; Osteogenesis; Phosphorylation; RNA, Messenger; Stem Cells; Tissue Engineering

Aortic valve calcification (AVC) is an active process characterized by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free β-amino acid and plays important physiological roles including protective effect of cardiovascular events. To evaluate the possible role of taurine in AVC, we isolated human AVICs from patients with type A dissection without leaflet disease. We demonstrated that the cultured AVICs express SM α-actin, vimentin and taurine transporter (TAUT), but not CD31, SM-myosin or desmin. We also established the osteoblastic differentiation model of the AVICs induced by pro-calcific medium (PCM) containing β-glycerophosphate disodium, dexamethasone and ascorbic acid in vitro. The results showed that taurine attenuated the PCM-induced osteoblastic differentiation of AVICs by decreasing the alkaline phosphate (ALP) activity/expression and the expression of the core binding factor α1 (Cbfα1) in a dose-dependent manner (reaching the maximum protective effect at 10 mM), and taurine (10 mM) inhibited the mineralization level of AVICs in the form of calcium content significantly. Furthermore, taurine activated the extracellular signal-regulated protein kinase (ERK) pathway via TAUT, and the inhibitor of ERK (PD98059) abolished the effect of taurine on both ALP activity/expression and Cbfα1 expression. These results suggested that taurine could inhibit osteoblastic differentiation of AVIC via the ERK pathway.

Topics: Adult; Alkaline Phosphatase; Aortic Valve; Ascorbic Acid; Bicuspid Aortic Valve Disease; Biomarkers; Calcinosis; Calcium; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Dexamethasone; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression; Glycerophosphates; Heart Defects, Congenital; Heart Valve Diseases; Humans; MAP Kinase Signaling System; Membrane Glycoproteins; Membrane Transport Proteins; Myocytes, Smooth Muscle; Osteoblasts; Protein Kinase Inhibitors; Taurine

To investigate the capability of human placenta-derived adherent cells to differentiate into osteocytes and adipocytes.. Placenta-derived adherent cells were isolated by type IV collagenase digestion of a single freshly obtained human placenta and cultured under standard conditions. Cell surface markers of adherent cells from passages 3 - 9 were analysed by flow cytometry. Osteocytic differentiation was induced with β-glycerol phosphate, vitamin C and dexamethasone and confirmed by Alizarin red staining. Adipocytic differentiation was induced with dexamethasone and insulin and confirmed by oil red O staining.. Placenta-derived adherent cells were positive for high levels of CD44 and CD105 and very low levels of CD29 but were negative for CD34, CD45 and CD19. This pattern of cell surface markers is identical to human mesenchymal stem cells. Alizarin red-positive cells were detected 10 days after the induction of osteocyte differentiation. Oil red O-positive cells were detected 7 days after the induction of adipocyte differentiation.. Placenta-derived adherent cells can differentiate into either osteocytes or adipocytes in vitro. The human placenta may provide an alternative source of mesenchymal stem cells for basic research and clinical use.

Topics: Adipocytes; Adipogenesis; Antigens, CD; Antigens, CD19; Antigens, CD34; Ascorbic Acid; Cell Differentiation; Cells, Cultured; Dexamethasone; Endoglin; Female; Glycerophosphates; Humans; Hyaluronan Receptors; Integrin beta1; Leukocyte Common Antigens; Mesenchymal Stem Cells; Osteocytes; Placenta; Pregnancy; Receptors, Cell Surface

Ascorbic acid (AA) and β-glycerophosphate (βG) are considered in vitro osteogenic factors important to the differentiation of osteoblastic progenitor and dental pulp cells into mineralized tissue-forming cells. So, the present study investigated in vitro if these mineralizing inducible factors (AA and βG) could influence differentiation of human gingival fibroblasts when compared with human pulp cells and osteogenic cells derived from rat calvaria cultured. The expression of osteopontin (OPN) and osteoadherin (OSAD) was analyzed by indirect immunofluorescence, immunocytochemistry as well as Western-blotting. In addition, the main ultrastructural aspects were also investigated. No mineralized matrix formation occurred on gingival fibroblasts induced with AA+βG. On these cells, no expression of OPN and OSAD was observed when compared with pulp cells, pulp cells induced with AA+βG as well as osteogenic cells. Ultrastructure analysis additionally showed that gingival fibroblasts exhibited typical fibroblast morphology with no nodule formation. The present findings showed that AA and βG could not promote a mineralized cell differentiation of human gingival fibroblasts and confirm that human dental pulp cells, as the osteogenic cells, are capable to form a mineralized extracellular.

Topics: Animals; Animals, Newborn; Ascorbic Acid; Cell Differentiation; Cells, Cultured; Dental Pulp; Extracellular Matrix Proteins; Fibroblasts; Gingiva; Glycerophosphates; Humans; Osteopontin; Proteoglycans; Rats; Rats, Wistar

Previous studies have conflicting views on the effect of platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) signaling on osteogenesis. The current study investigated the effect of PDGF receptor-beta (PDGFR-β) inhibition by AG-1295 on the osteogenic differentiation of the mouse pre-osteoblastic cell line MC3T3-E1. Osteogenic differentiation was induced by treatment with β-glycerophosphate, ascorbic acid, and dexamethasone along with or absent AG-1295. Results showed that AG-1295 significantly increased alkaline phosphatase (ALP) activity and enhanced the formation of mineralized nodules in a dose-dependent manner. Furthermore, treatment with AG-1295 resulted in up-regulated mRNA expression of the osteogenic marker genes collagen type I (Col1A), runt-related transcription factor 2 (Runx2), osterix (Osx), tissue-nonspecific alkaline phosphatase (Tnap), and osteocalcin (Ocn). Consistent with its effect on osteoblast differentiation, AG-1295 also significantly suppressed the phosphorylation of Erk1/2 in MC3T3-E1 cells. In conclusion, findings suggest that blocking the PDGFR-β pathway with AG1295 markedly promotes osteoblast differentiation and matrix mineralization in mouse osteoblastic MC3T3-E1 cells and that the Erk1/2 pathway might participate in this process.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Blotting, Western; Cell Differentiation; Cell Line; Collagen Type I; Collagen Type I, alpha 1 Chain; Core Binding Factor Alpha 1 Subunit; Dexamethasone; Dose-Response Relationship, Drug; Gene Expression Regulation; Glycerophosphates; MAP Kinase Signaling System; Mice; Osteoblasts; Osteocalcin; Osteogenesis; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptor, Platelet-Derived Growth Factor beta; Sp7 Transcription Factor; Transcription Factors; Tyrphostins

This present study investigated the potential of Morinda citrifolia leaf aqueous extract to induce osteogenic differentiation and matrix mineralization of human periodontal ligament (hPDL) cells. Human periodontal ligament cells were cultured in complete medium, ascorbic acid with β-glycerophosphate, or Morinda citrifolia leaf aqueous extract. Morinda citrifolia leaf aqueous extract significantly increased alkaline phosphatase activity compared to culturing in complete medium or ascorbic acid with β-glycerophosphate. Matrixcontaining mineralized nodules were formed only when the cells were cultured in the presence of Morinda citrifolia leaf aqueous extract. These nodules showed positive alizarin red S staining and were rich in calcium and phosphorus according to energy dispersive X-ray analysis. In conclusion, Morinda citrifolia leaf extract promoted osteogenic differentiation and matrix mineralization in human periodontal ligament cells, a clear indication of the therapeutic potential of Morinda citrifolia leaves in bone and periodontal tissue regeneration.

Topics: 3T3 Cells; Adolescent; Adult; Alkaline Phosphatase; Animals; Anthraquinones; Ascorbic Acid; Calcification, Physiologic; Calcium; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Coloring Agents; Culture Media; Extracellular Matrix; Glycerophosphates; Humans; Mice; Microscopy, Electron, Scanning; Morinda; Osteoblasts; Osteogenesis; Periodontal Ligament; Phosphorus; Plant Extracts; Plant Leaves; Spectrometry, X-Ray Emission; Young Adult

Despite numerous reports on the ability of ascorbic acid and β-glycerophosphate (AA/β-GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential substrates) for protein kinases and traditional biochemical techniques to examine the signaling pathways modulated during AA/β-GP-induced osteoblast differentiation. The kinomic profile obtained after 7 days of treatment with AA/β-GP identified 18 kinase substrates with significantly enhanced or reduced phosphorylation. Peptide substrates for Akt, PI3K, PKC, BCR, ABL, PRKG1, PAK1, PAK2, ERK1, ERBB2, and SYK showed a considerable reduction in phosphorylation, whereas enhanced phosphorylation was observed in substrates for CHKB, CHKA, PKA, FAK, ATM, PKA, and VEGFR-1. These findings confirm the potential usefulness of peptide microarrays for identifying kinases known to be involved in bone development in vivo and in vitro and show that this technique can be used to investigate kinases whose function in osteoblastic differentiation is poorly understood.

Topics: Animals; Ascorbic Acid; Cell Differentiation; Cells, Cultured; Glycerophosphates; Mice; Osteoblasts; Phosphorylation; Signal Transduction

MC3T3-E1 cells grown in the presence of ascorbic acid and β-glycerophosphate (AA/β-GP) express alkaline phosphatase and produce an extensive collagenous extracellular matrix. Differentiated MC3T3-E1 cells are more sensitive to hydrogen peroxide-induced oxidative stress than undifferentiated cells. In this study, we compared the profile of antioxidant enzymes and molecular markers of apoptosis in undifferentiated and differentiated MC3T3-E1 cells (cell differentiation was induced by treatment with AA/β-GP). Differentiated osteoblasts showed lower expression and activity of catalase, glutathione S-transferase and glutathione peroxidase. The total superoxide dismutase activity and the expression of Cu/Zn superoxide dismutase were also lower, while the expression of Mn superoxide dismutase was higher in differentiated osteoblasts. The level of malondialdehyde, a widely used marker for oxidative stress, was lower in the AA/β-GP group compared with control cells, but this difference was not significant. Western blotting showed that treatment with AA/β-GP increased the Bax/Bcl-2 ratio used as an index of cellular vulnerability to apoptosis. In addition, the activities of caspases 3, 8 and 9 and cleaved poly (ADP) ribose polymerase were significantly higher in differentiated cells. These findings provide new insights into how changes in the activities of major antioxidant enzymes and in the signaling pathways associated with apoptosis may influence the susceptibility of bone cells to oxidative stress.

Topics: Animals; Antioxidants; Ascorbic Acid; Blotting, Western; Cell Differentiation; Cell Line; Glycerophosphates; Hydrogen Peroxide; Mice; Osteoblasts

Numerous previous studies have investigated the production of mineralised tissues by transplanting human dental pulp cells with calcium based scaffolds. The potential of alternative setups remains largely uninvestigated, therefore in this study, human dental pulp cells were encapsulated into non-calcium based biomaterial - self-assembling peptide nano-fibre hydrogel. The cell-gel constructs were cultured in full medium for 2 weeks. Then they were cultured in full medium supplemented with β-glycerophosphate, dexamethasone and l-ascorbic acid for 2 more weeks. These cell-gel constructs and plain-gel constructs (with no cells) were transplanted subcutaneously into five nude mice. The gel constructs were retrieved 4 weeks after surgery. The plain-gel constructs were all completely resorbed with no new tissue formation. The cell-gel constructs were transformed into tissue pieces that were mineralised and contained blood capillaries. Immunohistochemistry analysis confirmed the expression of multiple bone markers (osteopontin, osteocalcin, osteonectin and parathyroid hormone receptor) in these tissue pieces. Computerised analysis of the contact radiographs gave the mean radio-opaque area percentage as 78% (N=5, P<0.001 compared with the 0% of the control). The results demonstrate good prospects for using human dental pulp cell plus self-assembling peptide nano-fibre hydrogel to produce mineralised tissue pieces for clinical use.

Topics: Animals; Antioxidants; Ascorbic Acid; Calcification, Physiologic; Capillaries; Cell Culture Techniques; Cell Survival; Culture Media; Dental Pulp; Dexamethasone; Glucocorticoids; Glycerophosphates; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Mice; Mice, Nude; Nanofibers; Osteocalcin; Osteonectin; Osteopontin; Peptides; Pilot Projects; Receptor, Parathyroid Hormone, Type 1; Stem Cells; Subcutaneous Tissue; Time Factors; Tissue Engineering; Tissue Scaffolds

The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells.. Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4),β-glycerophosphate and L-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5mM) and TEGDMA (0.05-0.25mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2mM) and TEGDMA (1mM) were also evaluated.. DTSCs cultures were positive for STRO-1 (7.53±2.5%), CD146 (91.79±5.41%), CD34 (11.87±3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated.. These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.

Topics: Alkaline Phosphatase; Antigens, CD34; Antigens, Surface; Ascorbic Acid; Biomarkers; Calcification, Physiologic; CD146 Antigen; Cell Differentiation; Cell Movement; Cells, Cultured; Child, Preschool; Dental Pulp; Dexamethasone; Down-Regulation; Extracellular Matrix Proteins; Glucocorticoids; Glycerophosphates; Humans; Infant; Integrin-Binding Sialoprotein; Leukocyte Common Antigens; Methacrylates; Odontogenesis; Osteocalcin; Phosphates; Phosphoproteins; Polyethylene Glycols; Polymethacrylic Acids; Potassium Compounds; Sialoglycoproteins; Stem Cells; Tooth, Deciduous

Despite the frequent use of primary dental pulp cells in dental regenerative research, few systematic studies of stemness for osteogenic and dentinogenic differentiation of human adult pulp cells have been reported. To investigate the stemness of human adult dental pulp cells, pulp tissues were obtained from extracted third molars and used as a source of pulp cells. In FACS analysis and immunophenotyping, the general mesenchymal stem cell markers CD44, CD90, and CD146 were highly expressed in early passages of the pulp cell culture. The stem cell population was dramatically decreased in an expansion culture of human dental pulp cells. When pulp cells were treated with additives such as β-glycerophosphate, ascorbic acid, and dexamethasone, nodule formation was facilitated and mineralization occurred within 2 weeks. Expression of osteogenic markers such as alkaline phosphatase, osteocalcin, and osteonectin was relatively low in undifferentiated cells, but increased significantly under differentiation conditions in whole passages. Dentinogenic markers such as dentin sialophosphoprotein and dentin matrix protein-1 appeared to decrease in their expression with increasing passage number; however, peak levels of expression occurred at around passage 5. These data suggested that stem cells with differentiation potential might exist in the dental pulp primary culture, and that their phenotypes were changed during expansion culture over 8-9 passages. Under these conditions, a dentinogenic population of pulp cells occurred in limited early passages, whereas osteogenic cells occurred throughout the whole passage range.

Topics: Adult; Alkaline Phosphatase; Ascorbic Acid; Biomarkers; Blotting, Western; CD146 Antigen; Cell Differentiation; Cells, Cultured; Dental Pulp; Dentinogenesis; Dexamethasone; Extracellular Matrix Proteins; Flow Cytometry; Glycerophosphates; Humans; Hyaluronan Receptors; Immunophenotyping; Molar, Third; Osteocalcin; Osteonectin; Phosphoproteins; Polymerase Chain Reaction; Sialoglycoproteins; Thy-1 Antigens

Matrix vesicles (MVs) are cell-derived membranous entities crucial for mineral formation in the extracellular matrix. One of the dominant groups of constitutive proteins present in MVs, recognised as regulators of mineralization in norm and pathology, are annexins. In this report, besides the annexins already described (AnxA2 and AnxA6), we identified AnxA1 and AnxA7, but not AnxA4, to become selectively enriched in MVs of Saos-2 cells upon stimulation for mineralization. Among them, AnxA6 was found to be almost EGTA-non extractable from matrix vesicles. Moreover, our report provides the first evidence of annexin-binding S100 proteins to be present in MVs of mineralizing cells. We observed that S100A10 and S100A6, but not S100A11, were selectively translocated to the MVs of Saos-2 cells upon mineralization. This observation provides the rationale for more detailed studies on the role of annexin-S100 interactions in MV-mediated mineralization.

Topics: Annexins; Ascorbic Acid; Bone Matrix; Calcification, Physiologic; Calcinosis; Cell Fractionation; Cell Line; Cell Line, Tumor; Cytoplasmic Vesicles; Cytoskeletal Proteins; Glycerophosphates; Humans; Protein Transport; S100 Proteins

For bone regeneration therapy using stem cells, well-defined ex vivo protocols to expand mesenchymal stromal cells (MSC), as well as assays to show their potential differentiation into the osteogenic lineage, are needed. Aim of this study was to analyze the role of the biochemical osteogenic inducers, i.e. ascorbic acid, dexamethasone, and ß-glycerophosphate, employed in the current protocols for osteogenic differentiation of MSC in vitro, to address the requirements for reliable differentiation systems.. MSC were isolated from the bone marrow of donors (46-73 years of age) undergoing total hip replacement, and expanded in vitro. At confluence, MSC were cultured under four different conditions: α-MEM plus serum (basal medium or C1), basal medium plus ascorbate (C2), basal medium plus ascorbate and dexamethasone (C3), or basal medium plus ascorbate, dexamethasone and ß-glycerophosphate (C4). Morphology, proliferation, mineralization, alkaline phosphatase, collagen and expression of bone-related genes of MSC under the different media were analyzed at fixed time points.. MSC proliferation and the number of colony forming units were increased by ascorbic acid, whereas dexamethasone enhanced the proportion of ALP-positive CFU and was critical for mineral deposition. Runx-2 and type I collagen gene expression decreased along with additive-induced MSC differentiation, i.e. from C1 to C4, while ALP and osteocalcin were differently regulated.. Our findings support the role of different inducers on the sequential stages of MSC expansion and osteogenic differentiation in vitro, suggesting the addition of DEX following proliferation to ensure mineralization, as an index of in vivo osteogenic potency of human mesenchymal cells.

Topics: Aged; Alkaline Phosphatase; Ascorbic Acid; Cell Differentiation; Cell Proliferation; Cells, Cultured; Collagen Type I; Dexamethasone; Female; Glycerophosphates; Humans; Male; Mesenchymal Stem Cells; Middle Aged; Osteogenesis

Cementum and bone are rather similar hard tissues, and osteocytes and cementocytes, together with their canalicular network, share many morphological and cell biological characteristics. However, there is no clear evidence that cementocytes have a function in tissue homeostasis of cementum comparable to that of osteocytes in bone. Recent studies have established an important role for the secreted glycoprotein sclerostin, the product of the SOST gene, as an osteocyte-derived signal to control bone remodelling. In this study, we investigated the expression of sclerostin in cementocytes in vivo as well as the expression of SOST and sclerostin in periodontal ligament cell cultures following induction of mineralization.. Immunolocalization of sclerostin was performed in decalcified histological sections of mouse and human teeth and alveolar bone. Additionally, periodontal ligament cells from human donors were cultured in osteogenic conditions, namely in the presence of dexamethasone, ascorbic acid and beta-glycerophosphate, for up to 3 wk. The induction of calcified nodules was visualized by von Kossa stain. SOST mRNA was detected by real-time PCR, and the presence of sclerostin was verified using immunohistochemistry and western blots.. Expression of sclerostin was demonstrated in osteocytes of mouse and human alveolar bone. Distinct immunolocalization in the cementocytes was shown. In periodontal ligament cultures, following mineralization treatment, increasing levels of SOST mRNA as well as of sclerostin protein could be verified.. The identification of SOST/sclerostin in cementocytes and mineralizing periodontal ligament cells adds to our understanding of the biology of the periodontium, but the functional meaning of these findings can only be unravelled after additional in vitro and in vivo studies.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Adult; Aged; Alveolar Process; Animals; Ascorbic Acid; Blotting, Western; Bone Morphogenetic Proteins; Calcification, Physiologic; Cell Differentiation; Cells, Cultured; Child; Dental Cementum; Dexamethasone; Fibroblasts; Genetic Markers; Glycerophosphates; Glycoproteins; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Mice; Middle Aged; Osteocytes; Osteogenesis; Periodontal Ligament; Polymerase Chain Reaction; Young Adult

Culture of expanded mesenchymal stem cells (MSCs) seeded on biomaterials may represent a clinical alternative to autologous bone graft in bone regeneration. Foetal bovine serum (FBS) is currently used for MSC expansion, despite risks of infectious disease transmission and immunological reaction due to its xenogenic origin. This study aimed to compare the osteogenic capacities of clinical-grade human MSCs cultured with FBS or allogenic human platelet lysate (PL). In vitro, MSCs cultured in PL both accelerate the expansion rate over serial passages and spontaneously induce osteoblastic gene expression such as alkaline phosphatase (ALP), bone sialoprotein (BSP), osteopontin (Op) and bone morphogenetic protein-2 (BMP-2). In vivo, ectopic bone formation is only observed on ceramics seeded with MSCs grown in PL medium implanted under the skin of immunodeficient mice for 7 weeks. In conclusion, allogenic human PL accelerates MSC proliferation and enhances MSC osteogenic differentiation.

