transforming-growth-factor-beta and Dentin--Secondary

Pulp tissue responds to dentin damage by laying down a tertiary dentin matrix (reactionary or reparative) beneath the site of injury. Reactionary dentin is secreted by surviving odontoblasts in response to environmental stimuli, leading to an increase in metabolic activities of the cells. The inductive molecules that determine the success of the pulp healing may be released from the damaged dentin as well as from the pulp tissue subjacent to the injury. This paper will schematically consider two major growth factors probably implicated in the control of odontoblast activity: TGF beta-1 released from demineralized dentin and NGF from pulp. To analyze their role with an in vitro system that mimics the in vivo situation, we have used thick-sliced teeth cultured as described previously. The supply of factors was accomplished by means of a small tube glued onto the dentin. The tube was filled with TGF beta-1 (20 ng/mL) or NGF (50 ng/mL), and slices were cultured for 4 or 7 days. Results showed that TGF beta-1 binding sites are strongly detected on odontoblasts in the factor-rich zone. A strong expression of alpha 1(I) collagen transcripts was also detected. In the NGF-rich environment, p75NTR was re-expressed on odontoblasts and the transcription factor NF-kappa B activated. Modifications in the odontoblast morphology were observed with an atypical extension of the cell processes filled with actin filaments. These results suggest that odontoblasts respond to influences from both dentin and pulp tissue during pulp repair.

Topics: Actin Cytoskeleton; Actins; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Culture Techniques; Dental Pulp; Dentin; Dentin, Secondary; Dentinogenesis; Humans; Molecular Biology; Nerve Growth Factor; NF-kappa B; Odontoblasts; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Receptors, Transforming Growth Factor beta; Tooth Demineralization; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

Trans-dentinal stimulation of tertiary dentinogenesis has long been recognized, and has traditionally been ascribed to diffusion of irritant substances arising during injury and restorative treatment. Identification of bio-active components, especially growth factors including TGF-beta s, sequestered within dentin matrix provides a new explanation for cellular signaling during tertiary dentinogenesis. Both isolated dentin matrix components and pure growth factors (TGF-beta s) have been shown to signal cellular events leading to reactionary and reparative tertiary dentinogenesis. Release of these bio-active components from dentin matrix may arise during carious attack and other injury to the tissue, and also during subsequent surgical intervention and restoration of the tooth. Both cavity-conditioning agents and leaching from restorative materials may contribute to release of these components. Distance of diffusion, as determined by cavity residual dentin thickness, and other restorative parameters may influence the signaling process after release of these components. Careful consideration of the interplay between tissue injury and surgical and restorative material factors is required for optimum exploitation of the exquisite regenerative capacity of dentin-pulp for more biological approaches to clinical treatment of dental disease.

Topics: Animals; Dental Caries; Dental Cavity Lining; Dental Materials; Dental Pulp; Dental Restoration, Permanent; Dentin; Dentin, Secondary; Dentinogenesis; Diffusion; Growth Substances; Humans; Irritants; Odontoblasts; Regeneration; Signal Transduction; Transforming Growth Factor beta

Molecular biology is providing opportunities to develop new strategies or agents for the treatment of a wide variety of diseases. The availability of large amounts of highly purified proteins produced by recombinant DNA techniques is an obvious example. Recent evidence has implicated proteins belonging to the bone morphogenetic protein (BMP) subgroup of the transforming growth factor beta supergene family in tooth formation and dentinogenesis. It has long been known that bone and dentin contain bone morphogenetic protein activity. Recently, recombinant human BMP-2, -4, and -7 (also known as OP-1), have been shown to induce reparative dentin formation in experimental models of large direct pulp exposures in permanent teeth. The manner in which these agents act appears unique. New reparative dentin replaces the stimulating agents applied directly to the partially amputated pulp. Hence, the new tissue forms contiguous with, largely superficial to, and not at the expense of the remaining vital pulp tissue. This suggests a therapeutic approach permitting the induction of a predetermined and controlled amount of reparative dentin. Additionally, OP-1 has been associated with the formation of reparative dentin after application to a freshly cut but intact layer of dentin. These findings may provide future clinicians with additional options for the treatment of substantially damaged or diseased vital teeth.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Dental Pulp; Dental Pulp Diseases; Dentin; Dentin, Secondary; Dentinogenesis; Growth Substances; Humans; Molecular Biology; Odontogenesis; Proteins; Recombinant Proteins; Transforming Growth Factor beta

During the treatment of dental caries that has not penetrated the tooth pulp, maintenance of as much unaffected dentine as possible is a major goal during the physical removal of decayed mineral. Damage to dentine leads to release of fossilized factors (transforming growth factor-β [TGF-β] and bone morphogenic protein [BMP]) in the dentine that are believed to stimulate odontoblasts to secrete new "tertiary" dentine (reactionary dentine). This is formed on the pulpal surface of existing dentine and rethickens the dentine. We have previously shown that activation of Wnt/β-catenin signaling is pivotal for tooth repair in exposed pulp injury, and the pathway can be activated by small-molecule GSK-3 antagonists, resulting in enhanced reparative dentine formation. Here, we use a nonexposed pulp injury model to investigate the mechanisms of reactionary dentine formation in vivo, using small molecules to modulate the Wnt/β-catenin, TGF-β, and BMP pathways. We found that a local increase of Wnt activation at the injury site enhances reactionary dentine secretion. In addition, inhibition of TGF-β, BMP, or Wnt pathways does not impede reactionary dentine formation, although inhibition of TGF-β and/or BMP signaling does result in more disorganized, nontubular reactionary dentine. This suggests that Wnt/β-catenin signaling plays no major role in the formation of reactionary dentine, but in common with reparative dentine formation, exogenous elevation of Wnt/β-catenin signaling can enhance tertiary dentine formation. Release of latent TGF-β or BMPs from dentine is not required for the deposition of mineral to form reactionary dentine but does play a role in its organization.