Topics: Animals; Ascorbic Acid; Blood Platelets; Calcification, Physiologic; Calcium Phosphates; Cell Differentiation; Cell Extracts; Cell Proliferation; Cell Shape; Cells, Cultured; Culture Media; Durapatite; Gene Expression Regulation; Glycerophosphates; Humans; Mesenchymal Stem Cells; Mice; Osteoblasts; Osteogenesis; Phenotype; Tissue Scaffolds

In this study, in vitro osteogenesis was successfully achieved in human mesenchymal stem cells (hMSCs) by controlled release of the osteogenesis-inducing drugs dexamethasone, ascorbic acid (AA) and beta-glycerophosphate (GP) from poly(lactic-co-glycolic acid) (PLGA) sintered microsphere scaffolds (SMS). We investigated the osteogenesis of human MSCs (hMSCs) on dexamethasone laden PLGA-SMS (PLGA-Dex-SMS), and dexamethasone, AA and GP laden PLGA-SMS (PLGA-Com-SMS). hMSCs cultured on the microsphere systems, which act as drug release vehicles and also promote cell growth/tissue formation-displayed a strong osteogenic commitment locally. The osteogenic commitment of hMSCs on the scaffolds were verified by alkaline phosphatase (ALP) activity assay, calcium secretion assay, real-time PCR and immunohistochemistry analysis. The results indicated hMSCs cultured on PLGA-Com-SMS exhibited superior osteogenic differentiation owing to significantly high phenotypic expression of typical osteogenic genes-osteocalcin (OC), type I collagen, alkaline phosphatase (ALP), and Runx-2/Cbfa-1, and protein secretion of bone-relevant markers such as osteoclast and type I collagen when compared with PLGA-Dex-SMS. In conclusion, by promoting osteogenic development of hMSCs in vitro, this newly designed controlled release system opens a new door to bone reparation and regeneration.

Topics: Ascorbic Acid; Biocompatible Materials; Cell Proliferation; Cells, Cultured; Delayed-Action Preparations; Dexamethasone; Drug Carriers; Glycerophosphates; Humans; Lactic Acid; Mesenchymal Stem Cells; Microspheres; Osteogenesis; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Tissue Engineering; Tissue Scaffolds

The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined.. We evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and beta-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers (receptor activator of nuclear factor kappa B ligand [RANKL], bone sialoprotein [BSP], osteopontin [OPN]).. Dasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity.. Our results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism.

Topics: Adolescent; Adult; Alkaline Phosphatase; Ascorbic Acid; Bone Marrow Cells; Calcification, Physiologic; Cell Differentiation; Cell Proliferation; Cell Separation; Cells, Cultured; Child; Child, Preschool; Dasatinib; Dexamethasone; Dose-Response Relationship, Drug; Extracellular Matrix; Gene Expression Regulation; Glycerophosphates; Humans; Integrin-Binding Sialoprotein; Kinetics; Mesenchymal Stem Cells; Middle Aged; Osteoblasts; Osteogenesis; Osteopontin; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Mas; Pyrimidines; RANK Ligand; Sialoglycoproteins; src-Family Kinases; Thiazoles; Young Adult

This study was designed to investigate the potential merits of the combined use of bone morphogenetic protein (BMP)-2 or BMP-6 and osteogenic supplements (OS) [dexamethasone, ascorbic acid (AA), and β-glycerophosphate] on osteogenic differentiation of periodontal ligament cells (PDLCs). Osteogenic differentiation was evaluated by quantitative alkaline phosphatase (ALP) assay, alizarin red staining, quantitative calcium assay, and the qRT-PCR analysis for the expression of collagen type I, runt-related transcription factor-2, osteopontin (OPN), and osteocalcin in PDLCs. Culture with BMP-2 or BMP-6+AA increased ALP activity of PDLCs, suggesting their osteo-inductive effects. However, longer duration of culture showed neither of the BMPs induced in vitro mineralization. In contrast, OS were able to increase ALP activity and OPN expressions, and also induced in vitro mineralization. The mineralization ability was not enhanced by the addition of BMP-2 or BMP-6. These findings suggest that the addition of BMP-2 or BMP-6 to OS may not enhance an osteogenic differentiation of hPDLCs.

Topics: Alkaline Phosphatase; Anthraquinones; Ascorbic Acid; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 6; Calcification, Physiologic; Calcium; Cells, Cultured; Collagen Type I; Core Binding Factor Alpha 2 Subunit; Dexamethasone; Glycerophosphates; Humans; Osteocalcin; Osteogenesis; Osteopontin; Periodontal Ligament; Polymerase Chain Reaction; RNA, Messenger

Notch signalling is of fundamental importance to various processes during embryonic development and in adults. The possible role of Hey1, an important Notch signalling component, in odontoblast differentiation was evaluated in this study. Primary cultured dental pulp cells, derived from upper incisors of 5-week-old Wistar rats, were placed in alpha-modification of Eagle's minimal essential medium supplemented with 10% Fetal Bovine Serum (FBS), and ascorbic acid (AA) and beta-glycerophosphate (beta-GP), with or without dexamethasone, and cultured on dishes coated with collagen type IA for 7 days. Conventional and real-time Polymerase Chain Reaction (PCR) was performed to determine the expression of Notch-related genes and dentin sialophosphoprotein as a marker of odontoblast differentiation. Dentin sialophosphoprotein and Hey1 expression was significantly increased and decreased in the presence of AA + beta-GP compared with controls, respectively. These findings suggest that Hey1 may be a negative regulator in odontoblast differentiation.

Topics: Animals; Ascorbic Acid; Basic Helix-Loop-Helix Transcription Factors; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Collagen Type I; Culture Media; Dental Pulp; Dexamethasone; Extracellular Matrix Proteins; Glucocorticoids; Glycerophosphates; Helix-Loop-Helix Motifs; Homeodomain Proteins; Male; Odontoblasts; Phosphoproteins; Rats; Rats, Wistar; Receptor, Notch1; Receptor, Notch2; Receptor, Notch3; Receptors, Notch; Repressor Proteins; Sialoglycoproteins; Signal Transduction; Transcription Factor HES-1

Primordial germ cells (PGCs) are useful for producing transgenic chickens and preserving genetic material in avian species. In this study, we investigated the in vitro differentiation potential of chicken PGCs into different cell types. For differentiation into adipocytes, chicken PGCs were cultured for 21 days in induction media containing dexamethasone, insulin and/or 3-isobutyl-1-methylxanthine (IBMX), and differentiation rates ranging from 74% to 91% were identified by oil red-O and alkaline phosphatase (ALP) staining. For differentiation into neuron-like cells, chicken PGCs were cultured for 3 or 7 days in the induction media containing retinoic acid (RA) and IBMX, and differentiation rates ranging from 71% to 87% were identified by toluidine blue staining and immunohistochemical staining. For differentiation into osteoblasts, chicken PGCs were cultured for 15 or 21 days in the induction media containing desamethasone, beta-glycerol phosphate and/or vitamin C, and differentiation rates ranging from 47% to 79% were confirmed by Von Kossa, cytochemical and immunohistochemical staining. These data suggest that, like mammalian PGCs, chicken PGCs can differentiate into different cell types in vitro.

Topics: 1-Methyl-3-isobutylxanthine; Adipocytes; Adipogenesis; Alkaline Phosphatase; Animals; Ascorbic Acid; Cell Culture Techniques; Cell Differentiation; Chickens; Dexamethasone; Embryonic Stem Cells; Germ Cells; Glycerophosphates; Insulin; Neurogenesis; Neurons; Osteoblasts; Osteogenesis; Tretinoin

Voltage gated chloride channels (ClCs) play an important role in the regulation of intracellular pH and cell volume homeostasis. Mutations of these genes result in genetic diseases with abnormal bone deformation and body size, indicating that ClCs may have a role in chondrogenesis. In the present study, we isolated chicken mandibular mesenchymal cells (CMMC) from Hamburg-Hamilton (HH) stage 26 chick embryos and induced chondrocyte maturation by using ascorbic acid and β-glycerophosphate (AA-BGP). We also determined the effect of the chloride channel inhibitor NPPB [5-nitro-2-(3-phenylpropylamino) benzoic acid] on regulation of growth, differentiation, and gene expression in these cells using MTT and real-time PCR assays. We found that CLCN1 and CLCN3-7 mRNA were expressed in CMMC and NPPB reduced expression of CLCN3, CLCN5, and CLCN7 mRNA in these cells. At the same time, NPPB inhibited the growth of the CMMC, but had no effect on the mRNA level of cyclin D1 and cyclin E (P>0.05) with/without AA-BGP treatment. AA-BGP increased markers for early chondrocyte differentiation including type II collagen, aggrecan (P<0.01) and Sox9 (P<0.05), whilst had no effect on the late chondrocyte differentiation marker type X collagen. NPPB antagonized AA-BGP-induced expression of type II collagen and aggrecan (P<0.05). Furthermore, NPPB downregulated type X collagen (P<0.05) with/without AA-BGP treatment. We conclude that abundant chloride channel genes in CMMC play important roles in regulating chondrocyte proliferation and differentiation. Type X collagen might function as a target of chloride channel inhibitors during the differentiation process.

Topics: Aggrecans; Animals; Antioxidants; Ascorbic Acid; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chick Embryo; Chloride Channels; Chondrocytes; Chondrogenesis; Collagen Type II; Collagen Type X; Cyclin D1; Cyclin E; Gene Expression Regulation, Developmental; Glycerophosphates; Mandible; Mesoderm; Muscle Proteins; Nitrobenzoates; SOX9 Transcription Factor

Osteogenic supplements are a requirement for osteoblastic cell differentiation during in vitro culture of human calvarial suture-derived cell populations. We investigated the ability of ascorbic acid and beta-glycerophosphate with and without the addition of dexamethasone to stimulate in vivo-like osteoblastic differentiation. Cells were isolated from unfused and prematurely fused suture tissue from patients with syndromic and non-syndromic craniosynostosis and cultured in each osteogenic medium for varying lengths of time. The effect of media supplementation was investigated with respect to the ability of cells to form mineralised bone nodules and the expression of five osteodifferentiation marker genes (COL1A1, ALP, BSP, OC and RUNX2), and five genes that are differentially expressed during human premature suture fusion (GPC3, RBP4, C1QTNF3, WIF1 and FGF2). Cells from unfused sutures responded more slowly to osteogenic media but formed comparable bone nodules to fused suture-derived cells after 16 days of culture in either osteogenic media. However, gene expression differed between unfused and fused suture-derived cells, as did expression in each osteogenic medium. When compared to expression in the explant tissue of origin, neither medium induced a level or profile of gene expression similar to that seen in vivo. Overall, our results demonstrate that cells from the same suture that are isolated during different stages of morphogenesis in vivo, despite being de-differentiated to a similar level in vitro, respond uniquely and differently to each osteogenic medium. Further, we suggest that neither cell culture medium recapitulates differentiation via activation of the same genetic cascades as occurs in vivo.

Topics: Ascorbic Acid; Base Sequence; Cell Differentiation; Cells, Cultured; Cranial Sutures; Craniosynostoses; Culture Media; Dexamethasone; DNA Primers; Fibroblast Growth Factor 2; Gene Expression; Gene Expression Profiling; Glycerophosphates; Humans; In Vitro Techniques; Infant; Male; Osteoblasts; Osteogenesis; Phenotype; Tumor Necrosis Factors

Characterization of directed differentiation protocols is a prerequisite for understanding embryonic stem cell behavior, as they represent an important source for cell-based regenerative therapies. Studies have investigated the osteogenic potential of human embryonic stem cells (HESCs), building upon those using pre-osteoblastic cells, however no consensus exists as to whether differentiating HESCs behave in a similar manner to the traditionally used osteoblastic progenitors. Thus, the aim of the current investigation was to define the gene expression pattern of osteoblastic differentiating HESCs, treated with ascorbic acid phosphate, beta-glycerophosphate and dexamethasone over a 25 day period. Characterization of the gene expression dynamics revealed a phasic pattern of bone-associated protein synthesis. Collagen type I and osteopontin were initially expressed in proliferating immature cells, whereas osterix was up-regulated at the end of active cellular proliferation. Subsequently, mineralization-associated proteins, bone sialoprotein and osteocalcin were detected. In light of this dynamic expression pattern, we concluded that two distinguishable phases occurred during osteogenic HESC differentiation; first, cellular proliferation and secretion of a pre-maturational matrix, and second the appearance of osteoprogenitors with characteristic extracellular matrix synthesis. Establishment of this model provided the foundation of a time-frame for the additional supplementation with growth factors, BMP2 and VEGF. BMP2 induced the expression of principle osteogenic factors, such as osterix, bone sialoprotein and osteocalcin, whereas VEGF had the converse effect on the gene expression pattern.

Topics: Ascorbic Acid; Bone Matrix; Bone Morphogenetic Protein 2; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cells, Cultured; Dexamethasone; Embryonic Stem Cells; Gene Expression Regulation; Glycerophosphates; Humans; Models, Biological; Osteogenesis; Vascular Endothelial Growth Factor A

Fourteen stable subclones derived from the murine chondrogenic cell line MC615 were established and characterised regarding their differentiation stages and responsivity to BMP2. Based on their gene expression profiles which revealed remarkable variances in Col2a1 and Col10a1 expression, subclones could be grouped into at least three distinct categories. Three representative subclones (4C3, 4C6 and 4H4) were further characterised with respect to gene expression pattern and differentiation capacity. These subclones resembled (i) weakly differentiated chondrogenic precursors, strongly responding to BMP2 stimulation (4C3), (ii) collagen II expressing chondrocytes which could be induced to undergo maturation (4C6) and (iii) mature chondrocytes expressing Col10a1 and other markers of hypertrophy (4H4). Interestingly, BMP2 administration caused Smad protein phosphorylation and stimulated Col10a1 expression in all clones, but induced Col2a1 expression only in precursor-like cells. Most remarkably, these clones maintained a stable gene expression profile at least until the 30th passage of subconfluent culture, but revealed reproducible changes in gene expression and differentiation pattern in long term high density cultures. Thus, the newly established MC615 subclones may serve as a potent new tool for investigations on the regulation of chondrocyte differentiation and function.

Topics: Animals; Ascorbic Acid; Biomarkers; Bone Morphogenetic Protein 2; Cell Differentiation; Cell Line; Cell Proliferation; Cell Shape; Chondrocytes; Chondrogenesis; Clone Cells; Collagen; Extracellular Matrix; Gene Expression Regulation; Glycerophosphates; Insulin; Mice; Models, Biological; Molecular Mimicry; Phenotype

To examine the effect of carbon dioxide laser irradiation on mineralization in dental pulp cells.. Rat dental pulp cells were irradiated with a carbon dioxide laser at 2 W output power for 20, 40 and 60 s, and were cultured in ascorbic acid and beta-glycerophosphate containing media. Cell viability was examined 24 h after laser irradiation by a modified MTT assay. Alizarin Red S staining was performed 10 days after laser irradiation. The amounts of secreted collagen from the cells after irradiation were quantified following Sirius Red staining. The expression levels of collagen type I and HSP47, collagen-binding stress protein, were analysed by real-time PCR. HSP47 protein expression was examined by Western blotting. Statistical analysis was performed using one-way analysis of variance (anova) followed by the Tukey's multiple comparison test.. The cell viability was not affected by laser irradiation at 2 W for up to 40 s. However, it was significantly decreased by 20% at 60 s (P < 0.05). The amount of mineralization after 10 days of irradiation at 2 W for 40 s was significantly increased in comparison to the other conditions (P < 0.05). The extracellular collagen production was significantly increased by 73% on day 2 and 38% on day 4 after laser irradiation (P < 0.05). Although collagen type I gene expression was not changed by laser irradiation, HSP47 gene and protein expression was induced within 12 and 24 h, respectively.. These results suggested that carbon dioxide laser irradiation stimulated mineralization in dental pulp cells. The laser irradiation also increased HSP47 expression but not collagen gene expression.

Topics: Animals; Anthraquinones; Ascorbic Acid; Calcification, Physiologic; Cell Survival; Cells, Cultured; Collagen; Collagen Type I; Coloring Agents; Culture Media; Dental Pulp; Female; Glycerophosphates; HSP47 Heat-Shock Proteins; Lasers, Gas; Rats; Rats, Wistar; Tetrazolium Salts; Thiazoles; Time Factors

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stroma cells which can provide a potential therapy for diabetes mellitus. But the mechanism is still controversial. Also, the status of BM-MSCs under hyperglycemia is not known. In the present study, we investigated the status of BM-MSCs in experimental-diabetic rat and demonstrated the rescue of experimental diabetes by diabetic MSCs transplantation.. BM-MSCs were cultured and the potential of multiple-differentiation was identified through induction into osteoblasts. MSCs of passage 3 were used for the following experiment. The MSCs were labeled with 5-bromo-2?-deoxyuridine (BrdU). Diabetes in rats was induced by STZ injection. The rats were divided into three groups: normal control group (no DM, rats treated with saline through tail vein, n=10); DM control group (DM, no transplantation of MSCs, n=20); experimental group (DM and transplantation of MSCs, n=20). Body weight and blood glucose of the rats were monitored during the experiment after transplantation of MSCs. Paraffin sections of pancreas were obtained from rats of each group. Immuno-histochemistry analysis and double immunofluorescence were used to detect the BM-MSCs in the pancreatic tissue and their differentiating state.. MSCs were 89.5% labeled by BrdU and DAPI, which was green/blue double stained under fluorescent microscopy. Transplantation of diabetic MSCs resulted in a reduction of hyperglycemia on day 45 in experimental diabetic rats compared with control rats (17.7 mM +/-3.9 vs 27.8 mM +/- 2.1, P < 0.05), There was also a difference between MSC-treated experimental diabetic rats and control rats in body weight (232.7 g +/-19.7 vs 133.3g +/-13.1, P < 0.05). Histological and morphometric analysis of the pancreas of experimental diabetic rats showed the presence and differentiation of transplanted MSCs into insulin-producing cells which evidenced by double-staining of anti-BrdU and insulin. Also, there were many small islets throughout the sections. Their mean area and diameter analysis revealed that they were smaller than control islets (1835.7 +/- 175.8 microm2 vs 13257.2 +/- 1457.6 microm2; 43.5 +/- 3.7 microm vs 119.9 +/- 5.8 microm, respectively, P < 0.05).. Allogeneic MSCs transplantation can reduce blood glucose level in recipient rats. A relatively small quantity of transplanted diabetic MSCs survive and transdifferentiate into insulin-producing cells in the pancreas of recipient rats. Upon transplantation these cells initiate endogenous pancreatic regeneration by neogenesis of islet of recipient origin. The present study demonstrates that diabetic MSCs retains its stemness and potential to induce pancreatic regeneration on transplantation.

Topics: Animals; Ascorbic Acid; Blood Glucose; Body Weight; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Dexamethasone; Diabetes Mellitus, Experimental; Glycerophosphates; Immunohistochemistry; Insulin; Insulin-Secreting Cells; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Osteoblasts; Rats; Rats, Wistar; Streptozocin; Transplantation, Homologous

The primary goal of periodontal treatment is regeneration of the periodontium. Current theories suggest that the periodontal ligament (PDL) cells have the capacity to participate in restoring connective and mineralized tissues, when appropriately triggered. We evaluated whether human PDL cell sheets could reconstruct periodontal tissue.. To obtain the cell sheet, human PDL cells were cultured on temperature-responsive culture dishes with or without osteogenic differentiation medium. The cell sheets were transplanted on periodontal fenestration defects of immunodeficient rats. Forty rats were divided in two groups: in one group, cell sheets cultured with control medium were transplanted and in the other, cell sheets cultured with osteogenic differentiation medium were transplanted. The defects were analysed histologically and histomorphologically after healing.. Most of the experimental group exhibited a new cementum-like layer and new attachment of collagen fibres to the layer. Histomorphological analyses indicated significant periodontal regeneration. The control group revealed dense extracellular matrix and fibre formation, but an obvious cementum layer was not observed.. Transplanted PDL cell sheets cultured with osteogenic differentiation medium induced periodontal regeneration containing an obvious cementum layer and Sharpey's fibres. Thus, the method could be feasible as a new therapeutic approach for periodontal regeneration.

Topics: Actins; Animals; Ascorbic Acid; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Cementogenesis; Culture Media, Conditioned; Dexamethasone; Glycerophosphates; Humans; Integrin-Binding Sialoprotein; Osteoblasts; Osteopontin; Periodontal Ligament; Rats; Rats, Nude; Regeneration; Sialoglycoproteins; Tooth Root

When rat bone marrow stromal (BMS) cells were seeded on aligned type I collagen scaffolds and cultured in osteogenic media, they underwent simultaneous maturation and differentiation into osteogenic and vascular cell lineages. In addition, these cells produced mineralized matricellular deposits. BMS cells were seeded in Petri dish or the collagen scaffold, cultured in osteogenic media for 3, 6, and 9 d and subsequently processed for immunohistochemical and cytochemical analysis. Immunolocalization of lineage-specific proteins were visualized using confocal microscopy and mRNA transcript analysis was performed by real-time quantitative polymerase chain reaction (RT-qPCR). The alkaline phosphatase activity and calcium content significantly increased over the observed period of time in an osteogenic medium. Sheets of abundant Pecam (CD31), Flk-1 (VEGFR-2), tomato lectin (TL/LEL), and alpha-smooth muscle actin (alpha-SMA) positive cells were observed in the collagen scaffolds. Nascent capillary-like vessels were also seen amidst the osteoblasts in osteogenic culture, augmenting the maturation and differentiation of BMS cells into osteoblasts. In our in vitro study, concurrent differentiation of BMS cells, a heterogeneous cell population with multilineage differentiation potential, to osteogenic and vascular lineages demonstrated that the substrates (three-dimensional (3-D), collagen type I, aligned fibrils) had a profound effect on guiding the differentiation pathway of BMS cells.

Topics: Actins; Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Marrow Cells; Cell Differentiation; Collagen Type I; Core Binding Factor Alpha 1 Subunit; Dexamethasone; Endothelial Cells; Gene Expression; Glycerophosphates; Immunohistochemistry; Male; Osteoblasts; Osteocalcin; Osteonectin; Osteopontin; Plant Lectins; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Stromal Cells; Vascular Endothelial Growth Factor Receptor-2

Hyaluronan (HA) plays a predominant role in tissue morphogenesis, cell migration, proliferation, and cell differentiation. The aims of the present study were to investigate whether (i) prolonged presence of high concentration (4.0 mg/mL) 800 KDa HA and (ii) pretreatment with HA can modify osteogenic differentiation of pig bone marrow stromal cells (pBMSC). Cell proliferation and mineralization were measured. Expression of differentiation-related genes was evaluated by means of real-time reverse transcription polymerase chain reaction (RT-PCR). HA increased cell proliferation on day 7. HA decreased the basal level of bone-related gene expression and increased the basal level of sox9 marginally during 7-day pretreatment with HA. HA increased calcium deposit on day 21. cbfa1, ALP, and type 1 alpha collagen (Col1) expression was increased when pBMSC were cultivated in osteogenic medium, whereas their expression was decreased in the presence of HA on day 7. On day 14, the addition of HA upregulated cbfa1 and ALP expression compared to osteogenic medium group; there was no significant difference in Col1 expression. At day 21, osteocalcin (OC) expression showed 2.5-fold upregulation over osteogenic medium. These results suggest that exogenous HA stimulates endogenous HA, which together may play a synergetic role in osteogenic differentiation under osteoinducing conditions although gene expression was inhibited at the early stage.

Topics: Adjuvants, Immunologic; Animals; Ascorbic Acid; Bone Marrow Cells; Calcification, Physiologic; Cell Differentiation; Cell Proliferation; Cells, Cultured; Dexamethasone; Female; Gene Expression; Glucocorticoids; Glucuronosyltransferase; Glycerophosphates; Hyaluronan Synthases; Hyaluronic Acid; Osteogenesis; RNA, Messenger; Swine; Vitamins

The objective of this study was to compare the osteogenic potential of human embryonic stem cells (hESCs) within two- and three-dimensional (2D and 3D) culture systems. hESCs of the H1 line (Wicell Inc., Madison, Wisc., USA) were induced to form embryoid bodies (EBs) through 5 days of suspension culture within non-adherent culture dishes. Following enzymatic dissociation, the EB-derived single cells were seeded on either novel 3D porous PLGA scaffolds or 2D culture dishes with the same total cell number. Osteogenic differentiation was induced through culture media supplemented with dexamethasone, L-ascorbic acid and beta-glycerophosphate. After 3 weeks of in vitro culture, quantitative and qualitative assays of osteogenic differentiation were conducted. Osteocalcin secretion and alkaline phosphatase (AP) activities were detected at significantly higher levels within 3D culture compared with the 2D system. Subsequently, the cell-scaffold constructs were implanted in iliac crest defects of immunosuppressed rabbits. After 4 weeks, the constructs were subsequently explanted and characterized by histology and X-ray analysis. Formation of new bone was detected within and around the implanted scaffolds. The results demonstrate that the osteogenic differentiation of human embryonic stem cells is enhanced in a 3D culture system compared to a 2D culture environment. Upon implantation in situ, the differentiating human embryonic stem cells can contribute positively to the repair and regeneration of bone defects.

Topics: Alkaline Phosphatase; Animals; Antigens, Surface; Ascorbic Acid; Bone Regeneration; Calcification, Physiologic; Cell Differentiation; Cell Line; Cell Proliferation; Dexamethasone; Embryonic Stem Cells; Glycerophosphates; Humans; Ilium; Implants, Experimental; Lactic Acid; Male; Microscopy, Confocal; Octamer Transcription Factor-1; Osteocalcin; Osteogenesis; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Rabbits; Tissue Culture Techniques; Tissue Engineering; Tissue Scaffolds

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.

Topics: Analysis of Variance; Ascorbic Acid; Bioreactors; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Culture Techniques; Chondrogenesis; Dexamethasone; Glycerophosphates; Humans; Immunohistochemistry; Insulin; Magnetic Resonance Imaging; Mesenchymal Stem Cells; Osteogenesis; Silk; Tissue Engineering; Transforming Growth Factor beta; Transforming Growth Factor beta1

LIM mineralization protein 1 (LMP-1), an intracellular signaling molecule, regulates osteoblast differentiation and maturation, as well as bone formation. However, the role of LMP-1 in the differentiation of human dental pulp cells and formation of dentin has not been determined. The study was to investigate the expression of LMP-1, the related proteins, such as bone morphogenetic proteins 2, 6 and 7 (BMP-2, BMP-6 and BMP-7), and core binding factor alpha 1 (Cbfa1) during the differentiation of cultured human dental pulp cells and the formation of mineralized nodules.. Differentiation of human dental pulp cells was induced by dexamethasone, asorbic acid and beta-glycerophosphate. The formation of mineralized nodules, was determined by Von Kossa staining and immunocytochemistry detection of dentin sialoprotein. Expression of LMP-1, the related proteins and the differentiation marker alkaline phosphatase (ALP) was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR).. The expression of LMP-1, BMP-2, BMP-6, BMP-7 and Cbfa1 was significantly increased in the process of dental pulp cells differentiation and the formation of mineralized nodules, while the pattern of the expression was distinct.. The elevated level of LMP-1, BMPs and Cbfa1 expression indicated they might play a role in the differentiation of human dental pulp cells and the formation of mineralized nodules.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Adult; Alkaline Phosphatase; Antioxidants; Ascorbic Acid; Biomarkers; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Cytoskeletal Proteins; Dental Pulp; Dexamethasone; Extracellular Matrix Proteins; Glucocorticoids; Glycerophosphates; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Phosphoproteins; Reverse Transcriptase Polymerase Chain Reaction; Sialoglycoproteins; Tooth Calcification; Transforming Growth Factor beta; Zinc Fingers

The anterior cruciate ligament (ACL) inserts into bone through a characteristic fibrocartilagenous interface, which is essential for load transfer between soft and hard tissues. This multi-tissue interface is lost post ACL reconstruction, and the lack of an anatomic fibrocartilage interface between graft and bone remains the leading cause of graft failure. Currently, the mechanism of interface formation is not known. As a fibrocartilage-like tissue is found within the bone tunnel post ACL reconstruction, we hypothesize that fibroblast-osteoblast interactions at the graft-to-bone junction play a role in fibrocartilage formation. To test this hypothesis, a co-culture model permitting osteoblast-fibroblast communications was used to determine the effects of heterotypic interactions on cell phenotype and the development of fibrocartilage-relevant markers in vitro. It was found that co-culture decreased cell proliferation and osteoblast-mediated mineralization, while inducing fibroblast-mediated mineralization. Moreover, the expression of interface-relevant markers such as collagen type II and aggrecan were detected. Our findings suggest that osteoblast-fibroblast interactions may lead to cell trans-differentiation and eventual fibrocartilage formation. These results provide new insight into the mechanism of fibrocartilage formation, which are critical for interface tissue engineering and achieving biological fixation of soft tissue grafts to bone.

Topics: Alkaline Phosphatase; Animals; Animals, Newborn; Anterior Cruciate Ligament; Ascorbic Acid; Biomarkers; Calcification, Physiologic; Cattle; Cell Proliferation; Cells, Cultured; Coculture Techniques; Culture Media; Fibroblasts; Glycerophosphates; Osteoblasts; Phenotype

This study investigated the effects of various components [vitamin D3 (VD3), beta-glycerophosphate (BGP), and ascorbic acid (AA)] on the potential of human adipose-derived progenitor cells (ADPCs) to transdifferentiate into osteoblast-like cells. ADPCs were induced under four different supplement groups: (1) VD3+BGP+AA, (2) VD3 alone, (3) BGP+AA, and (4) no VD3, BGP or AA. Mineralization studies and presence of bone matrix-related proteins by immunostaining showed that the Group 1 ADPCs showed their ability to undergo osteoblastic differentiation. Further evaluation was made by estimation of levels of RUNX-2 and TAZ genes. Group 1 ADPCs showed the consistent expression of RUNX-2 and TAZ levels over the study period of 28days. The study showed good correlation among various parameters evaluated to conclude that ADPCs could be an alternative source for generating osteoblast-like cells.

Topics: Adipose Tissue; Adult; Anthraquinones; Ascorbic Acid; Bone and Bones; Cell Differentiation; Cholecalciferol; DNA Primers; Female; Gene Expression Regulation; Glycerophosphates; Humans; Middle Aged; Osteoblasts; Osteocalcin; Osteonectin; Osteopontin; Stem Cells

Interleukin (IL)-23 and IL-27 are IL-6/IL-12 family members that play a role in the regulation of T helper 1 cell differentiation. Cytokines are known to be involved in the bone remodeling process, although the effects of IL-23 and IL-27 have not been clarified. In this study, we examined the possible roles of these cytokines on osteoblast phenotypes and osteoclastogenesis. We found that IL-27 induced signal transducers and activators of transcription 3 activation in osteoblasts. However, neither IL-23 nor IL-27 showed any significant effects on alkaline phosphatase activity, receptor activator of nuclear factor kappaB ligand (RANKL) expression, mRNA expression such as alkaline phosphatase type I procollagen, or the proliferation of osteoblasts. Osteoclastogenesis from bone marrow cells induced by soluble RANKL was partially inhibited by IL-23 and IL-27 with reduced multinucleated cell numbers, but these interleukins did not affect the proliferation of osteoclast progenitor cells. These results indicate that IL-23 and IL-27 could partly modify cell fusion or the survival of multinucleated osteoclasts. On the other hand, partially purified T cells, which are activated by 2 microg/ml anti-CD3 antibody, completely inhibited osteoclastogenesis by M-CSF/RANKL. On using T cells activated with 0.2 microg/ml anti-CD3 antibody, in which osteoclastogenesis was partially inhibited, the interleukins had additive effects for inhibiting osteoclastogenesis. Although the consequences of phosphorylated signals in osteoblasts have not been identified, IL-23 and IL-27, partly and indirectly through activated T cells, inhibited osteoclastogenesis, indicating that these interleukins may protect against bone destructive autoimmune disorders.

Topics: Animals; Ascorbic Acid; Blotting, Western; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Gene Expression; Glycerophosphates; Interferon-gamma; Interleukin-17; Interleukin-23; Mice; Osteoblasts; Osteoclasts; Receptor Activator of Nuclear Factor-kappa B; Receptors, Interleukin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Time Factors

Human bone marrow-derived mesenchymal stem cells (hBMMSCs) must differentiate into osteogenic cells to allow for successful bone regeneration. In this study, we investigated the effects of different combinations of three soluble osteogenic differentiation-inducing factors [L-ascorbic acid (AC), beta-glycerophosphate (betaG), and bone morphogenic protein-2 (BMP-2)] and the presence of a hydroxyapatite (HA) substrate on hBMMSC osteogenic differentiation in vitro. hBMMSCs were cultured in medium containing various combinations of the soluble factors on culture plates with or without HA coating. After 7 days of culture, alkaline phosphatase (ALP) activity, calcium deposition, and osteoprotegerin (OPG) and osteopontin (OPN) expression were measured. The effects of individual and combined factors were evaluated using a factorial analysis method. BMP-2 predominantly affected expression of early markers of osteogenic differentiation (ALP and OPG). HA had the highest positive effect on OPN expression and calcium deposition. The interaction between AC, betaG, and HA had the second highest positive effect on ALP activity.

Topics: Alkaline Phosphatase; Ascorbic Acid; Bone Marrow Cells; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcium; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Coated Materials, Biocompatible; Dose-Response Relationship, Drug; Durapatite; Glycerophosphates; Humans; Mesenchymal Stem Cells; Osteogenesis; Osteopontin; Osteoprotegerin; Time Factors; Transforming Growth Factor beta

Bone marrow is a complex tissue composed of hematopoietic and stromal stem cells with the potential to differentiate into adipogenic, fibroblastic, reticular, osteogenic and chondrogenic lineages. Identification of differentiation markers during transformation of stromal cells into osteoblasts in a time-dependent manner may be informative for cell-based tissue engineering. Therefore, we investigated the effects of osteogenic medium (OM) on the proliferation and differentiation of rat bone marrow stromal cells (BMSCs). BMSCs from adult male rat tibia and femur were collected and cultured in alpha-MEM medium with 10% fetal bovine serum, penicillin, streptomycin and gentamycin. After three days of culture, the medium covering the adherent cells in culture was changed to OM containing dexamethasone, Na-beta-glycerophosphate and ascorbic acid. As a control, cell culture was also continued in the original medium for the same time period. Differentiated osteoblast cells were collected after 7, 10, 14, 21 and 30 days of culture, fixed with 4% paraformaldehyde and their immunolabelling for osteoblast markers osteonectin (ON) and osteocalcin (OC) was assessed using an indirect immunoperoxidase technique. Immunolabelling of ON and OC was detectable from day 10 of culture, began to increase on day 14, and increased steadily through to day 21. Labelling was highest on day 30 and was more intense in cells cultured with OM compared to the culture without OM. The control cells cultured in the absence of OM produced negligible levels of both markers. In conclusion, our culture system facilitated differentiation of BMSCs into osteoblasts featuring osteoblast markers, and these cells may be useful in autologous bone implant for the treatment of bone wound healing.

Topics: Animals; Ascorbic Acid; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Dexamethasone; Glycerophosphates; Immunohistochemistry; Male; Microscopy, Electron, Scanning; Osteoblasts; Osteocalcin; Osteonectin; Rats; Rats, Wistar; Stromal Cells; Time Factors

We cultured rat mesenchymal stem cells (MSCs) in a medium containing beta-glycerophosphate, ascorbic acid, and dexamethasone to show in vitro osteogenic differentiation of the MSCs. The differentiation was enhanced by adding solubilized type I collagen to the medium as evidenced by higher alkaline phosphatase activity as well as more calcium deposition than that without collagen. The exogenous collagen integrated well with the mineralized bone matrix and maintained the native triple helical structure. These findings indicate that exogenously supplemented type I collagen acts as a component of the extracellular matrix of MSCs, and deposited type I collagen facilitates osteogenic differentiation followed by maturation of mineralized bone matrix.

Topics: Animals; Ascorbic Acid; Calcification, Physiologic; Cell Differentiation; Cells, Cultured; Collagen Type I; Dexamethasone; Extracellular Matrix; Glycerophosphates; Male; Mesenchymal Stem Cells; Microscopy, Confocal; Osteogenesis; Rats; Rats, Inbred F344; Swine

Adult mesenchymal stem cells have the proclivity to differentiate along multiple lineages giving rise to new bone, cartilage, muscle, or fat. Collagen, a normal constituent of bone, provides strength and structural stability and is therefore a potential candidate for use as a substrate on which to engineer bone and cartilage from their respective mesenchymal-derived precursors. In this study, a collagen- glycosaminoglycan scaffold was used to provide a suitable three-dimensional (3-D) environment on which to culture adult rat mesenchymal stem cells and induce differentiation along the osteogenic and chondrogenic lineages. The results demonstrate that adult rat mesenchymal stem cells can undergo osteogenesis when grown on the collagen-glycosaminoglycan scaffold and stimulated with osteogenic factors (dexamethasone, ascorbic acid, beta-glycerophosphate), as evaluated by the temporal induction of the bone-specific proteins, collagen I and osteocalcin, and subsequent matrix mineralization. The osteogenic factors were coupled to activation of the extracellular-regulated protein kinase (ERK), and this kinase was found to play a role in the osteogenic process. As well as supporting osteogenesis, when the cell-seeded scaffold was exposed to chondrogenic factors (dexamethasone and TGF-1beta), collagen II immunoreactivity was increased, providing evidence that the scaffold can also provide a suitable 3-D environment that supports chondrogenesis.