Topics: Animals; Bone Morphogenetic Proteins; Dental Pulp; Dentin, Secondary; Dentinogenesis; Glycogen Synthase Kinase 3; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Wnt Signaling Pathway

Conditioned media (CM) from human dental pulp cells (HDPC) was investigated for its effects on the proliferation and differentiation of HDPC and MG63 cells.. CM prepared from the primary culture of HDPC was used for the culture of HDPC and MG63. Cell growth, alkaline phosphatase (ALPase) activity, mRNA expression of differentiation-related genes, and mineralization of the HDPC and MG63 cells in the media containing CM were assessed.. HDPC CM increased the cell growth of HDPC but decreased MG63 cell growth. ALPase activity and the mineralization of both HDPC and MG63 were enhanced by HDPC CM. The CM also up-regulated the expressions of DSPP, DMP-1, and OCN mRNA in HDPC. Pretreatment of HDPC CM with a neutralizing antibody against TGF-beta completely eliminated the effect of CM on ALPase activity in HDPC.. HDPC was able to secrete odontogenic differentiation-inducing factors, in which TGF-beta seems to a key element of the CM effects.

Topics: Adolescent; Alkaline Phosphatase; Calcification, Physiologic; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Child; Culture Media, Conditioned; Dental Pulp; Dentin, Secondary; Extracellular Matrix Proteins; Gene Expression Regulation, Developmental; Humans; Odontoblasts; Osteocalcin; Phosphoproteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Up-Regulation

The purpose of this study was to determine whether human recombinant bone morphogenetic protein-2 (rhBMP-2), calcitonin gene-related peptide (CGRP), calcitonin (CT), or parathyroid hormone-related protein (PTHrP) promoted reparative tertiary dentin or osteodentin formation in ferret canines.. Ferrets had up to 4 pulpotomies performed under anesthesia. All pulps had sterile absorbable sponge of a standard size placed in contact with the pulp. The sponge contained sterile saline, rhBMP-2, CGRP, CT, or PTHrP. The opening was filled with an intermediate restorative material. After 6 weeks, the ferrets were anesthetized, and the pulpotomized teeth were extracted. The canines were fixed, decalcified, sectioned, and stained with hematoxylin-eosin. Sections were selected from the area of the opening, and the amount of reparative tertiary dentin and osteodentin was measured by using a digitizer.. Analysis of the photomicrographs showed that rhBMP-2 induced 0.58 +/- 0.19 mm(2) osteodentin and 0.56 +/- 0.18 mm(2) tertiary dentin. CGRP induced 0.46 +/- 0.05 mm(2) osteodentin and 0.38 +/- 0.04 mm(2) tertiary dentin. The amount of rhBMP-2-induced and CGRP-induced osteodentin and tertiary dentin was significantly (P < .001) more than that found in the sterile saline-treated teeth (0.29 +/- 0.03 mm(2) osteodentin and 0.14 +/- 0.03 mm(2) tertiary dentin) or CT (0.2 +/- 0.06 mm(2) osteodentin and 0.16 +/- 0.05 mm(2) tertiary dentin; P < .01). PTHrP significantly (P < .05) reduced the amount of osteodentin (0.17 +/- 0.02 mm(2)) observed in the saline-treated teeth but was not significantly different in the amount of tertiary dentin observed.. RhBMP-2 and CGRP promoted more pulpal healing than either CT or PTHrP.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcitonin; Calcitonin Gene-Related Peptide; Dental Pulp; Dentin, Secondary; Ferrets; Humans; Male; Parathyroid Hormone-Related Protein; Pulpotomy; Recombinant Proteins; Regeneration; Transforming Growth Factor beta

Primary pulp cell cultures are frequently used to study cellular responses, odontogenic potential and stem cell responses. Their isolation and expansion via a range of technical approaches are widely reported. The purpose of this study was to investigate the influence of isolation approach and extended expansion on cell phenotype and behaviour.. To determine viable cell isolation, enzymatic dissociation was performed on rodent incisor pulps using collagenase, trypsin, hyaluronidase and ficin. Extended expansion culture of released cells was performed in DMEM and alpha-MEM media. Cultures were subsequently analysed for gene expression, cell proliferation, cell morphology and differentiation capacity up to passage 20.. Data indicated that incubation of extirpated and mechanically minced rodent pulpal tissue with 0.25% Trypsin:EDTA and subsequent culture in alpha-MEM medium provided optimal conditions for maximal cell growth and expansion. Under these conditions, extended culture decreased cellular proliferative capacity up to passage 7, whilst higher passages demonstrated recovered growth rates. In general gene expression analysis of osteogenic and dentinogenic associated markers decreased with increasing passage number. Notably expression of TGFbetas-1, -2 and -3 increased up to passage 10 as did the stem cell and pericyte/myofibroblast markers, CD74, Neuroserpin and alpha-SMA. Analysis of molecular phenotypes indicated little difference in lineage differentiation capacity between earlier and later passages.. The present study characterizes conditions for primary pulp cell isolation and expansion and indicates that both earlier and later passages maintain differentiation capacity. Continued passage however may result in selection for cells with a pericyte/myofibroblast phenotype.

Topics: Adult Stem Cells; Animals; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cell Separation; Cell Survival; Cells, Cultured; Dental Pulp; Dentin, Secondary; Dentinogenesis; Extracellular Matrix Proteins; Gene Expression; Gene Expression Profiling; Male; Osteogenesis; Phenotype; Phosphoproteins; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Sialoglycoproteins; Transforming Growth Factor beta

This review summarizes the in vivo experiments carried out by our group after implantation of bioactive molecules (matricellular molecules) into the exposed pulp of the first maxillary molar of the rat or the mandibular incisor of rats and mice. We describe the cascade of recruitment, proliferation and terminal differentiation of cells involved in the formation of reparative dentin. Cloned immortalized odontoblast progenitors were also implanted in the incisors and in vitro studies aimed at revealing the signaling pathways leading from undifferentiated progenitors to fully differentiated polarized cells. Together, these experimental approaches pave the way for controlled dentin regenerative processes and repair.

Topics: Amelogenin; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Clone Cells; Dental Pulp Exposure; Dentin; Dentin, Secondary; Extracellular Matrix; Integrin-Binding Sialoprotein; Mice; Odontoblasts; Peptide Fragments; Rats; Regeneration; Sialoglycoproteins; Signal Transduction; Stem Cells; Transforming Growth Factor beta; Wound Healing

Recombinant human bone morphogenetic protein-7 (BMP-7) has been shown to stimulate new reparative dentin formation in animal models. However, little is known about whether BMP-7 could promote the odontoblast-like differentiation and the formation of mineralized nodules in human dental pulp cells. Here, we reported that the infection with adenovirus-BMP-7 (Ad-BMP-7), a BMP-7-expressing adenoviral vector, induced the expression of BMP-7 in primarily cultured human dental pulp cells in the long term with little effect on their proliferation and viability. Importantly, BMP-7 expression significantly increased alkaline phosphatase activity and induced the dentin sialophosphoprotein expression in a dose- and time-dependent manner, suggesting that BMP-7 promoted the odontoblast differentiation. Furthermore, BMP-7 expression stimulated the formation of many mineralized dentin-like calcified nodules. Our data suggest that Ad-BMP-7-mediated BMP-7 expression can promote the differentiation of human pulp cells into odontoblast-like cells and mineralization in vitro, which may provide insight for the design of new gene therapy for the pulp capping in the clinic.

Topics: Adenoviridae; Adult; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Differentiation; Cells, Cultured; Child; Cloning, Molecular; Dental Pulp; Dentin, Secondary; Extracellular Matrix Proteins; Genetic Vectors; Humans; Odontoblasts; Phosphoproteins; Recombinant Proteins; Sialoglycoproteins; Transforming Growth Factor beta

Calcium hydroxide is often used for induction of reparative dentin formation in endodontic treatment. However, little is known about the mechanism by which calcium hydroxide works. The calcium ion (Ca2+) is an important regulator of cell functions. In this study, we examined the effect of extracellular Ca2+ on gene expression of bone-related proteins in human cultured pulp cells in serum-free conditions. A Ca2+ level elevated by 0.7 mM induced an increase in mRNA expression of osteopontin and bone morphogenetic protein (BMP)-2. However, mRNA levels of BMP-4 and alkaline phosphatase decreased under the elevated Ca2+ culture condition. The same concentration of additional magnesium ions had little effect on expressions of the examined bone-related protein mRNAs. These findings suggest that Ca2+ in Ca(OH)2 specifically modulates osteopontin and BMP-2 levels during calcification in pulp.

Topics: Alkaline Phosphatase; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Calcium; Calcium Hydroxide; Cells, Cultured; Dental Pulp; Dentin, Secondary; Gene Expression; Humans; Magnesium; Osteopontin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta

The purpose of this study was to identify the hard tissue formed early in experimental pulp exposures capped with mineral trioxide aggregate (MTA) or bone morphogenetic protein (BMP)-7 using dentin sialoprotein (DSP) as a marker. The pulps of 35 maxillary first, second, and third molar teeth from 10 male rats were experimentally exposed. The pulps were capped with MTA alone as a pulp-capping agent and final restoration or with BMP-7 followed by restoration with MTA. Five teeth with class I occlusal preparations, no exposure, and no restoration served as positive controls. Five teeth that received pulp exposures and no restoration served as negative controls. Five untreated third molars served as additional controls. The animals were killed at 2 weeks. The specimens were prepared and evaluated histologically and with immunohistochemistry using polyclonal antibodies raised against rat DSP. Pulps capped with MTA formed hard tissue that demonstrated significantly more immunostaining for DSP compared with BMP-7 (p = 0.0031). MTA-capped pulps also showed significantly more complete bridge formation compared with BMP-7 (p = 0.0008). Pulps capped with BMP-7 demonstrated a hard tissue that was bone-like in appearance and devoid of DSP staining.