Topics: Animals; Ascorbic Acid; Biocompatible Materials; Cell Differentiation; Cells, Cultured; Chondrogenesis; Collagen; Collagen Type I; Collagen Type II; Dexamethasone; Glycerophosphates; Glycosaminoglycans; Materials Testing; Mesenchymal Stem Cells; Minerals; Mitogen-Activated Protein Kinase Kinases; Osteocalcin; Osteogenesis; Rats; Tissue Engineering; Transforming Growth Factor beta; Transforming Growth Factor beta1

Heat shock (HS)-induced stress response in human cells results in a variety of biological effects and is known to induce the transcription of heat-shock proteins, which help the cells to cope with different kinds of stress. We have studied the effects of HS on the differentiation of human mesenchymal stem cells (hMSCs) into osteoblastic cells. As a model for hMSCs we used a telomerase-immortalized hMSC line designated hMSC-TERT. Cells were exposed to 1 h HS at 41 degrees C, 42.5 degrees C, or 44 degrees C prior to incubation in a medium containing either 10(-8) M 1alpha,25-dihydroxy-vitamin-D(3) (calcitriol) or 10(-8) M calcitriol, 50 microg/mL L-ascorbic acid, and 10 mM beta-glycerophosphate followed by an analysis of induction of osteoblast differentiation and the formation of mineralized matrix, respectively. Our results indicate that the exposure of cells to mild heat stress enhances the extent of differentiation of hMSCs by 12% to 42%. These effects are an expression of the phenomenon of mild stress-induced hormesis.

Topics: Adult; Ascorbic Acid; Bone Marrow Cells; Calcitriol; Cell Differentiation; Cell Line, Transformed; Culture Media; DNA-Binding Proteins; Dose-Response Relationship, Drug; Female; Glycerophosphates; Heat-Shock Response; Hot Temperature; Humans; Ilium; Male; Mesenchymal Stem Cells; Osteoblasts; Telomerase; Time Factors

The objectives of this work were to: (1) establish methodology for pretreating osteoblast-like cells in vitro with dexamethasone to cause glucocorticoid-induced osteoporosis, (2) perform quantitative and qualitative assessments of cellular attachment of osteoporosis-like osteoblasts when grown on implant surfaces of differing roughness, (3) and explore the hypothesis that dexamethasone-treated osteoblasts have altered cell attachment properties by focal adhesion disassembly and decreased tyrosine phosphorylation of the focal adhesion tyrosine kinase.. Osteoblasts were cultured with dexamethasone (10(-7) and 10(-6) M) for up to 4 days of incubation to induce osteoporosis-like conditions. Cellular attachment assays demonstrated the effect of dexamethasone treatments on cellular attachment properties of osteoblasts. Qualitative data were obtained utilizing immunofluorescent microscopy and Western blotting. Focal adhesion kinase (FAK) immunoprecipitation and tyrosine-phosphorylation Western blots were obtained from dexamethasone-treated human embryonic palatal mesenchymal- 1486 osteoblast cultures supplemented with ascorbate and beta-glycerol phosphate medium.. Cellular attachment was significantly greater (P < 0.05) with non-dexamethasone-treated osteoblasts (92%) as compared to dexamethasone-treated osteoblasts after 1 (72%), 2 (63%), and 4 days (53%) of exposure. Dexamethasone-treated osteoblasts were viable and capable of proliferation, suggesting that the reduction of cellular attachment may be related to these cell adhesion processes. Immunofluorescent microscopy of both dexamethasone-treated osteoblasts and non-dexamethasone-treated osteoblasts failed to show any relative difference in the disassembly of focal adhesions and actin filaments. Extended dexamethasone treatment periods (up to 3 weeks) showed changes in the levels of FAK and FAK-phosphotyrosine in human embryonic palatal mesenchymal-1486 osteoblasts.. The protocol used in this study demonstrated a glucocorticoid-induced osteoporosis-like suppression of osteoblasts. FAK disassembly was not a significant factor in short period; however, FAK protein levels and phosphotyrosine signaling on FAK were affected after 1-week exposure to dexamethasone. Phosphorylated FAK was not associated with the rise in the level of FAK, further indicating the possibility of FAK involvement in reduced cell attachment.

Topics: 3T3 Cells; Animals; Antioxidants; Ascorbic Acid; Blotting, Western; Cell Adhesion; Cell Line; Cell Proliferation; Dental Implants; Dexamethasone; Focal Adhesion Protein-Tyrosine Kinases; Glucocorticoids; Glycerophosphates; Humans; Immunoprecipitation; Mice; Microscopy, Electron, Scanning; Osteoblasts; Osteoporosis; Phosphorylation; Surface Properties; Titanium; Tyrosine

The loss of bone mass observed in aging enhances the risk of fractures. The process of bone repair in aging is slow and limited due to reduced activity of the osteoblasts. Bone marrow stem cells (MSCs) residing in the bone marrow are the progenitors for osteoblasts. The ability to enhance healing of bone defect in aging by MSCs can contribute in the prevention of the complications resulting from long-term immobilization that are especially fatal in old age. Our aim was to test the ability of MSCs inserted into a biological scaffold to enhance bone defect repair. Osteoprogenitor cells were selected from rat bone marrow stem cells cultured in DMEM medium supplemented with FCS, antibiotics, ascorbic acid, beta-glycerophosphate, and dexamethasone. The selected osteogenic subpopulation was identified by osteocalcin immunohistochemistry as well as Alizarin red S and von Kossa staining which are specific for bone matrix and mineral deposition. Committed osteoprogenitor cells cultured on the hydrogel scaffold were transplanted into the area of a rat tibia segmental bone defect and examined after 6 weeks. Radiology images revealed that 6 weeks post-implantaion, calcified material was present in the site of the defect, indicating new bone formation. It is concluded that committed osteogenic MSCs contained in a biocompatible scaffold can provide a promising surgical tool for enhancement of bone defect healing that will minimize the complications of bone repair in aging and disease.

Topics: Aging; Animals; Anti-Bacterial Agents; Ascorbic Acid; Blood Proteins; Bone and Bones; Bone Marrow Cells; Cells, Cultured; Dexamethasone; Fracture Healing; Glycerophosphates; Growth Substances; Hydrogel, Polyethylene Glycol Dimethacrylate; Immunohistochemistry; Mesenchymal Stem Cells; Mesoderm; Microscopy, Electron, Scanning; Models, Biological; Osteoblasts; Osteocalcin; Osteogenesis; Rats; Rats, Sprague-Dawley; Stem Cells; Time Factors; Wound Healing

We have been studying the potential of human fibroblastic cells (HFC) from periapical granulation tissue to form a calcified matrix. Recently, we reported that inflamed periapical granulation tissue contains osteogenic cells. In the present study, we tested the hypothesis that HFC, cultured with decalcified bone (DB) of rat, might form much greater calcified matrices than with rat decalcified boiled bone (DBB), which was originally prepared as a negative control. HFC were cultured with DB or DBB in the presence or absence of 2 mM beta-glycerophosphate (beta-GP) and 50 microg/ml ascorbic acid. After six weeks of culture, a number of von Kossa-positive globular structures were unexpectedly observed inside DBB, but not DB. Without HFC, such structures were never seen in DBB incubated with 2 mM beta-GP and 50 microg/ml ascorbic acid. DB cultured with HFC under the same conditions did not show these structures. Electron-microscopic observation revealed that matrix vesicles aggregated on collagen fibrils around globular structures in DBB. Energy dispersive X-ray microanalysis confirmed that these structures were calcified matrices composed of calcium and phosphate. These results suggest that human periapical granulation tissue contains cells responsible for the formation of calcified matrices in DBB, and that DBB could serve as an excellent scaffold for the calcification of HFC, rather than DB.

Topics: Animals; Ascorbic Acid; Bone and Bones; Calcification, Physiologic; Cell Culture Techniques; Cells, Cultured; Electron Probe Microanalysis; Extracellular Matrix; Fibroblasts; Glycerophosphates; Granulation Tissue; Humans; Periapical Tissue; Rats; Rats, Inbred Strains

Bone marrow stromal cells (BMSCs) are multipotent progenitor cells with a capacity to form bone tissue in vivo, and to differentiate into the osteoblastic lineage in vitro. Drawing on evidence that bone is mechanosensitive and mechanical stimuli are anabolic, we postulate that proliferation and osteoblastic differentiation of BMSCs may be stimulated by mechanical forces. In this study, BMSCs cultured in the presence of osteogenic factors (dexamethasone, beta-glycerophosphate, and ascorbate) were stimulated repeatedly (every second day) with shearing flow (1.6 dyn/cm(2)) for 5, 30, or 120 min, and assayed for systematic changes in cell number and phenotypic markers of osteoblastic differentiation. Cells exposed to fluid flow on days 2 and 4 after the addition of osteogenic factors and assayed at day 6 exhibited a modest decrease in cell number and increase in normalized alkaline phosphatase activity, suggesting the detachment of a non-osteogenic subpopulation. Cells exposed to fluid flow on days 6, 8, 10, and 12 and assayed at day 20 demonstrated maximal expression of osteopontin and bone sialoprotein mRNA with 30 min duration of flow. Concurrently, at day 20 expression of the adipogenic marker, lipoprotein lipase, was minimal with a 120-min duration of flow. These results indicate that repeated application of shear stress stimulates late phenotypic markers of osteoblastic differentiation of BMSCs in a manner that depends on the duration of stimulus. Finally, accumulation of prostaglandin E(2) in culture medium in response to shearing flow systematically decreased with repeated exposure to 30 and 120 min of shear stress (from day 6 to day 12), suggesting an adaptation of the cells to fluid flow.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Marrow Cells; Cell Count; Cells, Cultured; Dexamethasone; Dinoprostone; Gene Expression Regulation; Glycerophosphates; Integrin-Binding Sialoprotein; Lipoprotein Lipase; Microfluidics; Osteopontin; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sialoglycoproteins; Stromal Cells; Time Factors

X-box-binding protein 1 (XBP-1) is a basic-region leucine zipper protein in the cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors involved in different cell-differentiation processes. We have investigated the expression of XBP-1 in differentiating MC3T3-E1 osteoblastic cells. Cultures were treated with ascorbic acid (AA) and beta-glycerophosphate (BGP) to induce differentiation. Under these conditions, the basal transcription of xbp-1 increases at day 2 following induction, peaks at day 5 and decreases thereafter. This result showed that xbp-1 gene is differentially expressed during MC3T3-E1 cell differentiation. Detection of XBP-1 by immunofluorescence at days 0 (control culture without AA and BGP), 8 and 21 showed that the protein has a major cytoplasmic perinuclear location. In addition, xbp-1 is transcriptionally upregulated by parathyroid hormone within 2.5 h of treatment and decreases thereafter.

Topics: Animals; Ascorbic Acid; Blotting, Northern; Cell Differentiation; Cell Nucleus; Cytoplasm; DNA-Binding Proteins; Fluorescent Antibody Technique; Gene Expression Regulation; Glycerophosphates; Mice; Nuclear Proteins; Osteoblasts; Parathyroid Hormone; Regulatory Factor X Transcription Factors; RNA, Messenger; Trans-Activators; Transcription Factors; X-Box Binding Protein 1

We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca2+ channel function.. During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs.. Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique.. mRNA for the pore-forming alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization.. These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.

Topics: Adult; Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Marrow; Bone Marrow Cells; Calcium; Calcium Channels, L-Type; Calcium Channels, T-Type; Carrier Proteins; Cell Differentiation; Cell Line; Culture Media; Dexamethasone; DNA Primers; Flow Cytometry; Glycerophosphates; Glycoproteins; Humans; Membrane Glycoproteins; Mesenchymal Stem Cells; Microscopy, Phase-Contrast; Middle Aged; Nerve Tissue Proteins; Nifedipine; Osteoblasts; Osteopontin; Osteoprotegerin; Patch-Clamp Techniques; Phosphates; Polymerase Chain Reaction; RANK Ligand; Rats; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Sialoglycoproteins

Retinoic acid (RA) exerts a wide variety of effects on development, cellular differentiation and homeostasis in various tissues. However, little is known about the effects of RA on the differentiation of periodontal ligament cells. In this study, we investigated whether RA can affect the dexamethasone-induced differentiation of periodontal ligament cells.. Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate for mineralized nodule formation, as characterized by von Kossa staining. Continuous treatment with all-trans-RA inhibited the mineralization in a dose-dependent manner, with complete inhibition over 1 microm RA. Other RA analogs, 9-cis-RA and 13-cis-RA, were also effective. Furthermore, addition of RA for just the first 4 days completely inhibited the mineralization; however, as RA was added at later stages of culture, the inhibitory effect was diminished, suggesting that RA had a phase-dependent inhibition of mineralization. RA receptor (RAR)-alpha agonist (AM-580), but not retinoid X receptor agonist (methoprene acid), inhibited the mineralization, and reverse transcription-polymerase chain reaction analysis revealed that RAR-alpha was expressed on the cells, suggesting that RAR-alpha was involved in the inhibitory mechanism. This inhibition was accompanied by inhibition of alkaline phosphatase activity; however, neither expression of platelet-derived growth factor (PDGF) receptor-alpha, PDGF receptor-beta, or epidermal growth factor (EGF) receptor, nor phosphorylation of extracellular signal-regulated kinases triggered by PDGF-ascorbic acid or PDGF-BB was changed, as assessed by flow cytometry or western blot analyses.. These findings suggest that RA is a potential negative regulator for differentiation of human periodontal ligament cells.

Topics: Alitretinoin; Alkaline Phosphatase; Ascorbic Acid; Benzoates; Cell Differentiation; Cells, Cultured; Cementogenesis; Dexamethasone; Enzyme Inhibitors; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glycerophosphates; Humans; Isotretinoin; Periodontal Ligament; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Tetrahydronaphthalenes; Time Factors; Transcription Factors; Tretinoin

To investigate the alkaline phosphatase activity of enzymatically released human dental pulp cells with collagenase I in vitro.. We cultivated human dental pulp cells from connective tissue explants digested with collagenase I. Immunocytochemical staining was performed for characterization. When the subcultured cells grew in multilayers, the activity of alkaline phosphatase was examined by enzyme histochemical staining,and the ability of mineralization was detected.. Cultures of human primary dental pulp cells became confluent after 15-20 days. Subcultured cells proliferated to multilayers in long-term cultures, and the staining of alkaline phosphatase was noted to be regional on culture slides. The pulp cells cultured in the presence of beta-glycerophosphate and L-ascorbic acid formed mineralization nodules.. These results suggest that human dental pulp cells can be cultivated preferably from tissue explants digested with collagenase I in vitro. Enzyme histochemistry is useful for studying the biological behavior of dental pulp cells.

Topics: Alkaline Phosphatase; Ascorbic Acid; Cells, Cultured; Collagenases; Culture Media; Dental Pulp; Glycerophosphates; Humans

Bone defects caused by periapical inflammation can be treated and improved by endodontic therapy. However, the mechanism for osseous healing of periapical lesions after root canal treatment is unclear. In this study we examined whether fibroblastic cells from human periapical granulation tissue could produce calcified matrix in vitro. Periapical lesions from three patients were dissected in endodontic surgery, and fibroblastic cells (HFC) migrating from these lesions in vitro were used in this study. The HFC were cultured with or without beta-glycerophosphate (beta-GP) and ascorbic acid (AA), and the expression of human runt-related transcription factor-2 (Runx2), osterix (Osx), osteopontin (Opn), and osteocalcin (Ocn) mRNA, and alkaline phosphatase (ALPase) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) or by an enzyme-cytochemical technique. Furthermore, calcification in the cells was investigated by von Kossa staining. At the beginning of the culture, HFC expressed Runx2 mRNA faintly, but neither Opn mRNA nor ALPase activity. Immunocytochemical study also showed HFC expressed Runx2 more weakly, compared to SaOS2. However, the expression levels of ALPase, and Runx2, Osx, and Opn mRNA, were stimulated by 2 mM beta-GP and 50 microg/ml AA. After 4 weeks of culture with 2 mM beta-GP and 50 microg/ml AA, HFC formed von Kossa staining-positive calcified deposits on culture dishes, and also expressed Ocn mRNA. These results suggest that inflamed periapical granulation tissue contains osteogenic cells that have the potential to differentiate into mature osteoblastic or cementoblastic cells, and that such cells might contribute to osseous healing after root canal treatment.

Topics: Aged; Alkaline Phosphatase; Ascorbic Acid; Calcification, Physiologic; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Enzyme Activation; Female; Fibroblasts; Glycerophosphates; Granulation Tissue; Humans; Male; Middle Aged; Neoplasm Proteins; Osteocalcin; Osteopontin; Periapical Tissue; RNA, Messenger; Sialoglycoproteins; Transcription Factors

Human bone marrow-derived mesenchymal stem cells (MSCs) represent an ideal source for cell therapy for inherited and degenerative diseases, bone and cartilage repair, and as target for gene therapy. The role of the combination of human parathyroid hormone (PTH) and vitamin D(3) in bone formation and mineralization has been established in several osteoblast cell culture studies. The aim of the present study was to evaluate the role of this hormonal combination alone and in the presence of bone morphogenetic protein-4 (BMP-4) or-6 (BMP-6) in inducing osteogenic differentiation of human MSC. Human MSC derived from adult normal bone marrow that are positive for CD29, CD44, CD105, and CD166 and negative for CD14, CD34, and CD45, were treated with the PTH and 1,25-dihydroxyvitamin D(3) in the presence and absence of recombinant human BMP-4 or BMP6. PTH and vitamin D(3) induced high levels of expression of two key markers of bone formation: osteocalcin and alkaline phosphatase by MSCs. BMP-6 but not BMP-4 increased osteocalcin expression induced by PTH and vitamin D(3). Both BMPs enhanced calcium formation in MSC cultures and this response was potentiated by PTH and vitamin D(3). The present results revealed a novel potent effect of PTH and vitamin D(3) plus BMPs in inducing bone development by human MSCs. These results may facilitate therapeutic utility of MSCs for bone disease and help clarify mechanisms involved in stem cell-mediated bone development.

Topics: Adult; Alkaline Phosphatase; Antigens, CD; Ascorbic Acid; Bone Marrow Cells; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 6; Bone Morphogenetic Proteins; Calcium; Cell Differentiation; Cells, Cultured; Cholecalciferol; Dexamethasone; Glycerophosphates; Humans; Interleukin-6; Mesenchymal Stem Cells; Osteoblasts; Osteocalcin; Osteogenesis; Parathyroid Hormone

Matrix extracellular phosphoglycoprotein (MEPE) is an extracellular matrix protein that was first detected in tumor-induced osteomalacia (TIO). Investigations in mice revealed that MEPE is expressed in bone and teeth in a maturation-dependent manner, reaching its maximum during mineralization. However, from knockout experiments, although it has become clear that MEPE might function as a mineralization inhibitor, the exact mechanism of action is still unclear. Even less is known about the regulation of MEPE in men. Therefore, we have studied the time- and maturation-dependent expression of MEPE in two human osteoblast culture systems, the osteosarcoma cell line HOS 58 and primary trabecular osteoblasts. Cells were cultured for up to 29 days, and the influence of beta-glycerophosphate (bGP), ascorbate, transforming growth factor beta (TGF-beta), BMP-2, and dexamethasone was studied. HOS 58 cells showed no significant effect on MEPE gene expression up to 5.0 mM, but a significant inhibition was revealed at 10 and 20 mM, when osteocalcin (OC) expression was maximal. Under the same conditions, primary human osteoblasts showed no effect on MEPE gene expression. However, when cultured in the presence of 5 mM beta-glycerophosphate, ascorbate, and dexamethasone for 29 days, which are similar conditions to those described by Owen in his differentiation model in rat osteoblasts, a progressive inhibition of MEPE gene expression to 20% of the maximum was observed. Increasing osteocalcin expression indicated advancing differentiation. In conclusion, in contrast to the results in mice, when MEPE was maximally expressed during mineralization, in the human system, this factor seems to be maximally active in the proliferation and early matrix maturation phase. It was, however, strongly suppressed, associated with the mineralization phase.

Topics: Adult; Aged; Ascorbic Acid; Base Sequence; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line, Tumor; Dexamethasone; DNA Primers; Down-Regulation; Extracellular Matrix Proteins; Glycerophosphates; Glycoproteins; Humans; Middle Aged; Osteoblasts; Phosphoproteins; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Although best known for its role in cholinergic signalling, a substantial body of evidence suggests that acetylcholinesterase (AChE) has multiple biological functions. Previously, we and others identified AChE expression in areas of bone that lacked expression of other neuronal proteins. More specifically, we identified AChE expression at sites of new bone formation suggesting a role for AChE as a bone matrix protein. We have now characterised AChE expression, secretion and adhesive function in osteoblasts. Using Western blot analysis, we identified expression of two AChE species in osteoblastic cells, a major species of 68 kDa and less abundant species of approximately 55 kDa. AChE colocalised with the Golgi apparatus in osteoblastic cells and was identified in osteoblast-conditioned medium. Further analyses revealed differentiation-dependent secretion by osteoblasts, with AChE secretion levels corresponding with alkaline phosphatase activity. AChE expression by osteoblastic cells was also found to be regulated by mechanical strain both in vitro and in vivo. Finally, we investigated the possibility of a functional role for AChE in osteoblast adhesion. Using specific inhibitors, blockade of sites thought to be responsible for AChE adhesive properties caused a concentration-dependent decrease in osteoblastic cell adhesion, suggesting that AChE is involved in regulating cell-matrix interactions in bone.

Topics: Acetylcholinesterase; Animals; Ascorbic Acid; Cell Differentiation; Cells, Cultured; Cholinesterase Inhibitors; Dexamethasone; Glycerophosphates; Humans; Mice; Osteoblasts; Rats

Embryonic stem cells are pluripotent cells derived from the inner cell mass of mouse blastocysts that have been shown to differentiate spontaneously into cell types representing all three germ layers. This study shows that ES cells were induced to differentiate in vitro into mineralized osteoblasts under the influence of ascorbic acid, beta-glycerophosphate and 1alpha,25-OH vitamin D3. The activity of alkaline phosphatase, an early osteoblast marker, was found to be increased around day 12 of culture. Mineralized cells were clearly identified by histochemical staining, which detects mineralized calcium. The major noncollagenous component of bone matrix, osteocalcin, was localized to the mineralized cells by immunofluorescence. The expression of bone-specific genes was analyzed by real-time quantitative PCR. Osteocalcin and bone sialoprotein (BSP) were identified as early as in the fourth week of embryonic stem cell culture, both being characteristic for late stages of osteoblastic differentiation, indicating that at this time of culture the identified cells represent "mature" osteoblasts. The osteoblast-specific transcription factor Cbfa1 was induced a few days earlier. The expression of osteopontin and osteonectin, both being involved in binding calcium ions and hydroxyapatite during mineralization processes, as well as of collagen type I, representing by far the most predominant collagen in vertebrate organisms, is enhanced at the beginning of the second culture week upon addition of supplements. In the third week of culture, treated cells showed a second peak of osteopontin, osteonectin and collagen type I expression, osteopontin and osteonectin being stimulated 3-4-fold and collagen type I being induced 6-fold over control values. Alkaline phosphatase (ALP) expression was enhanced at the beginning of the third week of culture and was found to be increased again at later stages of culture at days 27-34. The in vitro differentiation of mouse embryonic stem cells into osteoblasts may provide a suitable model for studying the molecular processes of osteoblastic development in vivo.

Topics: Animals; Antioxidants; Ascorbic Acid; Calcification, Physiologic; Cell Differentiation; Cells, Cultured; Cholecalciferol; Glycerophosphates; Integrin-Binding Sialoprotein; Mice; Osteoblasts; Osteocalcin; Sialoglycoproteins; Stem Cells

Numerous techniques are currently used to characterize biological mineralization in intact tissues and cell cultures; the von Kossa staining method, electron microscopic analysis (EM), X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR) are among the most common. In this study, we utilized three of these methods to compare the mineralization of cultured fetal rat calvarial cells (FRC) and the osteoblast cell lines 2T3 and MC3T3-E1 with the in vivo mineral of rat calvarial bone. The cells were cultured with or without ascorbic acid (100 microg/ml) and beta-glycerophosphate (2.5, 5, or 10 mM betaGP), and harvested between 16 and 21 days (FRC cells and 2T3 cells) or at 30 days of culture (MC3T3-E1 cells). In the FRC cultures, maximal von Kossa staining was observed with 2.5 and 5 mM betaGP in the presence of 100 microg/ml ascorbate. FRC cells also showed some von Kossa staining when cultured with bGP alone. In contrast, maximal von Kossa staining for MC3T3-E1 cells was observed with 10 mM betaGP. Only the cultures of MC3T3-E1 cells that received both ascorbate and betaGP produced von Kossa positive structures. The 2T3 cultures produced von Kossa positive staining only upon treatment with ascorbic acid and betaGP, which was greatly accelerated by bone morphogenic protein-2 (BMP-2). FTIR was performed on the mineral and matrix generated in FRC, MC3T3, and 2T3 cultures, and the results were compared with spectra derived from 16-day-old rat calvaria. The mineral-to-matrix ratios calculated from FTIR spectra for rat calvaria ranged from 2.97 to 7.44. FRC cells made a bonelike, poorly crystalline apatite, and, with increasing betaGP, there was a statistically significant (P

Topics: Animals; Ascorbic Acid; Calcification, Physiologic; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Drug Combinations; Glycerophosphates; Osteoblasts; Osteogenesis; Rats; Skull; Spectroscopy, Fourier Transform Infrared; Staining and Labeling

Bacterial effects on in vitro mineralization of human periodontal fibroblasts (HPF) have not yet been examined in great detail. In our study, we investigated the effects of soluble extracts of the periodontopathic bacteria Porphyromonas gingivalis, Bacteroides forsythus and, Treponema denticola on cell proliferation, mineralization, as well as on osteoblastic markers present in HPF cultured in vitro, such as alkaline phosphatase (ALP) activity and collagen content. Periodontal fibroblasts stimulated by B-glycerophosphate, ascorbic acid and dexamethasone (BAD) or by dexamethasone and ascorbic acid (DA) were compared to unstimulated cells. During the cultivation period, the stimulation of HPF by combined dexamethasone and ascorbic acid (DA) had a strong inductive effect on proliferation, ALP activity and collagen formation. The extracts obtained from the periodontal pathogens had a suppressing effect on the proliferation rate of HPF. The extracts from P. gingivalis, B. forsythus and T. denticola caused a decrease in ALP activity within 24 h of application. While extracts obtained from P. gingivalis and B. forsythus induced a reduction in collagen content in BAD- and DA-stimulated HPF cells, T. denticola extracts led to an increase in collagen. Our data suggest that specific periodontopathic bacteria may suppress tissue regeneration in vivo not only by activating host defense mechanisms but also directly via a suppression of growth and differentiation of HPF and a reduction in the extracellular collagen matrix. For the process of pocket formation, not even the direct influence of viable bacteria seems to be necessary. Additionally, long-distance effects of bacteria harboured in periodontal pockets or in root canals may be of importance.

Topics: Ascorbic Acid; Bacteroides; Cell Division; Cells, Cultured; Collagen; Culture Media, Conditioned; Dexamethasone; Drug Combinations; Fibroblasts; Glycerophosphates; Humans; Periodontal Ligament; Porphyromonas gingivalis; Statistics, Nonparametric; Treponema

Using a culture system that facilitates osteogenic differentiation of bone marrow-derived human mesenchymal stem cells, we analyzed gene-expression profiles during the mineralization process by means of a cDNA microarray system consisting of 23,040 genes. We compared expression profiles of the cells at days 3, 15, and 27 of incubation in media containing either a combination of 0.1 microM dexamethasone, 0.05 mM ascorbic acid-2-phosphate, and 10 mM beta-glycerophosphate, dexamethasone only, ascorbic acid-2-phosphate plus beta-glycerophosphate, or medium without any of these osteogenic supplements. Histochemical analysis revealed osteogenic differentiation of cells incubated in the presence of all three agents, but not in the other cultures. Comparison of the expression profiles disclosed transcriptional stimulation of 55 genes and repression of 82 genes among more than 20,000 examined. A set of differentially expressed genes we report here should contribute to a better understanding of the process of mineralization in the matrix surrounding human mesenchymal stem cells.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Ascorbic Acid; Cell Differentiation; Dexamethasone; DNA, Complementary; Down-Regulation; Glycerophosphates; Humans; Mesoderm; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Time Factors; Transcription, Genetic; Up-Regulation

To investigate the growth and osteogenesis characteristics of cultured rat marrow stromal cells(rMSCs) under bone induction condition.. rMSCs were isolated from adult rat using density gradient separation method. The rMSCs attachment formed soon after seeding and grew into colonies with the appearance of fibroblastic cells. The osteogenic induction compound of Dex(10(-8) mol/L), beta-GP(10 mmol/L) and AA(50 micrograms/ml) was added to different passaged rMSCs and the proliferation and osteogenic differentiation of them was observed.. The induction compound had strong effect on promoting proliferation, especially on that of further passaged rMSCs, and its stimulation effect on osteogenesis was also well proved with the expression of alkaline phosphatase (ALP) rising after one-week induction. Further subculturing from P1 to P6 led to the increase of proliferation activity of rMSCs. After three-week induction, mineral deposits appeared in the culture.. The lower expression of ALP in P1 rMSCs and the increase of it in further passages or under induction condition suggest the rMSCs in our culture system are mainly undifferentiated osteoprogenitors.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Development; Bone Marrow Cells; Cells, Cultured; Dexamethasone; Glycerophosphates; Osteogenesis; Rats; Rats, Wistar; Stromal Cells

To observe the effect of leptin on osteoblast.. Human osteoblast primary culture was carried out, and the morphology and function of osteoblast were observed. The effects of different levels of leptin on osteoblast in different days were assessed by MTT colorimetry. Osteocalcin production was measured also.. Human osteoblasts were fusiform in shape and were positive for alkaline phosphatase by histochemical staining, positive for osteocalcin by immunofluorescence staining, and positive by Alizarin Reds staining after mineralized upon supplementation with ascorbate and beta-glycerophosphate. On the first, second and third days, the proliferation of osteoblast, cultured with different concentrations of leptin, had no changes. The leptin-stimulated synthesis of osteocalcin of cells was found to be dose-dependent (P < 0.05), but not time-dependent (P > 0.05).. The above data indicated that there were no evidences for the effects of leptin on the proliferation of human osteoblast, but leptin could enhance the function of human osteoblast.

Topics: Ascorbic Acid; Cell Division; Cells, Cultured; Glycerophosphates; Humans; Leptin; Male; Middle Aged; Osteoblasts; Osteocalcin

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.

Topics: Animals; Antioxidants; Ascorbic Acid; Bone and Bones; Cell Differentiation; Cell Line; Cell Lineage; Cells, Cultured; Coculture Techniques; Dexamethasone; Embryo, Mammalian; Glucocorticoids; Glycerophosphates; Humans; Immunohistochemistry; Mice; Microscopy, Confocal; Osteoblasts; Osteogenesis; Stem Cells; Tretinoin

Embryonic stem (ES) cells have the capacity to differentiate into various cell types in vitro. In this study, we show that retinoic acid is important for the commitment of ES cells into osteoblasts. Culturing retinoic acid treated ES cells in the presence of the osteogenic supplements ascorbic acid and beta-glycerophosphate resulted in the expression of several osteoblast marker genes, osteocalcin, alkaline phosphatase, and osteopontin. However, there was only a slight amount of mineralized matrix secretion. Addition of bone morphogenic protein-2 or compactin, a drug of the statin family of HMG-CoA reductase inhibitors, resulted in a greatly enhanced formation of bone nodules. Compactin did not modify the expression of osteogenic markers, but at the late stage of differentiation promoted an increase in BMP-2 expression. These results establish ES-cell derived osteogenesis as an effective model system to study the molecular mechanisms by which the statin compactin promotes osteoblastic differentiation and bone nodule formation.

Topics: Alkaline Phosphatase; Animals; Antigens, Differentiation; Ascorbic Acid; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cells, Cultured; Glycerophosphates; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Mice; Osteoblasts; Osteocalcin; Osteogenesis; Osteopontin; RNA, Messenger; Sialoglycoproteins; Stem Cells; Transforming Growth Factor beta; Tretinoin

The mechanisms controlling the initiation of mineralization of bone matrix are not clear. To examine this process, we established a cell line called MLO-A5 that mineralizes in sheets, not nodules, within 3 days of culture in the presence of beta-glycerophosphate (beta-GP) and ascorbic acid and within 7 days in the absence of beta-GP and ascorbic acid. The mineral formed in both cases was shown to be bonelike apatite by Fourier transformed infrared (FTIR) spectroscopy. Mineral-to-matrix ratios (min/matrix) calculated from the FTIR data, which are related directly to ash weight, were approximately 0.4 in the absence of beta-GP and ascorbic acid and approximately 1.2 in the presence of beta-GP and ascorbic acid. By comparison, these ratios in fetal rat calvarial cells without beta-GP equal 0 and with beta-GP 1.9. This cell line and three others (MLO-A2, -D1, and -D6) were isolated from the long bones of transgenic mice expressing the large T-antigen driven by the osteocalcin promoter, the same mice from which the osteocyte-like cell line MLO-Y4 was isolated.(1) The cell lines were selected based on a dendritic or stellate morphology. MLO-A5 cells express high alkaline phosphatase, collagen type 1, parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor, bone sialoprotein (BSP), and osteocalcin (767 ng/10(6) cells compared with <1-2.2 ng/10(6) cell for primary mouse osteoblasts and five osteoblast cell lines). The single unique feature of the MLO-A5 cells compared with the other three nonmineralizing cell lines is the high expression of messenger RNA (mRNA) for BSP. These cell lines may represent stages of osteocyte differentiation and the MLO-A5 cells represent the postosteoblast, preosteocyte responsible for triggering mineralization of osteoid.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Biomarkers; Calcification, Physiologic; Cell Adhesion Molecules; Cell Culture Techniques; Cell Division; Cell Line; Collagen; Gene Expression; Glycerophosphates; Mice; Mice, Transgenic; Osteoblasts; Osteocalcin; Osteocytes; Osteopontin; Receptor, Parathyroid Hormone, Type 1; Receptors, Parathyroid Hormone; Sialoglycoproteins; Stem Cells

Although basic fibroblast growth factor (FGF-2) had been shown to inhibit type I collagen gene expression in osteoblast, its inhibitory mechanism is unknown. In the present study, we investigated the underlying mechanisms by which growth factors downregulate type I collagen gene expression. Treatment of mouse osteoblastic MC3T3-E1 cells with okadaic acid (40 ng/ml), an inhibitor of phosphoserine/threonine-specific protein phosphatase and activator of ERK1/2, for 24 h and 48 h completely inhibited steady-state mRNA levels of type I collagen. FGF-2 (30 ng/ml), platelet-derived growth factor-BB (PDGF-BB), 30 ng/ml, and serum, which activate ERK mitogen-activated protein kinase (MAPK) pathway also inhibited collagen type I gene expression, suggesting that the activation of ERK pathway mediates inhibition of type I collagen mRNA. This observation was further confirmed by experiments using inhibitors of the ERK pathway (i.e., PD and U0126), which increased type I collagen mRNA in MC3T3-E1 cells, indicating that the inhibition of ERK pathway upregulates type I collagen gene expression. Low serum (0.3%) markedly increased type I collagen mRNA. MEK inhibitor PD inhibited c-fos induction by FGF-2 and PDGF-BB, suggesting that c-fos is the downstream target of ERK pathway. Our data have clearly demonstrated for the first time that the ERK MAPK pathway play an important role in the regulation of type I collagen gene expression in osteoblastic cells. Results also showed that one of the mechanisms by which FGF-2 and PDGF-BB downregulate type I collagen gene expression in the osteoblast is through the activation of ERK signaling pathway.

Topics: 3T3 Cells; Animals; Ascorbic Acid; Becaplermin; Butadienes; Down-Regulation; Enzyme Inhibitors; Fibroblast Growth Factor 2; Flavonoids; Genes, fos; Glycerophosphates; Humans; Mice; Mitogen-Activated Protein Kinases; Nitriles; Okadaic Acid; Osteoblasts; Platelet-Derived Growth Factor; Procollagen; Proto-Oncogene Proteins c-sis; Recombinant Proteins; RNA, Messenger; Signal Transduction

Bone morphogenetic protein-7 (BMP-7) affects differentiation of preosteoblasts enabling the resultant cells to respond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-mobilizing inositol 1,4,5-trisphosphate (InsP3) receptor are integral to stimulus-secretion coupling in osteoblasts, the hypothesis that BMP-7 affects InsP3 receptor expression was examined in the G-292 human osteosarcoma cell line and in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein, alkaline phosphatase, alpha1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured long term in the presence of ascorbic acid and beta-glycerophosphate, G-292 cells underwent further osteoblastic differentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with an increase in InsP3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292 cells increased InsP3 receptor density 2.4-fold with no apparent change in affinity. Immunoblot analysis with antibodies specific for type I, type II, and type III InsP3 receptors revealed that BMP-7 (50 ng/ml) treatment resulted in a specific increase (206+/-8%) in the type I receptor. Reverse transcription-polymerase chain reaction and Northern blot analyses of G-292 and primary human osteoblasts confirmed an increase in type I InsP3 receptor mRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase InsP3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca2+-mobilizing secretory hormones during osteoblast differentiation.

Topics: Alkaline Phosphatase; Ascorbic Acid; Blotting, Northern; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Calcification, Physiologic; Calcium Channels; Cell Differentiation; Cells, Cultured; Collagen; ErbB Receptors; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glycerophosphates; Humans; Immunoblotting; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Integrin-Binding Sialoprotein; Osteoblasts; Osteocalcin; Osteosarcoma; Phosphatidylinositols; Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Growth Factor; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Cells, Cultured

This work analyses the proliferation/differentiation behaviour of human bone marrow cells cultured in alpha-minimum essential medium supplemented with 10% foetal bovine serum (standard medium) and in the presence of ascorbic acid (AA, 50 microg ml(-1)), beta-glycerophosphate (betaGP, 10 mmol) and dexamethasone (Dex, 10 nmol) under selected experimental conditions. Cultures were compared concerning cell morphology, cell growth, ALP activity and ability to form calcium phosphate deposits. Cells growing in the various experimental conditions proliferated gradually with the incubation time and presented high ALP activity. Cultures grown in standard medium and in the presence of either AA or Dex failed to form calcium phosphate deposits. Cultures grown in the presence of betaGP, betaGP + AA and betaGP + AA + Dex, i.e. in the presence of a source of phosphate ions, showed the formation of a mineralised extracellular matrix. The presence of Dex resulted in a significant induction in the ALP activity and ability to form mineral deposits. The behaviour of the various cell cultures is in agreement with previous studies stating a reciprocal and functionally coupled relationship between proliferation and differentiation, i.e. cultures grown in a medium containing betaGP presented a less proliferative but more differentiated osteoblastic cell population, as compared to cultures lacking the mineralisation process.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Biocompatible Materials; Bone and Bones; Cattle; Cell Differentiation; Cell Division; Cells, Cultured; Culture Media; Dexamethasone; Glycerophosphates; Humans; Materials Testing; Osteoblasts

The contraction of connective tissue cells may facilitate their production and maintenance of extracellular matrix architecture and can benefit healing through wound closure. However, aberrant cell contraction can result in pathological contracture. Implants may stimulate this process leading to contracture of the surrounding fibrous capsule. In the case of compliant porous matrices used for tissue engineering the cell contraction may cause the constriction of pores and the distortion of the implant. The objective of this study was to determine if osteoblasts expressed a specific muscle actin, alpha-smooth muscle actin (SMA), that could provide for their contraction. Immunohistochemistry revealed the presence of SMA in some cells in all of three human and three canine trabecular bone specimens. The majority of the cells of an osteoblastic cell line, MC3T3-E 1, also expressed this actin isoform in two-dimensional culture and when seeded into a collagen-glycosaminoglycan (GAG) matrix. These SMA-containing cells were found to cause contraction of the collagen-GAG analog of extracellular matrix. These findings demonstrate that osteoblasts can display contractile behavior that might help to explain the mechanism by which they impart architecture to bone matrix, including that at implant interfaces. An understanding of this process could also guide the development of matrices for bone tissue engineering.