Topics: Aluminum Compounds; Animals; Biomarkers; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcium Compounds; Dental Pulp Capping; Dental Pulp Exposure; Dentin, Secondary; Drug Combinations; Extracellular Matrix Proteins; Immunohistochemistry; Male; Oxides; Phosphoproteins; Protein Precursors; Rats; Rats, Sprague-Dawley; Root Canal Filling Materials; Sialoglycoproteins; Silicates; Transforming Growth Factor beta

Bone morphogenetic protein 7 (BMP 7), also termed osteogenic protein 1, a member of the transforming growth-factor superfamily, was examined for its efficacy in inducing reparative dentinogenesis in the exposed pulps of rat molars. To determine if the reaction was dose-dependent, collagen pellets containing 1, 3 or 10 microgram of recombinant BMP 7 were inserted in intentionally perforated pulps (10-12 pulps per group) in the deepest part of half-moon class V-like cavities cut in the mesial aspect of upper first molars. As controls, the collagen carrier (CC group) alone and calcium hydroxide (Ca group) were used as capping agents. All cavities were then restored with a glass-ionomer cement. Half of the animals were killed after 8 days and the other half after 28 days, by intracardiac perfusion of fixative. The molars were processed for histological evaluation by light microscopy. No difference in effect could be detected between the three concentrations of BMP 7 groups at either time interval. After 8 days, all groups showed varying inflammation, from mild of severe, and the Ca group demonstrated early formation of a reparative dentine bridge. At 28 days the CC group displayed irregular osteodentine formation, leaving some unmineralized areas at the exposure site and interglobular unmineralized areas containing pulp remnants. In the Ca-treated pulps, the initial formation of thick reparative osteodentine bridges that sealed more or less completely the pulp perforation was followed, in the deeper part, by irregular tubular dentine. In most BMP 7-treated specimens, the initial inflammation has resolved at 8 days and at 28 days heterogeneous mineralization or osteodentine filled the mesial coronal pulp. They also had complete filling of the radicular pulp by homogenous mineralization in the mesial root; this reaction was found in 11 teeth in the BMP 7 group, one tooth in the CC group an none of the Ca group. These results emphasize the biological differences the coronal and radicular parts of the pulp, and the potential of bioactive molecules such as BMP 7 to provide an a alternative conventional endodontic treatments.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcium Hydroxide; Collagen; Dental Pulp; Dental Pulp Capping; Dental Pulp Exposure; Dentin, Secondary; Dose-Response Relationship, Drug; Humans; Models, Animal; Molar; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Stimulation, Chemical; Tooth Crown; Tooth Root; Transforming Growth Factor beta

This is the first in a series of four papers aimed at understanding human pulpal responses to tissue injury, cavity preparation and restorative events. This article provides an insight into the exquisite regenerative potential of the dentine-pulp complex which underpins the success of restorative dentistry.

Topics: Acid Etching, Dental; Biology; Cell Differentiation; Cell Survival; Dental Cavity Preparation; Dental Pulp; Dental Restoration, Permanent; Dentin; Dentin Solubility; Dentin, Secondary; Dentinogenesis; Humans; Odontoblasts; Regeneration; Signal Transduction; Stem Cells; Tooth Diseases; Transforming Growth Factor beta

Transforming growth factor beta, TGF-beta, is expressed during tooth formation and can induce pre-odontoblast differentiation and formation of functional odontoblast-like cells in vitro. In addition, exogenous TGF-beta can increase reparative dentin formation, presumably by acting on odontoblasts. In this study, we examined the tooth phenotype of transgenic mice, in which TGF-beta 2 expression is directed by the osteocalcin promoter. Previous studies have shown that these mice have a bone phenotype that resembles that of human osteoporosis, including the existence of spontaneous fractures. Microhardness testing of the enamel and dentin showed no differences in the molars of these transgenic mice as compared with those of their wild-type littermates. Consistent with the increase in bone mineral apposition rate previously reported in these mice, the dentin apposition rate appeared to be increased in the TGF-beta 2-overexpressing mice. Thus, in teeth, as in bone, TGF-beta 2 appears to stimulate the synthesis and deposition of matrix. Further studies are needed to understand the effect of TGF-beta 2 on distinct mineralized tissues (bone, dentin, and cementum) and to determine whether exogenous TGF-beta 2 may be useful for tooth repair.

Topics: Animals; Calcification, Physiologic; Cell Differentiation; Cementogenesis; Dental Enamel; Dentin; Dentin, Secondary; Dentinogenesis; Disease Models, Animal; Hardness; Humans; Image Processing, Computer-Assisted; Male; Mice; Mice, Transgenic; Odontoblasts; Osteocalcin; Osteoporosis; Phenotype; Promoter Regions, Genetic; Tooth Calcification; Transforming Growth Factor beta; Transforming Growth Factor beta2

Several growth factors and extracellular matrix molecules, which are expressed during embryonic tooth development, are re-expressed in dental tissues under pathological conditions. Pathological conditions such as caries lesions and dental injuries are often lethal to the odontoblasts, which are then replaced by other pulp cells. These cells are able to differentiate into odontoblast-like cells and produce a reparative dentin. Here we demonstrate the in vivo distribution of several molecules in human permanent teeth under normal and pathological conditions. The intermediate filament protein nestin, which is a marker of young odontoblasts, is absent from old permanent teeth. Similarly, the Notch protein, which is involved in cell fate specification and is localized in the sub-odontoblastic cell layer during odontogenesis, is not detected in adult dental tissues. In carious and injured teeth, nestin is expressed in a selective manner in odontoblasts surrounding the injury site, while Notch is expressed in the sub-odontoblastic layer of cells. We reproduced this physiological event in an in vitro culture system. Pulp cells cultured in the presence of beta-glycerophosphate formed mineralization nodules. As odontoblasts, pulp cells contributing to the nodule formation express type I collagen, osteonectin, dentin sialophosphoprotein, and nestin. In this in vitro assay system, nestin is up-regulated after local application of Bone Morphogenetic Protein 2 and 4. Fourier transform infrared microspectroscopy showed that both the organic and the mineral compositions of the nodules have the characteristics of human dentin and differ from those of enamel and bone. These findings show that both the molecular and the mineral characteristics of the human dentin matrix are respected in the in vitro culture conditions.