Topics: 3T3 Cells; Actins; Animals; Ascorbic Acid; Bone and Bones; Cell Differentiation; Collagen; Dogs; Glycerophosphates; Glycosaminoglycans; Humans; Immunohistochemistry; Mice; Movement; Muscle Contraction; Muscle, Smooth; Osteoblasts; Polymers

The role of epidermal growth factor receptors (EGF-R) in osteogenic cell differentiation was investigated using preosteoblastic MC3T3-E1 (MC3T3) cells and osteoblast-like ROS 17/2.8 (ROS) cells. When cultured in the presence of beta-glycerophosphate (GP) and ascorbic acid (AA), MC3T3 cells underwent spontaneous differentiation into osteoblasts which was confirmed as they expressed osteoblast markers such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). Interestingly, the number of EGF-binding sites decreased during their differentiation into osteoblasts, and the osteogenic protein-1 (OP-1) treatment, which accelerated their differentiation, lowered the number of EGF-binding sites even further. On the other hand, ROS cells with high expression levels of osteoblast markers and no EGF-R, after being transfected with human EGF-R cDNA (EROS cells), expressed numerous EGF-binding sites as well as EGF-R mRNA and protein; in the process, they ceased to express osteoblast markers, indicating their dedifferentiation into osteoprogenitor cells. Both MC3T3 and EROS cells showed increased cell growth in response to EGF, whereas ROS cells did not. These results imply that the EGF/EGF-R system in osteogenic cells has a crucial function in osteoblast phenotype suppression and osteogenic cell proliferation.

Topics: Alkaline Phosphatase; Ascorbic Acid; Blotting, Northern; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line; DNA, Complementary; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Glycerophosphates; Humans; Integrin-Binding Sialoprotein; Osteoblasts; Osteocalcin; RNA, Messenger; Sialoglycoproteins; Transformation, Genetic; Transforming Growth Factor beta

To achieve new insights into the coordinate regulation of gene expression during osteoblast differentiation we utilized an approach involving global analysis of gene expression to obtain the identities of messenger RNAs (mRNAs) expressed using an established in vitro model of bone development. MC3T3-E1 osteoblast-like cells were induced to differentiate by the addition of beta-glycerophosphate (beta-GP) and ascorbic acid. RNA samples derived from induced and uninduced control MC3T3-E1 cells were used to prepare complementary DNA (cDNA) for serial analysis of gene expression (SAGE). A preliminary SAGE database was produced and used to prepare a hybridization array to further facilitate the characterization of changes in the expression levels of 92 of the SAGE-mRNA assignments after induction of osteoblast differentiation, specifically after 6 days and 14 days of ascorbate treatment. SAGE-array hybridization analysis revealed coordinate induction of a number of mRNAs including Rab24, calponin, and calcyclin. Levels of MSY-1, SH3P2, fibronectin, alpha-collagen, procollagen, and LAMPI mRNAs, present at day 6 postinduction, were markedly reduced by day 14 postinduction. A number of unanticipated and potentially important developmental genes were identified including the transforming growth factor beta (TGF-beta) superfamily member Lefty-1. Lefty-1 transcript and translation product were found to be induced during the course of MC3T3-E1 cell differentiation. We present evidence, using transient transfection and antibody neutralization approaches, that Lefty-1 modulates the induction of alkaline phosphatase (ALP) after treatment of MC3T3-E1 cells with ascorbate and beta-GP. These data should provide useful new information for future analysis of transcriptional events in osteoblast differentiation and mineralization.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Calcification, Physiologic; Cell Differentiation; Cell Line; Cloning, Molecular; Databases, Factual; Enzyme Induction; Expressed Sequence Tags; Gene Expression Regulation, Developmental; Glycerophosphates; Left-Right Determination Factors; Mice; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Osteoblasts; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Transforming Growth Factor beta

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.Cells incubated in psi-CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi-CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta-glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.

Topics: Adipocytes; Alkaline Phosphatase; Animals; Ascorbic Acid; Azacitidine; beta-Galactosidase; Bone and Bones; Bone Marrow Cells; Cell Adhesion; Cell Differentiation; Cell Division; Cell Separation; Cells, Cultured; Dexamethasone; Drug Resistance; Glucocorticoids; Glycerophosphates; Mesoderm; Mice; Mice, Inbred BALB C; Neomycin; Osteogenesis; Retroviridae; Stem Cell Transplantation; Stem Cells; Transfection; Vascular Cell Adhesion Molecule-1

The mechanism by which interleukin-1beta (IL-1) inhibits the formation of mineralized tissue nodules by periodontal ligament (PDL) cells in vitro was investigated through the processes of morphological analysis, immunoprecipitation, and Northern blot analysis. PDL cells were obtained from a 2-day-old coagulum in tooth socket and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bone serum (FBS) and antibiotics. Confluent cells were grown for up to 3 weeks in the presence of ascorbic acid (AA), beta-glycerophosphate (GP), and dexamethasone (Dex), or IL-1. PDL cells cultured in the presence of GP and AA did not differentiate, but those treated with Dex, GP, and AA (Dex group) underwent differentiation, showing four stages (confluent, multilayer, nodule, and mineralization) of disparate morphological characteristics. In contrast, the cells treated with IL-1, Dex, GP, and AA (IL-1 group) did form multilayers but failed to form mineralized nodules. Electron microscopy demonstrated that the Dex-induced mineralized nodules contain multilayers of fibroblastic cells, numerous collagen fibrils, and dense globular as well as fused electron dense patches that are associated with numerous apatite crystals. The nodule-like structures in the IL-1 group were also comprised of multilayered fibroblastic cells, but they contained only a small number of collagen fibrils, and no dense globular or fused patches. Von Kossa staining confirmed the presence of numerous mineralized nodules in the Dex group and their scarceness in the IL-1 group. Northern blot analysis of IL-1-treated cells, however, revealed the presence of mRNAs for type I collagen (Col I), secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC), whose expression patterns and levels were comparable to those of the Dex group. Immunoprecipitation analysis of OPN and BSP in the cell/matrix layers and the culture media after [35S]-methionine labeling showed their deposition primarily in the mineralized nodules of the Dex group, and their release into the media in the IL-1 group. Immunogold labeling demonstrated the location of OPN and BSP in mineralized nodules of the Dex group, but no significant labeling occurred in the nodule-like structures from the IL-1 group. Interestingly, IL-1 treatment increased the expression of collagenase mRNA by sevenfold, compared with that of the Dex group. These data sugge

Topics: Animals; Ascorbic Acid; Blotting, Northern; Calcification, Physiologic; Cells, Cultured; Collagen; Collagenases; Dexamethasone; Extracellular Matrix Proteins; Fibroblasts; Glycerophosphates; Integrin-Binding Sialoprotein; Interleukin-1; Osteocalcin; Osteonectin; Osteopontin; Periodontal Ligament; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sialoglycoproteins

In order to investigate the significance of hydroxyapatite based microporous composite (HA/mica composite) surfaces and a macroporous synthetic hydroxyapatite, rat marrow cell culture, which shows osteogenic differentiation, was carried out on six different culture substrata (two control culture dishes, two identical HA/mica composites, and two identical macroporous synthetic hydroxyapatites). A culture period of two weeks in the presence of beta-glycerophosphate (BGP), ascorbic acid, and dexamethasone resulted in abundant mineralized nodule formations that were positive for alkaline phosphatase (ALP) stain. The stain on the macroporous synthetic hydroxyapatite and the HA/mica composites were intense, the enzyme activity being about double that of control culture dishes. These data indicate that the synthetic macroporous hydroxyapatite surface and the HA/mica composite surface promotes osteoblastic differentiation.

Topics: Alkaline Phosphatase; Aluminum Silicates; Animals; Ascorbic Acid; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Coloring Agents; Culture Media; Dexamethasone; Durapatite; Glucocorticoids; Glycerophosphates; Hematopoietic Stem Cells; Osteoblasts; Osteogenesis; Porosity; Rats; Surface Properties; Time Factors

Vitamin C or ascorbate is important in wound healing due to its essential role in collagen synthesis. To study wound healing in the periodontium, cells adherent to expanded polytetrafluoroethylene (ePTFE) augmentation membranes, recovered from edentulous ridge augmentation procedures, have been established in culture in our laboratories. The objective of this study was to determine whether treatment of these cells with a calcium ascorbate, which contains vitamin C metabolites (metabolite-supplemented ascorbate), would increase the production of collagenous protein and mineralized tissue in vitro, as compared to unsupplemented calcium ascorbate (ascorbate).. Cells derived from ePTFE membranes were cultured with beta-glycerophosphate and the test agents for 2 to 5 weeks, and the surface areas of the cell cultures occupied by mineralized nodules were measured using computerized image analysis. One experiment tested the effects of calcium threonate, one of the vitamin C metabolites in metabolite-supplemented ascorbate. Incorporation of radioactive proline and glycine was used as a measure of total protein (radioactivity precipitated by trichloracetic acid) and collagenase-digestible protein (radioactivity released by collagenase digestion.) Co-localization of collagen and fibronectin was examined by immunofluorescence.. In vitro treatment of these cells with metabolite-supplemented ascorbate increased the area of the cell cultures occupied by mineralized nodules after 5 weeks. Cell cultures treated with metabolite-supplemented ascorbate also exhibited significant increases in total protein. The increase in collagenous proteins in these cultures accounted for 85% of the increase in total protein. The greatest difference between treatment groups was observed in the cell-associated fraction containing the extracellular matrix. The additional collagen exhibited normal co-distribution with fibronectin. In cultures treated with ascorbate spiked with calcium threonate, the area of mineralized tissue was significantly greater than in ascorbate-treated cultures, but was less than that observed in cultures treated with metabolite-supplemented ascorbate.. In vitro treatment with ascorbate containing vitamin C metabolites enhanced the formation of mineralized nodules and collagenous proteins. Calcium threonate may be one of the metabolites influencing the mineralization process. Identifying factors which facilitate the formation of mineralized tissue has significant clinical ramifications in terms of wound healing and bone regeneration.

Topics: Alveolar Ridge Augmentation; Antioxidants; Ascorbic Acid; Butyrates; Calcium; Cell Line; Cells, Cultured; Collagen; Culture Media; Fibroblasts; Fibronectins; Fluorescent Antibody Technique; Glycerophosphates; Glycine; Humans; Image Processing, Computer-Assisted; Jaw, Edentulous; Membranes, Artificial; Periodontium; Polytetrafluoroethylene; Proline; Protein Biosynthesis; Proteins; Radiopharmaceuticals; Wound Healing

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus large T-antigen. At the permissive temperature of 34 degrees C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5 degrees C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5 degrees C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10(-8) M Dex, gene expression for ALP, PTH-R, and LPL increased, but that for OC decreased. Stimulation with 10(-8) M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5 degrees C with ascorbate and beta1-glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.

Topics: Adipocytes; Antigens, Viral, Tumor; Ascorbic Acid; Bone Marrow Cells; Calcitriol; Cell Differentiation; Cell Division; Cell Line; Dexamethasone; Genetic Vectors; Glycerophosphates; Humans; Immunohistochemistry; Mutation; Osteoblasts; Osteogenesis; Retroviruses, Simian; RNA, Messenger; Stromal Cells; Transfection

We investigated the effects of the time course of addition of osteogenic supplements dexamethasone, beta-glycerolphosphate, and L-ascorbic acid to rat marrow stromal cells, and the exposure time on the proliferation and differentiation of the cells. It was the goal of these experiments to determine the time point for supplement addition to optimize marrow stromal cell proliferation and osteoblastic differentiation. To determine this, two studies were performed; one study was based on the age of the cells from harvest, and the other study was based on the duration of exposure to supplemented medium. Cells were seen to proliferate rapidly at early time points in the presence and absence of osteogenic supplements as determined by 3H-thymidine incorporation into the DNA of replicating cells. These results were supported by cell counts ascertained through total DNA analysis. Alkaline phosphatase (ALP) activity and osteocalcin production at 21 days were highest for both experimental designs when the cells were exposed to supplemented medium immediately upon harvest. The ALP levels at 21 days were six times greater for cells maintained in supplements throughout than for control cells cultured in the absence of supplements for both studies, reaching an absolute value of 75 x 10(-7) micromole/min/cell. Osteocalcin production reached 20 x 10(-6) ng/cell at 21 days in both studies for cells maintained in supplemented medium throughout the study, whereas the control cells produced an insignificant amount of osteocalcin. These results suggest that the addition of osteogenic supplements to marrow-derived cells early in the culture period did not inhibit proliferation and greatly enhanced the osteoblastic phenotype of cells in a rat model.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Marrow Cells; Cell Differentiation; Cell Division; Cells, Cultured; Culture Media; Dexamethasone; Glycerophosphates; Male; Osteoblasts; Osteocalcin; Phenotype; Rats; Rats, Sprague-Dawley; Stromal Cells; Thymidine; Time Factors

The small proteoglycan decorin had been localized previously at the d-band in the gap zone of collagen fibrils in nonmineralizing tissues. In bone matrix this zone is proteoglycan free and is at least in some species the place where mineralization along collagen fibrils starts. To study the metabolism of the small proteoglycans decorin and biglycan under mineralizing conditions, osteoblasts from human nasal bone were cultured for several weeks in the presence or absence of beta-glycerophosphate and ascorbate. An immediate consequence of the treatment was a reduced expression of decorin, as judged by immune precipitation, whereas the biosynthesis of biglycan was not affected. Pulse-chase experiments were performed with osteoblasts embedded in floating type I collagen gels. In the presence of beta-glycerophosphate and ascorbate, a more rapid turnover of both proteoglycans was noted; the one of biglycan reached statistical significance. Indirect evidence for an enhanced rate of proteoglycan endocytosis was obtained. This effect was not seen in cultured skin fibroblasts. Thus, osteoblasts respond rapidly to mineralizing conditions with alterations of small proteoglycan biosynthesis and turnover.

Topics: Adolescent; Adult; Ascorbic Acid; Biglycan; Cells, Cultured; Collagen; Decorin; Electrophoresis, Polyacrylamide Gel; Endocytosis; Extracellular Matrix Proteins; Fibroblasts; Gels; Glycerophosphates; Half-Life; Humans; Nasal Bone; Osteoblasts; Precipitin Tests; Proteoglycans; Skin

Postproliferative confluent cultures of primary rat tibial osteoblasts (ROB), cultured in medium supplemented with ascorbic acid and beta-glycerophosphate (AS-bGP, differentiation medium) express, in sequence, specific bone markers which identify a succession of maturation stages, and eventually form mineralized noduli. We report an investigation on the effect of extensive proliferation in vitro in unsupplemented medium on the osteogenic potential of mass cultures of ROB. The growth rates of the populations, derived from two independent primary cultures, was constant throughout 110 cumulative population doublings (CPD) in culture. Propagated cells maintained features similar to osteoblasts in primary cultures with respect to serum and anchorage dependence for growth and to the chemokinetic effect on endothelial cells exerted by their conditioned media (CM). Propagated populations, set at confluence in differentiation medium, were tested for the expression of early [alkaline phosphatase (AP)] and late [osteocalcin (OC); bone sialoprotein (BSP); 45Ca incorporation and mineralization] osteogenic markers. We observed an increase, parallel to the increase in CPD, in both the level of maximal expression of AP (enzyme/microgram cellular DNA) and in the frequency of nodules, reaching five- to sixfold (at 78 CPD) and eightfold (at 60 CPD), respectively, the levels of primary cultures. AP expression (enzyme and mRNA) persisted during mineralization and 45Ca incorporation. The time required by propagated cultures for the formation of nodules decreased with increase of CPD, and was reduced to less than one third at 87 CDP. Nodules became mineralized over a similar lapse of time as in primary cultures and were positive by histochemistry for BSP and OC. We also obtained osteogenic clones from two independent cultures after 72 CPD. 90% of these showed an osteoblast phenotype, expressing AP and forming nodules positive for OC and BSP, which mineralized. Timing of formation and frequency of nodules/plated cells in clones was similar to that found in propagated cultures of equivalent CPD. In summary, propagated ROB populations and derived clones showed enhanced osteoblast phenotype, possibly due to an increase in osteogenic cells and enrichment of proliferating mature osteoblasts, consequent to extended propagation in culture.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Blotting, Northern; Calcification, Physiologic; Calcium; Cell Differentiation; Cell Division; Cells, Cultured; Chemotaxis; Cloning, Molecular; Culture Media, Conditioned; DNA; Fluorometry; Glycerophosphates; Immunohistochemistry; Integrin-Binding Sialoprotein; Osteoblasts; Osteocalcin; Osteogenesis; Phenotype; Rats; Rats, Wistar; RNA, Messenger; Sialoglycoproteins; Tibia

Human dental papilla cells were enzymatically separated from deciduous tooth germs of an 8-month-old embryo legally aborted. The second passage cells were cultured up to 35 days in 3 groups. The beta-GP group was cultured in the Dulbecco MEM containing ascorbic acid and beta-glycerophosphate supplemented with 15% fetal bovine serum. The Dex group was in the same medium, in addition containing dexamethasone. The control group contained none of the 3 chemicals. Mineralized nodules were formed after 15 days in the beta-GP and Dex groups. Only in the presence of ascorbic acid and organic phosphate did they mineralize. The addition of dexamethasone caused a significant increase in the number of nodules. By electron microscopy, the nodules contained needle-shaped crystals associated with a network of collagen fibrils. Calcium and phosphorus were detected by energy-dispersive X-ray microanalysis in the nodules. Furthermore, the crystalline material exhibited a pattern consistent with hydroxyapatite and dentin when examined by X-ray diffractometry. Cells showed high levels of alkaline phosphatase activity, which was increased 2-3 times in the presence of the 3 chemicals. These results indicated that human dental papilla cells have the ability to form dentin in culture. The formation of mineralized nodules by human dental papilla in vitro provides a useful model for studying the morphogenesis and differentiation of dental papilla ectomesenchyme.

Topics: Alkaline Phosphatase; Ascorbic Acid; Calcium; Cell Differentiation; Cells, Cultured; Collagen; Crystallography; Culture Media; Dental Papilla; Dentin; Dentinogenesis; Dexamethasone; Durapatite; Ectoderm; Electron Probe Microanalysis; Glucocorticoids; Glycerophosphates; Humans; Male; Mesoderm; Microscopy, Electron; Morphogenesis; Phosphorus; Tooth Calcification; Tooth Germ; Tooth, Deciduous; X-Ray Diffraction

The periodontal ligament (PDL) is a fibrous and cellular connective tissue that mediates tooth attachment to bone, and it comprises fibroblastic and mineralized tissue-forming (MTF) progenitors. The MTF progenitors are believed to give rise to the cementoblastic and osteoblastic lineages. Cementum attachment protein (CAP) is a collagenous cementum-derived protein which binds strongly to osteoblasts, moderately to PDL cells, and weakly to gingival fibroblasts. The aim of the present study was to determine the relationship between the capacity of PDL progenitors to bind CAP and their potential to express alkaline phosphatase (ALP) and form mineralized-like tissue in culture. Cloned human PDL progenitor populations obtained from nine human donors were assayed for their constitutive capacity to bind CAP and express ALP, and for the dexamethasone-induced potential to form mineralized-like tissue in culture in the presence of ascorbic acid and beta-glycerophosphate. Forty percent of the progenitor clones produced mineralized-like tissue. Two patterns of mineralization were observed: a spread and flat pattern similar to that produced by human bone cells in culture and a nodular ridge-like type resembling that formed by human cementoma-derived cells. A direct correlation was found between the percentage of ALP positive cells in each progenitor clone and the amount of mineralized-like tissue formed (r = 0.565). Similar correlations were found between the number of ALP positive cells and the binding capacity of each clone (r = 0.392) and between the CAP binding capacity and mineralized-like tissue formation (r = 0.584). Multiple regression analysis indicated that the constitutive capacity of a clone to bind CAP and express ALP is directly correlated to its dexamethasone-induced potential to form mineralized tissue (r = 0.675). These results indicate that CAP binding and ALP expression can serve as markers for the identification of MTF progenitors in the heterogeneous cultured population of the human periodontal ligament. These data show for the first time that binding capacity to extracellular components of mineralized tissues can be a marker for mineralized tissue-forming progenitors.

Topics: Alkaline Phosphatase; Ascorbic Acid; Binding Sites; Calcification, Physiologic; Cell Adhesion Molecules; Cell Division; Cells, Cultured; Clone Cells; Dental Cementum; Dexamethasone; Gingiva; Glucocorticoids; Glycerophosphates; Humans; Periodontal Ligament; Stem Cells

Topics: 2,2'-Dipyridyl; Animals; Ascorbic Acid; Avian Proteins; Carrier Proteins; Cartilage; Cell Differentiation; Cells, Cultured; Chick Embryo; Cytokines; Extracellular Matrix; Gene Expression; Glycerophosphates; Intracellular Signaling Peptides and Proteins; Midkine; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Procollagen-Proline Dioxygenase; RNA, Messenger

The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and beta-glycerophosphate, and samples were collected from 0-26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using 125I-PTHrP. The numbers of receptors per microgram DNA were greatest at days 3 and 5 for MC3T3-E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-E1! cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-E1 cells had twice the PTH-stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro.

Topics: 3T3 Cells; Animals; Ascorbic Acid; Binding, Competitive; Cell Differentiation; Cells, Cultured; Collagen; Cyclic AMP; DNA; Extracellular Matrix; Gene Expression Regulation; Glycerophosphates; Mice; Osteoblasts; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proline; Proteins; Rats; Receptor, Parathyroid Hormone, Type 1; Receptors, Parathyroid Hormone; RNA, Messenger

To examine the effects that an organizing extracellular matrix might have on osteoblast precursors, we created MC3T3-E1 cell lines that stably incorporated a plasmid that expressed pro alpha 1 (I) collagen chains having a truncated triple helical domain. Cells that had incorporated the pro alpha 1 (I) expression plasmid (pMG155) efficiently secreted molecules with shortened pro alpha 1 (I) chains into culture media. Electron micrographs indicated that expression of the minigene dramatically interferes with normal type I collagen fibril architecture. The turnover of newly deposited collagenous matrix as measured by 3[H]-hydroxyproline release was 29% after a 14 day chase in cells expressing the minigene compared to essentially no turnover in control cultures. MC3T3-E1 cells in culture normally demonstrate a time dependent reduction of cell division followed by an increase in osteoblast characteristics. Cell number was consistently 20-25% higher than control in MC3T3-E1 cultures expressing the truncated pro alpha 1 (I) gene but ALP activity was only 45% of control. Secretion and steady state mRNA levels for osteocalcin were over fivefold higher than control cultures but expression of other extracellular matrix components was not changed. These findings demonstrate that osteoblasts require a normally structured collagenous matrix for inhibition of cellular proliferation and subsequent upregulation of ALP. However, in the presence of rapid turnover of osteoblast matrix, the gene for osteocalcin may be upregulated in response to local signals.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Cell Differentiation; Cell Division; Cell Line; Glycerophosphates; Mice; Mutation; Osteoblasts; Osteocalcin; Procollagen; Recombinant Fusion Proteins

Rat epiphyseal plat chondrocytes were grown on glass slides, as nonadhering monolayer cultures for up to 6 weeks. Chondrocyte growth, differentiation and maturation, matrix formation and mineralization, and the temporospatial distribution of the vitamin D-dependent calcium-binding proteins, calbindin-D9K and -D28K, and the 1,25(OH)2D3 receptor (VDR), were all monitored. Chondrocytes became confluent in 2.5 weeks, differentiated to acquire a chondrocyte (polygonal) morphology, produced extracellular matrix, and finally formed a true monolayer mineralizing cartilaginous tissue, with all the stages of chondrocyte development within a single culture. beta-Glycerophosphate promoted initial matrix mineralization in 4 weeks and accelerated cell differentiation. High nominal calcium and ascorbic acid were needed for abundant matrix formation. VDR occurred at all differentiation stages, in the nuclei and nucleoli and in the cytoplasm. Calbindin-D28K and -D9K were not coexpressed. Calbindin-D28K was found in prechondroblasts, chondroblasts, and in newly differentiated chondrocytes. It was cytoplasmic in prechondroblasts and subsequently also in the perinuclear region and in nuclei, suggesting migration to the nuclear chromatin. Calbindin-D28K was nuclear only in newly differentiated chondrocytes in vitro and was not found in mature chondrocytes. In contrast, calbindin-D9K was present in the cytoplasm of mature and hypertrophic chondrocytes only. It was first in the cell body and eventually migrated within and to the far end of long cell processes with a decreasing cytoplasmic concentration showed by decreased immunostaining intensity, and ultimately hypertrophy of chondrocytes in culture. These in vitro patterns of calbindins-D and VDR accurately reflect their in vivo distributions. The genomic action of vitamin D, in vitro, resulted in the synthesis of nuclear VDR and calbindins-D.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Ascorbic Acid; Bone Density; Calbindin 1; Calbindins; Calcification, Physiologic; Calcium; Cell Differentiation; Cells, Cultured; Coloring Agents; Extracellular Matrix; Glycerophosphates; Growth Plate; Immunohistochemistry; Male; Nerve Tissue Proteins; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Calcitriol; S100 Calcium Binding Protein G

Calcification is a common feature of advanced atherosclerotic lesions and is being reemphasized as a clinically significant element of vascular disease. However, the scarcity of in vitro models of vascular calcification preclude studying its molecular and cellular mechanism. In the present study, we describe an in vitro calcification in which diffuse calcification can be induced by culturing bovine vascular smooth muscle cells (BVSMC) in the presence of beta-glycerophosphate, ascorbic acid, and insulin in a manner analogous to in vitro mineralization by osteoblasts. Calcification was confirmed by von Kossa staining and 45Ca accumulation. Factor analysis revealed that beta-glycerophosphate is the most important factor for this calcification process, suggesting that alkaline phosphatase (ALP) may be involved. As predicted, high levels of ALP expression were detected by ALP assay and Northern blot analysis. Functional significance of ALP was confirmed by demonstrating that levamisole, a specific inhibitor of ALP, inhibited BVSMC calcification in a dose-dependent manner. Bisphosphonates such as etidronate and pamidronate potently inhibited BVSMC calcification, suggesting that hydroxyapatite formation may be involved. Importantly, expression of osteopontin mRNA was dramatically increased in calcified BVSMC compared with uncalcified control cells. These data suggest that beta-glycerophosphate can induce diffuse calcification by an ALP-dependent mechanism and that this in vitro calcification system is useful for analyzing the molecular and cellular mechanisms of vascular calcification.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Calcinosis; Calcium; Calcium Radioisotopes; Cattle; Cells, Cultured; Glycerophosphates; Insulin; Muscle, Smooth, Vascular

Bone marrow stromal cells comprise a heterogeneous population including fibroblastic, adipocytic, hemopoietic, and osteogenic cells. Although the conditions under which different lineages are regulated have not been fully elucidated, dexamethasone clearly stimulates osteogenic expression in stromal cultures. The purpose of this study was to begin to elucidate and quantify some of the subpopulations present when rat bone marrow stromal cells are grown with or without dexamethasone under conditions favoring bone formation. Bone marrow stromal cells from young adult rats were cultured with ascorbic acid, beta-glycerophosphate, and with or without dexamethasone for various periods of time. Culture dishes were then analyzed for cell counts, or stained with either histochemical or immunohistochemical stains, and colony types were quantitated, or cells were processed for flow cytometry. Dexamethasone significantly increased the number of alkaline phosphatase (AP) positive colonies, von Kossa positive bone nodules, alpha-naphthylbutyrate esterase positive colonies, and ED2 positive (macrophage) colonies. The number of adipocytic foci was largely unaffected in these experiments. Flow cytometry confirmed colony counts and showed stimulation by dexamethasone of AP positive cells and macrophages, and in addition, the reduction of hemopoietic cells expressing leukocyte common antigen. These data show conclusively that when rat bone marrow stromal populations are grown under conditions stimulating osteoprogenitor differentiation and bone formation, the stromal subpopulation make-up, including expression of hemopoietic lineages, is markedly altered.

Topics: Adipocytes; Animals; Antigens; Ascorbic Acid; Bone Development; Bone Marrow; Bone Marrow Cells; Cell Count; Cells, Cultured; Dexamethasone; Femur; Flow Cytometry; Glycerophosphates; Hematopoietic Stem Cells; Immunohistochemistry; Macrophages; Male; Osteogenesis; Rats; Rats, Wistar; Stromal Cells

TGF beta has opposing effects on osteoblasts which are thought to be differentiation stage dependent; however, little is known concerning the effects of TGF beta on osteoblastic characteristics at different stages of maturation. The purpose of this study was to characterize the pattern of mRNA expression for the PTH/PTHrP receptor during normal osteoblastic differentiation in vitro, and evaluate the effects of TGF beta 1 on PTH/PTHrP receptor and osteocalcin (OCN) steady-state mRNA at different stages of osteoblastic differentiation. MC3T3-E1 preosteoblasts were plated at low density and induced to differentiate with ascorbic acid and beta-glycerophosphate. The first group served as a vehicle control and the remaining five groups received a single 48 h TGF beta 1 (3.0 ng/ml)-pulse staggered on a weekly basis for 30 days. Cell cultures were harvested weekly and evaluated for: steady-state PTH/PTHrP receptor and OCN mRNA levels via northern analysis, calcium and phosphorous levels, bone nodules via Von Kossa staining, alkaline phosphatase enzyme levels, and hydroxyproline levels. Group 1 (control) samples followed a normal pattern of proliferation, extracellular matrix deposition, and mineralization. PTH/PTHrP receptor and OCN mRNA expression increased 8-fold and 10-fold respectively, over the collection periods. When TGF beta 1 was administered during the first 48 h period (group 2) while cells were rapidly proliferating, there was a persistent inhibition of PTH/PTHrP receptor expression and a striking reduction in OCN mRNA expression at all time points. There was also a down-regulation of PTH/PTHrP receptor and OCN expression when TGF beta 1 was administered later during osteoblast differentiation (groups 3-6); however, these effects were not persistent. In addition there was a total lack of bone nodule formation in group two cultures, whereas groups 3-6 had increasing bone nodule formation because the TGF beta 1 was administered later in the culture period. These studies indicate that expression of the PTH/PTHrP receptor increases with osteoblastic differentiation and suggest that TGF beta 1 inhibits osteoblastic maturation with more persistent effects found in less differentiated osteoblastic cells.

Topics: 3T3 Cells; Animals; Ascorbic Acid; Blotting, Northern; Cell Differentiation; Glycerophosphates; Mice; Osteoblasts; Osteocalcin; Receptor, Parathyroid Hormone, Type 1; Receptors, Parathyroid Hormone; RNA, Messenger; Transforming Growth Factor beta

When grown in medium containing ascorbic acid and beta-glycerol phosphate, mouse MC3T3-E1 cells express an osteoblast phenotype and produce a highly mineralized extracellular matrix. The purpose of this study was to independently examine the role of the collagenous matrix and functional osteoblasts on the mineralization process. Cultures with and without an extensive collagenous matrix were prepared by growing MC3T3-E1 cells in the presence and absence of ascorbic acid. Matrix-rich cultures mineralized at much lower calcium phosphate ion products than nonmatrix cultures. At higher ion products, spontaneous precipitation in the medium and cell layers of nonmatrix cultures were observed. In contrast, mineral in matrix-rich cultures was still exclusively associated with collagen fibrils and not with ectopic sites in the cell layer or medium. To examine the effect of cell viability on matrix mineralization, cells were grown 8 or 16 days in the presence of ascorbic acid, then killed and incubated in a mineralizing medium. Significant mineralization was not observed in the collagenous matrix of 8-day killed cultures or age-matched controls. At 16 days mineral was associated with collagen fibrils at specific foci in the matrix of both viable and killed cultures. This observation is consistent with the concept that collagenous matrices must undergo a maturation process before they can support a mineral induction and growth. It further shows that osteoblast-like cells are not required for mineralization of mature matrices, but are required for matrix maturation.

Topics: 3T3 Cells; Animals; Ascorbic Acid; Calcification, Physiologic; Cell Division; Cell Survival; Cells, Cultured; Extracellular Matrix; Glycerophosphates; Mice; Microscopy, Electron; Osteoblasts

Mineralization occurred both in fetal rat calvarial cells and UMR 106 osteoblastic cells when they were cultured in medium containing L-ascorbate and beta-glycerophosphate as evidenced by von Kóssa staining as well as deposition of calcium ions and inorganic phosphate in the cells. When compared with corresponding non-mineralized cell cultures, both the mineralized cultures of calvarial cells and UMR 106 cells did not exhibit any change in intracellular bone-specific alkaline phosphatase activities which were measured by wheatgerm lectin precipitation method. Our results support the hypothesis that mineralization may not exert any direct negative feedback on matrix protein synthesis in osteoblasts during bone formation.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Calcification, Physiologic; Calcium; Culture Media; Fetus; Glycerophosphates; Osteoblasts; Osteosarcoma; Phosphates; Rats; Skull; Tumor Cells, Cultured

Matrix vesicles, media vesicles, and plasma membranes from three well-characterized, osteoblast-like cells (ROS 17/2.8, MG-63, and MC-3T3-E1) were evaluated for their content of enzymes capable of processing the extracellular matrix. Matrix vesicles were enriched in alkaline phosphatase specific activity over the plasma membrane and contained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collagenase, lysozyme, hyaluronidase, and tissue inhibitor of metalloproteinases (TIMP) were not found in any of the membrane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 micrograms/ml), beta-glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcification. Ascorbate or beta-glycerophosphate alone had no effect, but in combination produced significant increases in both active and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix vesicles being the most dramatic. This correlated with an increase in the formation of von Kossa-positive nodules. The results of the present study indicate that osteoblast-like cells produce matrix vesicles enriched in proteoglycan-degrading metalloproteinases. In addition, the observation that matrix vesicles contain significantly increased metalloproteinases under conditions favorable for mineralization in vitro lends support to the hypothesis that matrix vesicles play an important role in extracellular matrix processing and calcification in bone.

Topics: 3T3 Cells; Animals; Ascorbic Acid; Calcification, Physiologic; Cell Line; Cell Membrane; Cells, Cultured; Drug Synergism; Extracellular Matrix; Glycerophosphates; Glycoproteins; Metalloendopeptidases; Mice; Organelles; Osteoblasts; Tissue Inhibitor of Metalloproteinases

Rat bone marrow stromal cells comprise a heterogeneous mixture of cell lineages including osteoblastic cells. When grown in the presence of ascorbic acid, beta-glycerophosphate and 10(-8) M dexamethasone, osteoprogenitor cells within the population divide and differentiate to form bone nodules (Maniatopoulos et al., 1988, Cell Tissue Res., 254:317-330; Aubin et al., 1990, J. Bone Miner. Res., 5:S81) providing a useful model to investigate temporal and spatial changes in expression of osteoblastic markers. Immunocytochemistry was combined with Northern blotting, enzymatic assay, and radioimmunoassay to analyze the expression of bone-related proteins during the growth and differentiation sequence. By mRNA levels, protein production and/or enzymatic activity, expression of osteocalcin, bone sialoprotein, and alkaline phosphatase increased concomitantly with the development of bone nodules, while osteopontin mRNA levels decreased and those of SPARC/osteonectin did not change significantly. In older cultures with mineralizing nodules, mRNA levels for alkaline phosphatase and bone sialoprotein, but not osteocalcin, declined. Immunolabeling revealed that cells in early cultures stained poorly for SPARC/osteonectin and strongly for thrombospondin. Later, SPARC/osteonectin staining increased in most cells, while thrombospondin staining could be seen in both matrix and in cells, but with marked intercellular variability in intensity. At all time points studied, osteoblasts within bone nodules stained homogeneously for thrombospondin and alkaline phosphatase, and with marked heterogeneity of intensity amongst cells for SPARC/osteonectin and osteocalcin. Labelling with RCC455.4, a monoclonal antibody raised against rat calvaria cells which intensely labels osteoblasts and osteocytes (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339-1352), co-localized with osteocalcin. Alkaline phosphatase activity and the amount of osteocalcin determined by both radioimmunoassay and immunolabelling decreased in very late cultures, a time corresponding to appearance of fully mineralized nodules. These studies indicate that the bone marrow stromal cell system is a useful model to study the temporal and spatial expression of bone-related proteins during osteogenesis and formation, mineralization, and maturation of bone nodules. Further, immunolabelling at the individual cell and single bone nodule level allowed discrimination of marked variability of expression of osteoblast mark

Topics: Alkaline Phosphatase; Animals; Antibodies, Monoclonal; Antibody Specificity; Ascorbic Acid; Biomarkers; Blotting, Northern; Bone Marrow; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Cross Reactions; Dimethyl Sulfoxide; Glycerophosphates; Immunohistochemistry; Integrin-Binding Sialoprotein; Male; Membrane Glycoproteins; Osteoblasts; Osteocalcin; Osteoclasts; Osteogenesis; Osteonectin; Osteopontin; Phenotype; Radioimmunoassay; Rats; Rats, Wistar; RNA, Messenger; Sialoglycoproteins; Thrombospondins; Time Factors

Bone formation during mechanical unloading is reduced, mainly as a result of osteoblastic hypofunction. At the same time, the total number of osteoblasts per long bone is also markedly reduced. We tested the hypothesis that the number of osteogenic precursors present in the bone marrow stroma was concomitantly diminished by using an in vitro cell culture system in which femoral adherent bone marrow cells differentiate into active osteoblasts and produce bone-like nodules. Hindlimbs of 32-day-old male rats were either immobilized (unloaded) by sciatic neurectomy (immo) or sham operated (sham) and animals were killed after 11 days. Femora were either ashed to determine bone mass or used to generate bone marrow cultures. Adherent marrow cells were cultured in the presence of ascorbic acid, beta-glycerophosphate, and dexamethasone. Bone mass was significantly reduced in unloaded femora (by 16%) and tibiae (by 18%). The number of adherent cells (determined on day 6) was reduced by 50% in the immo group. Reduced cell number did not result from slower proliferation in culture since [3H]thymidine incorporation on days 4 and 6 was similar in the two groups. The osteogenic potential in vitro of marrow from unloaded bones was diminished compared with that from loaded ones as evidenced by (1) lower alkaline phosphatase (ALP) activity per mg protein (by 25-40%, examined on days 6 and 12), and (2) reduced nodule formation (by 70%, expressed as percentage of the dish area stained with Alizarin Red S on day 21). None of these changes occurred in the contralateral limb of operated (immobilized) animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Ascorbic Acid; Bone Marrow; Bone Marrow Cells; Cell Count; Cell Differentiation; Cells, Cultured; Dexamethasone; Femur; Glycerophosphates; Male; Osteoblasts; Osteogenesis; Rats; Stem Cells; Stromal Cells; Weight-Bearing

Osteogenetic protein-1 (OP-1), a member of the TGF-beta superfamily, induces endochondrial bone formation at subcutaneous sites in vivo and stimulates osteoblastic phenotypic expression in vitro. Primary cultures of newborn rat calvarial cells contain a spectrum of osteogenic phenotypes ranging from undifferentiated mesenchymal osteoprogenitor cells to parathyroid hormone (PTH)-responsive osteoblasts. We examined whether treatment of this cell population with recombinant human osteogenic protein-1 could induce chondrogenesis in vitro. Markers of chondroblastic versus osteoblastic differentiation included alcian blue staining at pH 1, alkaline phosphatase-specific activity, osteocalcin radioimmunoassay, and expression of collagen mRNAs. 6 d of treatment (culture days 1-7) with 4-100 ng OP-1/ml caused dose-dependent increases in alcian blue staining intensity and alkaline phosphatase activity (4.7- and 3.4-fold, respectively, at 40 ng/ml), while osteocalcin production decreased twofold. Clusters of round, refractile, alcian blue-stained cells appeared by day 3, increased in number until day 7, and then became hypertrophic and gradually became less distinct. Histochemically, the day 7 clusters were associated with high alkaline phosphatase activity and became mineralized. mRNA transcripts for collagen types II and IX were increased by OP-1, peaking at day 4, while type X collagen mRNA was detectable only on day 7 in OP-1-treated cultures. Delay of OP-1 exposure until confluence (day 7) amplifies expression of the normal osteoblastic phenotype and accelerates its developmental maturation. In contrast, early OP-1 treatment commencing on day 1 strongly amplifies chondroblastic differentiation. In the same protocol, TGF-beta 1 alone at 0.01-40 ng/ml fails to induce any hypertrophic chondrocytes, and in combination with OP-1, TGF-beta 1 blocks OP-1-dependent chondroinduction. OP-1 is believed to act on a subpopulation of primitive osteoprogenitor cells to induce endochondrial ossification, but does not appear to reverse committed osteoblasts to the chondrocyte phenotype.