Topics: Adult; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Differentiation; Collagen Type I; Culture Techniques; Dental Caries; Dental Pulp; Dentin; Dentin, Secondary; Dentinogenesis; Extracellular Matrix Proteins; Fetus; Humans; Intermediate Filament Proteins; Membrane Proteins; Nerve Tissue Proteins; Nestin; Odontoblasts; Odontogenesis; Osteonectin; Phosphoproteins; Protein Precursors; Receptors, Notch; Sialoglycoproteins; Spectroscopy, Fourier Transform Infrared; Tooth Calcification; Tooth Diseases; Tooth Injuries; Trans-Activators; Transforming Growth Factor beta; Up-Regulation

The nature and specificity of the mechanisms by which the amputated dentin-pulp interface is therapeutically healed determine the properties of the barrier at this site and play a critical role in the outcome of vital pulp therapy. Healing of the dentin-pulp complex proceeds either by natural repair-which results in defensive hard-tissue formation, or therapeutically regulated dentin regeneration, which aims to reconstitute the normal tissue architecture at the pulp periphery. Progress in biomedical research opens new directions for the design of biologically effective pulp therapies. Application of biocompatible and biodegradable carrier vehicles for local delivery of signaling molecules in pulp-capping situations showed induction of fibrodentin/reparative dentin formation, but often at the expense of underlying pulp tissue. An alternative pre-clinical model aiming to reconstitute normal tissue architecture directly at the dentin-pulp interface should be designed on the basis of the direct induction of odontoblast-like cell differentiation and reparative dentin formation at the pulp-capping material interface. Experimental data clearly showed that pulpal cells can differentiate directly into odontoblast-like cells in association with specific extracellular matrices (dentinal or fibrodentinal matrix) or TGF beta 1-containing artificial substrates. Dentin-induced dentinogenesis can be used as a master plan for the achievement of new therapeutic opportunities. In the present study, several short-term experimental studies on dog teeth for potential direct induction of odontoblast-like cell differentiation at the surface of rhTGF beta 1-containing artificial substrates (Millipore filters, hydroxyapatite granules, calcium hydroxide, pure titanium) failed to induce any specific reparative dentinogenic effects.

Topics: Animals; Biocompatible Materials; Calcium Hydroxide; Cell Differentiation; Dental Pulp; Dental Pulp Capping; Dental Pulp Exposure; Dentin; Dentin, Secondary; Dentinogenesis; Dogs; Drug Carriers; Drug Delivery Systems; Durapatite; Extracellular Matrix; Membranes, Artificial; Micropore Filters; Odontoblasts; Regeneration; Surface Properties; Titanium; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

To evaluate the effects of bioactive molecules in pulpal wound healing, we carried out experiments using the rat upper molars as an in vivo model. Cavities were prepared on the mesial aspect, and pulp perforation was accomplished by the application of pressure with the tip of a steel probe. After the pulp-capping procedure, the cavities were filled with a glass-ionomer cement. Comparison was made between and among: (1) sham-operated controls with dentin and predentin fragments implanted in the pulp during perforation after 8, 14, and 28 days; (2) carrier without bioactive substance; (3) calcium hydroxide; (4) Bone Sialoprotein (BSP); (5) different concentrations of Bone Morphogenetic Protein-7 (BMP-7), also termed Osteogenic Protein-1 (OP-1); and (6) N-Acetyl Cysteine (NAC), an anti-oxidant agent preventing glutathione depletion. Histologic and morphometric comparison, carried out among the first 4 groups on demineralized tissue sections, indicated that, at 28 days after implantation, BSP was the most efficient bioactive molecule, inducing homogeneous and well-mineralized reparative dentin. BMP-7 gave reparative dentin of the osteodentin type in the coronal part of the pulp, and generated the formation of a homogeneous mineralized structure in the root canal. These findings indicate that the crown and radicular parts of the pulp bear their own specificity. Both BSP and BMP-7 were superior to calcium hydroxide in their mineralization-inducing properties, and displayed larger areas of mineralization containing fewer pulp tissue inclusions. The overall mineralization process to these molecules appeared to proceed by mechanisms that involved the recruitment of cells which differentiate into osteoblast-like cells, producing a mineralizing extracellular matrix. We also provide preliminary evidence that NAC induces reparative dentin formation in the rat molar model. Pulp-capping with bioactive molecules provides new prospects for dental therapy.

Topics: Acetylcysteine; Animals; Antioxidants; Biocompatible Materials; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcium Hydroxide; Dental Pulp; Dental Pulp Capping; Dental Pulp Cavity; Dental Pulp Exposure; Dental Restoration, Permanent; Dentin; Dentin, Secondary; Dentinogenesis; Free Radical Scavengers; Glass Ionomer Cements; Integrin-Binding Sialoprotein; Male; Pharmaceutical Vehicles; Rats; Rats, Sprague-Dawley; Sialoglycoproteins; Time Factors; Transforming Growth Factor beta; Wound Healing

In vivo and ex vivo gene transfer are being developed for localized skeletal regeneration. These strategies for tissue regeneration were tested in an adult ferret model of vital pulp therapy. In this model a reversible pulpitis was induced first. Then after 3 d, the pulps were directly infected with recombinant virus or implanted with ex vivo transduced autologous dermal fibroblasts. The genome of the recombinant adenovirus contained a full-length cDNA encoding mouse bone morphogenetic protein (BMP)-7 (AdBMP7) or bacterial beta-galactosidase cDNA (AdlacZ). The BMP-7, but not lacZ, ex vivo transduced dermal fibroblasts induced reparative dentinogenesis with apparent regeneration of the dentin-pulp complex. In vivo infection with AdBMP-7 failed to produce reparative dentin in all cases. E. vivo gene transfer of BMP-7 may be an effective method for inducing dentin regeneration in teeth with reversible pulpitis.

Topics: Adenoviridae; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Dentin, Secondary; Ferrets; Fibroblasts; Gene Transfer Techniques; Male; Mice; Pulpitis; Regeneration; Transforming Growth Factor beta

Recombinant human BMP-7 (bone morphogenetic protein-7, osteogenic protein-1) is osteogenic, dentinogenic and cementogenic when implanted into the appropriate tissue in vivo. However, most studies characterizing the induction of these tissues have implanted BMP-7 into freshly surgerized, clinically healthy tissues. To determine if BMP-7 is dentinogenic in inflamed dental pulps, we applied BMP-7 to inflamed ferret pulps. A single application of 5 microg of a commercial preparation of lipopolysaccharide (LPS) from Salmonella typhimurium directly to the coronal pulp induced a reversible mixed inflammatory exudate of moderate intensity within 3 d. Treatment with a single application of 2.5, 7.5 or 25 microg recombinant human BMP-7/mg collagen (2 mg total mass/tooth) induced reparative dentinogenesis in controls but not LPS treated dental pulps. These data reveal that a single application of up to 50 microg/tooth of exogenous recombinant BMP-7 is insufficient to induce reparative dentinogenesis in ferret teeth with reversible pulpitis. Given that pulp cells in the inflamed tissues likely retain the capacity to respond to exogenous BMP-7, it is possible that insufficient active recombinant protein is available to induce tissue formation in experimentally inflamed dental pulps.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Collagen; Dentin, Secondary; Dentinogenesis; Drug Carriers; Ferrets; Humans; Lipopolysaccharides; Pulpitis; Recombinant Proteins; Salmonella typhimurium; Transforming Growth Factor beta

Dental pulps contain sulfated glycosaminoglycans (GAGs), such as chondroitin 4-sulfate (CSA/4CS), dermatan sulfate (CSB/DS), and chondroitin 6-sulfate (CSC/6CS). Sulfated GAGs play important roles in mineralization and collagen fibrillogenesis during primary, secondary, and reparative dentin formations. Transforming growth factor-beta (TGF-beta) is a potent regulator for several extracellular matrix (ECM) components and modulates the proliferation and differentiation. Using rat clonal dental pulp cells (RPC-C2A), we investigated the constituents of GAGs synthesized by the cells and the effect of TGF-beta on their synthesis by measuring the radioactivity of [35S]sulfate incorporated into GAG fractions. Cellulose acetate electrophoresis analysis revealed that RPC-C2A cells synthesized CSA and CSB but not CSC and that 10 ng/ml of TGF-beta increased the production of CSA and CSB in the cell/ECM fraction. Measurement of [35S]sulfate incorporation showed a significant increase in the amount of GAGs by TGF-beta, 1.3-fold CSA, and 1.2-fold CSB in the cell/ECM fraction. In the medium fraction the most secreted GAG was CSA, whereas CSB was stored in the cell/ECM fraction. Secreted CSA in the medium was markedly increased by 10 ng/ml of TGF-beta (1.7-fold). These findings indicate that CSA and CSB are major sulfated GAGs synthesized by RPC-C2A cells and that TGF-beta acts as a stimulator of sulfated GAG synthesis in dental pulp cells.

Topics: Animals; Cell Differentiation; Cell Division; Cells, Cultured; Chondroitin Sulfates; Clone Cells; Collagen; Dental Pulp; Dentin, Secondary; Dentinogenesis; Dermatan Sulfate; Electrophoresis, Cellulose Acetate; Extracellular Matrix; Radiopharmaceuticals; Rats; Statistics as Topic; Sulfur Radioisotopes; Tooth Calcification; Transforming Growth Factor beta

To investigate the reparative dentine formation induced by the complex of recombinant human bone morphogenetic protein 2 (rhBMP2) and fibrin sealant (FS) in dog tooth.. Freshly exposed molars, premolars and cuspids were treated with the complex of rhBMP2/FS, rhBMP2 and Ca(OH)(2) paste which served as control.. The wound healing of exposed pulp treated with FS seemed to be better than that without FS. Dentine bridge formation was observed in pulp treated with the complex at 7 days, with rhBMP2, Ca(OH)(2) at 28 days. At 63 days after operation, the cases of tubular dentine bridge formation of the pulp treated with the complex were more than that of pulp treated with rhBMP2.. The synergistic effects of FS and the complex rhBMP2 exist and can be used as bioactive pulp capping agent.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Dental Pulp Capping; Dentin, Secondary; Dogs; Fibrin Tissue Adhesive; Recombinant Proteins; Transforming Growth Factor beta