Topics: Alkaline Phosphatase; Animals; Animals, Newborn; Ascorbic Acid; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage; Cell Differentiation; Cell Division; Glycerophosphates; Humans; Osteoblasts; Osteocalcin; Phenotype; Proteins; Rats; Rats, Sprague-Dawley; Skull; Stem Cells; Transforming Growth Factor beta

Bone marrow stromal cells contain colony forming cells with the potential to differentiate into osteoprogenitor (OPC) cells. OPC-stimulation medium, containing dexamethasone, ascorbate, and beta-glycerophosphate is widely used to recruit OPCs in culture. Cultures were incubated 24 h with rhodamine 123 (Rho), on different days, to examine the effect of the OPC-stimulation medium on the mitochondrial membrane potential of stromal cells. Cultures grown in both ordinary medium (DMEM with 15% FCS) and OPC-stimulation medium showed 2 Rho retention peaks on days 3-4 and 10-11. Between days 5 and 10 there was a drop in Rho retention/cell. OPC-stimulation medium increased Rho retention by at least twice the amount relative to ordinary medium, and has quadrupled it on day 7. Incubation with Rho concentrations above 5.0 micrograms/ml inhibited the portion of increased Rho retention which was contributed by the OPC-stimulation medium. Prolonged exposure to 0.1, 1.0, and 10.0 micrograms/ml Rho for 12 days only slightly increased day 12 ALP activity/cell, had no effect on day-21 mineralization and only the high dose, 10.0 micrograms/ml, doubled stromal cell proliferation. Under 24 h incubation Rho concentrations of 1.0 microgram/ml and below can serve as a marker for mitochondrial membrane potential in differentiating stromal cells. The results indicate that under both culture conditions stromal cell mitochondria undergo cycles of high and low membrane potential states and that the OPC-stimulation medium constantly maintains an elevated membrane potential relative to ordinary medium.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Marrow Cells; Calcification, Physiologic; Cell Differentiation; Cells, Cultured; Dexamethasone; Female; Fluorescent Dyes; Glycerophosphates; Microscopy, Fluorescence; Mitochondria; Osteoblasts; Rats; Rhodamine 123; Rhodamines; Stem Cells

The mesodermal clone C1 was derived from the multipotent embryonal carcinoma 1003 cell line transformed with the plasmid pK4 carrying SV40 oncogenes under the control of the adenovirus E1A promoter. We have shown that the C1 clone becomes committed to the osteogenic pathway when cultured in aggregates in the presence of mediators of the osteogenic differentiation. To further validate C1 as a model with which to study osteogenesis in vitro the kinetics of its differentiation was studied, focusing on the histology of the aggregates and on the expression of a set of genes corresponding to representative bone matrix proteins. The presence of ascorbic acid and beta- glycerophosphate specifically leads to mineralization in almost 100% of the aggregates. Transcription of the above genes, silent in exponentially growing cells, specifically occurred with the establishment of cell-cell contacts independently of the presence of ascorbic acid and inorganic phosphate. The latter, however, were absolutely required for matrix deposition and mineralization. In their presence, one observed an overall decline in type I collagen and alkaline phosphatase transcripts while osteocalcin and osteopontin transcripts preferentially accumulated in cells lining the mineralizing foci. Concomitantly, type I collagen and osteocalcin became extracellularly deposited. The osteogenic differentiation of C1 occurred while cells were still proliferating. The C1 clone thus behaves as a mesodermal stem cell, becoming committed to the osteogenic pathway upon: firstly, establishment of cellular contacts; and secondly, addition of ascorbate and beta-glycerophosphate. It therefore appears to be a promising in vitro system for deciphering the molecular basis of osteoblast ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Biomarkers; Cell Differentiation; Collagen; Gene Expression; Glycerophosphates; Immunohistochemistry; Mesoderm; Mice; Models, Biological; Osteoblasts; Osteocalcin; Osteogenesis; Osteonectin; Osteopontin; RNA, Messenger; Sialoglycoproteins; Teratocarcinoma; Transcription, Genetic; Tumor Cells, Cultured

We have previously reported the isolation of an osteogenic clonal cell line (C1) derived from mouse teratocarcinoma and immortalized by the SV 40 oncogenes. In this report we describe the kinetics of osteogenic differentiation of aggregated C1 cells by following the matrix deposition and mineralization and the expression of alkaline phosphatase. We show that after addition of beta-glycerophosphate and ascorbic acid, more than 95% of C1 aggregates synthesize a bone matrix which is deposited as early as 2 days and increases progressively with time in culture. Matrix calcification is evidenced by von Kossa staining and tetracycline incorporation into the mineral whereas no calcification appears in control cultures. Calcium is detectable in mineralizing aggregates at 2 days and calcium content increases linearly with time in culture, being 125-fold higher in mineralizing nodules than in control aggregates at 30 days. Aggregated C1 cells are characterized by a high activity of the bone type isoenzyme of alkaline phosphatase, a marker of osteoblast phenotype. Upon addition of inducers, alkaline phosphatase activity decreases by five-fold after the onset of mineralization and remains stable thereafter. The down-regulation of alkaline phosphatase activity is confirmed at the cellular level by histochemical staining. The mRNA levels for alkaline phosphatase decline during osteogenesis, following a pattern similar to the decrease in protein activity. Analysis of DNA synthesis by (3H)-thymidine incorporation and quantification of labelled nuclei on autoradiographs shows that C1 cells proliferation is not down-regulated during the time course of differentiation and that proliferating C1 cells still express alkaline phosphatase activity during osteogenic differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Biomarkers; Cell Differentiation; Cell Transformation, Viral; Clone Cells; DNA; Extracellular Matrix; Glycerophosphates; Isoenzymes; Kinetics; Mice; Minerals; Osteoblasts; Osteogenesis; RNA, Messenger; Simian virus 40; Teratoma; Tumor Cells, Cultured

We investigated the effects of 1,25-dihydroxyvitamin D3[1,25-(OH)2D3], on osteoprogenitor cell differentiation and bone nodule formation at various stages of differentiation by evaluating the effects on long term cultures of fetal rat calvaria (RC) cells. RC cells were plated at 3 x 10(4) cells/35-mm dish in alpha-minimal essential medium containing 15% fetal bovine serum, ascorbic acid, and beta-glycerophosphate (beta-GP), conditions under which bone nodules form. 1,25-(OH)2D3 inhibited bone nodule formation in a dose-dependent manner with total inhibition occurring at 1-10 nM and half-maximal inhibition occurring at approximately 0.06 nM. 1,25-(OH)2D3 also significantly stimulated RC cell growth in a dose-dependent manner in both the presence and absence of ascorbic acid. Addition of 1 nM 1,25-(OH)2D3 at different times after the start of culture inhibited nodule formation when added before and up to the early multilayering stage (up to day 11 of culture), but had no effect on nodule number when added later. When 1,25-(OH)2D3 was added at the start of the culture period and removed at the early multilayering stage, nodule formation was also inhibited. Pulses of 48-h duration also inhibited nodule formation, with maximal effect occurring between days 3 and 11. Thus, 1,25-(OH)2D3 inhibited osteoprogenitor cell differentiation during the earlier stages of culture before visible bone nodule formation occurred and the effect was not reversible upon removal of 1,25-(OH)2D3. In cultures grown to the multilayering stage in medium without ascorbic acid and beta-GP and then changed to medium with ascorbic acid and beta-GP, 1,25-(OH)2D3 inhibited when present before, but not after, the addition of ascorbic acid and beta-GP. Two other vitamin D3 metabolites, 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] and 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3] had inhibitory effects similar to 1,25-(OH)2D3. The effects were dose dependent for each metabolite tested and correlated with the biological effectiveness of these metabolites in other systems: i.e. 1,25-(OH)2D3 was more effective than 1,24,25-(OH)3D3 which in turn was more effective than 24,25-(OH)2D3. The data show that 1,25-(OH)2D3 inhibits osteoprogenitor cell differentiation at an early stage and at a time during which cell growth is stimulated.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Ascorbic Acid; Bone and Bones; Calcitriol; Cell Differentiation; Cells, Cultured; Glycerophosphates; Hydroxycholecalciferols; Rats; Rats, Wistar; Stem Cells

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The de

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Cell Differentiation; Cell Division; Cells, Cultured; Drug Synergism; Glycerophosphates; Mice; Models, Biological; Osteoblasts; Time Factors

Bone marrow stromal cells are a mixed population that contribute to the formation of the hematopoietic microenvironment. The osteogenic lineage includes populations of cells that, in culture, form discrete nodules of mineralized tissue when grown in the presence of ascorbic acid and sodium beta-glycerophosphate. We have used nodule formation to assay for the self-renewal capacity of osteoprogenitor cells in chick bone marrow cultures. To examine the regulatory influence of dexamethasone (Dx), first subcultures were grown continuously or split 1:1 at repeated subculture. Cells in continuous culture exhibited less than two population doublings, while cellular proliferation and alkaline phosphatase area were inhibited by 10(-8) mol/L Dx. Cells in split (redistributed) cultures exhibited up to 14 population doublings and cellular proliferation was also inhibited by Dx. In contrast with continuous cultures, redistributed cultures treated with Dx had increased alkaline phosphatase area and 15-fold larger amounts of mineralized tissue formation than controls. Osteogenesis was sustained for up to four subcultures and the ratio of mineralized tissue area to alkaline phosphotase positive cell area was at most 0.55. These data indicate that the osteogenic lineage of bone marrow stromal cells contains self-renewing progenitors that are recruited by Dx in culture and that at a maximum, only 55% of the alkaline phosphatase-positive cell population contributes to osteogenesis.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Bone and Bones; Bone Marrow Cells; Calcification, Physiologic; Cell Division; Cells, Cultured; Chick Embryo; Dexamethasone; Glycerophosphates; Osteogenesis

Osteoid nodules form but do not mineralize in fetal rat calvaria cell cultures grown in alpha-minimal essential medium with 10% fetal bovine serum in the absence of Na beta-glycerophosphate (beta-GP). To study factors involved in the initiation and progression of mineralization, cultures were treated with beta-GP and radiolabelled with 0.1-0.2 microCi/ml 45Ca after nodules had formed (17-19 days in medium without beta-GP). Concentrations of beta-GP from 1 to 14 mM induced a dose-dependent increase in 45Ca uptake. 45Ca uptake was restricted to nodule-containing cultures and did not occur in cultures without nodules. Continuous labelling over 72 h compared with 2 h pulses over the same time period showed that little mineralization occurred over the first 8-12 h and that the rate of mineralization was maximal and constant after 24 h exposure to beta-GP. Calcium uptake from medium was slow during the first 12 h of beta-GP exposure but increased rapidly thereafter until the medium calcium concentration reached a steady state of between 0.5 and 0.6 mM. Measurement of calcium concentration in the medium after mineralization had been initiated (24 h after beta-GP exposure) showed a linear calcium uptake into nodules (r = 0.990) over a 7 h period at a rate of 9.2 micrograms calcium/h/culture. Initiation of mineralization was prevented by 100 microM levamisole, but not by 100 microM dexamisole. When 100 microM levamisole was added 24 h after mineralization had been initiated by the addition of beta-GP, the progression of mineralization was unaffected. Similarly, after mineralization had been initiated for 24 h by 10 mM beta-GP, mineralization continued independent of the presence of beta-GP. The data show that the initiation and progression of mineralization are separate phenomena and that organic phosphate and alkaline phosphatase play a crucial role in the initiation of mineralization but are not required for the continuation of mineralization of bone nodules.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Bone and Bones; Calcification, Physiologic; Calcium; Cells, Cultured; Dexamethasone; Glycerophosphates; Image Processing, Computer-Assisted; Levamisole; Rats; Rats, Inbred Strains; Tetramisole

Marrow stromal cells are a heterogeneous population, comprising a variety of lineages including osteogenic cells. In the presence of ascorbic acid, sodium beta-glycerophosphate, and dexamethasone, rat bone marrow stromal cells form discrete nodules of mineralized, bonelike tissue. We used nodule formation by rat bone marrow stromal cells to assay for the self-renewal capacity of osteogenic progenitor cell populations. Cultures were subcultured every 5 days up to six times. Osteogenesis was assayed from second to sixth subcultures by counting the number and measuring the areas of mineralized nodules formed in cultures grown with 10(-8) mol/L dexamethasone. Nodule number and area decreased progressively between second and sixth subcultures. Alkaline phosphatase activity associated with individual cells and measured videodensitometrically decreased exponentially between the second and sixth subculture. The number of cells with alkaline phosphatase activity also decreased with progressive subculturing. The proportions of 3H-thymidine-labeled cells after continuous labeling from the beginning of the culture period showed 90% labeling for cells with alkaline phosphatase activity and fibroblastlike cells. Cultures labeled for only the first 3 days exhibited higher labeling of alkaline phosphatase-positive cells than fibroblastlike cells (P less than .05). Cultures that were flash-labeled at the end of the culture period demonstrated low labeling indices for cells with alkaline phosphatase activity and up to 10-fold higher labeling indices for fibroblastlike cells. Separate cultures treated with a cytocidal dose of high specific activity 3H-thymidine did not form nodules. These results indicate that osteogenic progenitor cells or another cell type required for nodules to develop must divide early in culture if nodule formation is to occur, and that osteoprogenitor cells express a limited capacity for self-renewal.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Marrow Cells; Cell Division; Cells, Cultured; Dexamethasone; Glycerophosphates; Osteoblasts; Osteogenesis; Rats; Rats, Inbred Strains; Stem Cells

The bone, liver, and kidney isozyme of alkaline phosphatase (ALP) has been measured in MG-63 human osteosarcoma cells after treatment with ascorbic acid (AA) and/or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Both compounds were required to achieve maximum ALP activity. When grown in the absence of 1,25-(OH)2D3 cells had low basal ALP activity regardless of whether media contained AA. In AA-free medium, 1,25-(OH)2D3 (10 nM) increased ALP activity fourfold. Addition of AA further increased levels of ALP activity induced by 1,25-(OH)2D3 to 10-15 times those found in -AA controls. The earliest effects of 1,25-(OH)2D3 were seen after 24-48 h, and ALP activity continued to increase for 6-8 days. AA and 1,25-(OH)2D3 had similar effects on ALP activity in ROS 17/2.8 rat osteosarcoma cells. In MG-63 cells the effects of AA and 1,25-(OH)2D3 could not be simply explained by the ability of these compounds to inhibit cell growth because another mitotic inhibitor, hydroxyurea, had a minimal effect on ALP activity. 1,25-(OH)2D3-specific induction of ALP +/- AA was totally blocked by inhibitors of protein and RNA synthesis. Maximal ALP induction was obtained when cells were plated at low density. Consistent with our previous report (Franceschi et al. 1988 J Biol Chem 263:18938-18945), 1,25-(OH)2D3 rapidly stimulated type I collagen synthesis and acid-precipitable hydroxyproline production in MG-63 cells and this stimulation was further increased by AA. These results suggest that induction of the osteoblast marker, ALP, is directly or indirectly coupled to collagen matrix synthesis and/or accumulation.

Topics: Alkaline Phosphatase; Ascorbic Acid; Bone and Bones; Calcitriol; Cell Count; Cell Division; Cell Line; Collagen; Glycerophosphates; Humans; Isoenzymes; Kinetics; Protein Biosynthesis; RNA

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Calcium; Cartilage; Cells, Cultured; Chick Embryo; Collagen; Enzyme Induction; Glycerophosphates; Kinetics; RNA, Messenger

Cultures of osteoblastlike cells obtained from the endosteal surfaces of rabbit long bones formed and mineralized an extracellular matrix when they were supplied daily with medium containing fresh ascorbate. No matrix formed without this supplementation. The matrix mineralized whether or not beta-glycerophosphate, a substrate of alkaline phosphatase, was added to the medium. The ion-transporting ATPase activities of untreated, ascorbate-treated, and ascorbate plus beta-glycerophosphate-treated cells were measured. Ascorbate-treated and ascorbate plus beta-glycerophosphate-treated cells had similar enzyme activities. The activities of the Ca2+-ATPase; Ca2+,Mg2+-ATPase; and alkaline phosphatase in treated cells were elevated over the activities in untreated cells. Na+,K+-ATPase activity was lower in treated than in untreated cells. HCO3--ATPase activity was not changed by treatment. Alkaline phosphatase activity was 20 times higher in freshly isolated osteoblastlike cells than in cells grown to confluence in primary culture. In addition, subculturing further reduced the activity of this osteoblast-marker enzyme. The activities of the ion-transporting ATPases and alkaline phosphatase in second passage cells were similar to the activities of these enzymes in fresh, noncalcifying tissues. Nevertheless, second passage cells retain the ability to mineralize an extracellular matrix, and their ion-transporting ATPase and alkaline phosphatase activities are altered when the cells mineralize a matrix.

Topics: Adenosine Triphosphatases; Alkaline Phosphatase; Animals; Ascorbic Acid; Bone and Bones; Bone Matrix; Calcium-Transporting ATPases; Cells, Cultured; Collagen; Cyclic AMP; Glycerophosphates; Osteoblasts; Parathyroid Hormone; Rabbits; Sodium-Potassium-Exchanging ATPase