Calcium hydroxide has been the material of choice for apexification. The purpose of this study was to compare the efficacy of osteogenic protein-1 and mineral trioxide aggregate with that of calcium hydroxide in the formation of hard tissue in immature roots of dogs. Sixty-four roots of premolars were used. After induction of periradicular lesions, the canals were debrided and filled with calcium hydroxide for 1 wk. After the removal of calcium hydroxide, the root canals received one of the treatment materials in a balanced design. The animals were euthanized 12 wk later. The degree of hard tissue formation and amount of inflammation were evaluated histomorphically. Data were statistically evaluated using ANOVA, chi 2, and Kruskal-Wallis. Mineral trioxide aggregate produced apical hard tissue formation with significantly greater consistency. The difference in the amount of hard tissue produced among the three test materials was not statistically significant. Furthermore, the degree of inflammation was not significantly different between the various test groups.

Topics: Aluminum Compounds; Analysis of Variance; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcium Compounds; Calcium Hydroxide; Chi-Square Distribution; Collagen; Dentin, Secondary; Dogs; Drug Combinations; Evaluation Studies as Topic; Oxides; Silicates; Statistics, Nonparametric; Tooth Apex; Transforming Growth Factor beta

Expression of transforming growth factor-beta (TGF-beta) isoforms by odontoblasts leads to their sequestration within dentine matrix. TGF-beta1 and -beta3 stimulate matrix secretion and also initiate odontoblast cytodifferentiation in vitro and in vivo. Using a recently established organ-culture model, the aim here was to examine the effects of TGF-beta isoforms on the response of the dentine-pulp complex during culture. Agarose beads were soaked in isoforms 1-3, and placed on the odontoblast area of slices of 28-day-old rat incisor tooth. The slices were maintained in Trowel-type cultures for 7 days. Both TGF-beta1 and -beta3 stimulated a local increase in predentine secretion at the site of the bead application when compared to control cultures. Mitogenic effects on the cells of the subodontoblast layer were also seen and occasionally small foci of newly differentiated odontoblast-like cells could be observed a little distant from the application site of TGF-beta3. TGF-beta2 had a minimal effect on the cultured tissues. These results demonstrate that TGF-beta1 and -beta3 can stimulate secretion of extracellular matrix by odontoblasts, are mitogenic to pulp cells, and that TGF-beta3 may have inductive effects on pulpal cells. Such activities might be important during reparative processes in the dentine-pulp complex after tissue injury.

Topics: Animals; Cell Differentiation; Dental Pulp; Dentin; Dentin, Secondary; Dentinogenesis; Extracellular Matrix; Gene Expression Regulation; Incisor; Mitogens; Odontoblasts; Organ Culture Techniques; Protein Isoforms; Rats; Rats, Wistar; Transforming Growth Factor beta

To investigate the reparative dentin formation induced by the complex of recombinant human bone morphogenetic protein 2 (rhBMP2) and fibrin sealant (FS) in dogs.. Freshly exposed pulp of molars, premolars, and canines were treated, respectively, with the complex of rhBMP2-FS, rhBMP2, and Ca(OH)2 paste, which served as control.. Wound healing of exposed pulp treated with the complex of rhBMP2 and FS seemed better than that with rhBMP2 alone. Dentin bridge formation was observed at 1 week when pulp was treated with the complex and at 4 weeks with rhBMP2 and Ca(OH)2. At 9 weeks after operation, more amount of tubular dentin bridge formation was found in pulp treated with the complex than with rhBMP2.. The results suggest that synergistic effects of fibrin and rhBMP2 existed and that rh-BMP2 with FS carrier can be used as bioactive pulp capping agent. Together, these agents can induce a large amount of dentin and enhance healing of exposed pulp.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Dental Pulp; Dentin, Secondary; Dentinogenesis; Dogs; Fibrin Tissue Adhesive; Humans; Recombinant Proteins; Transforming Growth Factor beta

Growth factors involved in normal wound healing may promote tissue repair when applied as a direct pulp capping medication. A minimal pulp exposure was made in rat molars, a pulp capping medication was placed, and the cavity was sealed. Epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor II, platelet-derived growth factor-BB, and transforming growth factor-beta 1 (TGF-beta 1)--each absorbed onto a sterile collagen membrane (BioMend; Calcitek, Carlsbad, CA)--were used separately as pulpal medicaments. Dycal, unimpregnated collagen membrane, and no medication were used as controls. Eight samples from each treatment regimen were collected 2 and 3 weeks after surgery. Pulpal soft and hard tissue responses were graded. Data were analyzed by one-way ANOVA and Tukey-Kramer tests. No significant differences were detected after 2 wk. Pulp treated with TGF-beta 1 showed significantly improved soft and hard tissue healing at week 3, compared with the procedure control. We conclude that TGF-beta 1 as a pulp-capping medication enhances reparative dentin formation in rat molars.

Topics: Analysis of Variance; Animals; Dental Pulp; Dental Pulp Capping; Dentin, Secondary; Dentinogenesis; Growth Substances; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Wound Healing

It was the aim of the present study to investigate the induction of dentin formation by recombinant human osteogenic protein-1 (hOP-1). In 4 adult miniature pigs a total of 16 teeth with artificially exposed dental pulps were capped with 3 mg of a complex of recombinant hOP-1 in collagen matrix (2.5 micrograms/mg), collagen matrix alone, or calcium hydroxide paste. Teeth were removed in block section after a healing period of 5 weeks. Decalcified sections were processed for light microscopy and histomorphometric analysis. In hOP-1 treated teeth substantial amounts of hard tissue formation (osteodentin and tubular dentin) had consistently led to a complete bridging of the defects. Less dentin formation was seen after calcium hydroxide application. In control defects collagen matrix alone failed to form complete dentin bridges. Recombinant human osteogenic protein-1 in a collagen carrier matrix appeared to be suitable as a bio-active capping agent for surgically exposed dental pulps.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcium Hydroxide; Collagen; Dental Pulp; Dental Pulp Capping; Dentin, Secondary; Dentinogenesis; Evaluation Studies as Topic; Humans; Recombinant Proteins; Swine; Swine, Miniature; Transforming Growth Factor beta; Wound Healing

Bone morphogenetic proteins (BMP) have been shown to induce reparative dentine formation experimentally but the cells responsible, which respond to BMPs, have not been identified. The BMP signal is probably mediated by interaction of type I and II BMP receptors (R). Here, the RNA of human adult dental pulp and pulp cells in culture was examined by reverse transcription (RT) polymerase chain reaction (PCR) for evidence of mRNA for BMPs. mRNAs for BMP-2, -4, osteogenic protein-1, ActR-1 (activin-like kinase receptor), BMPR-IA, -IB and -II were detected by RT-PCR. The 698-bp PCR fragment for BMPR-IB was used to probe pulp cells for expression of that receptor. Cell expression of BMPR-IB was detected by the hybridization probe. The findings suggest that resident pulp cells may be able to respond to BMPs to initiate tissue formation.

Topics: Activin Receptors, Type I; Adult; Base Sequence; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Chromosome Mapping; Dental Pulp; Dentin, Secondary; Dentinogenesis; Gene Expression; Humans; In Situ Hybridization; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, Growth Factor; RNA, Messenger; Transforming Growth Factor beta

Recombinant human osteogenic protein-1 (OP-1) when applied to freshly cut dentine stimulated significantly more reparative dentine than calcium hydroxide paste in permanent monkey teeth. The response to OP-1 was dependent upon the concentration applied to the tooth as a cavity liner as well as the thickness of the residual dentine. The response to calcium hydroxide was similarly dependent upon the thickness of the residual dentine. These data suggest that dental pulps contain cells, including perhaps mature odontoblasts, responsive to OP-1. Therefore OP-1 may be useful in the therapeutic induction of reparative dentine formation.

Topics: Analysis of Variance; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcium Hydroxide; Dental Pulp; Dentin; Dentin, Secondary; Humans; Macaca fascicularis; Male; Proteins; Recombinant Proteins; Stimulation, Chemical; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cattle; Dentin, Secondary; Haplorhini; Transforming Growth Factor beta

Recombinant human bone morphogenetic protein (BMP)-2, BMP-4 and transforming growth factor (TGF)-beta 1 combined with collagen matrix as a carrier were examined for their effects on pulp regeneration and dentine formation. Seventy days after implantation of 2 micrograms of BMP-2, mineralized osteodentine-like tissue containing embedded osteodentinocytes was seen in the cavity. Unmineralized fibrous tissue and pulp-like loose connective tissue were also found in the same cavity. In teeth implanted with 660 ng of BMP-2 only unmineralized fibrous and pulp tissues were seen. In teeth with 220 ng of BMP-2 or collagen alone, pulp tissue was seen. It is therefore likely that the cavity fills with pulp tissue and that spindle-shaped cells elaborate extracellular matrix that mineralizes to be osteodentine in a dose-dependent manner. Similar osteodentine was seen in teeth implanted with 4 micrograms of BMP-4 and collagen. No distinct tubular dentine was formed, unlike an earlier experiment in which BMP-2 or -4 was implanted with enriched, inactivated dentine matrix. These findings suggest that both BMP-2 and -4 induce osteodentine formation if combined with collagen matrix; some other matrix component present in inactivated dentine matrix might be essential for further differentiation into odontoblasts. In teeth implanted with TGF-beta 1, the carrier collagen remained in the cavity and little pulp tissue proliferation was seen, suggesting a possible inhibitory effect of TGF-beta 1 in pulp regeneration. It is likely that the response to growth and differentiation factors is dependent on the state of differentiation of pulp cells.

Topics: Animals; Bone Morphogenetic Proteins; Cell Differentiation; Collagen; Dental Pulp; Dentin, Secondary; Dentinogenesis; Dogs; Growth Substances; Proteins; Pulpotomy; Recombinant Proteins; Regeneration; Transforming Growth Factor beta

Recombinant human osteogenic protein-1 (hOP-1, BMP-7) induces cartilage and bone formation when implanted in extra- and intraskeletal sites in vivo. rOP-1 also preserves pulp vitality and stimulates reparative dentine formation when placed on partially amputated vital dental pulp tissue. The amount of dentine formed in 6 weeks was earlier found to be proportional to the total amount of rOP-1/carrier placed on the pulp and the capacity of the pulp to respond to rOP-1 appeared to be independent of the amount of coronal pulp removed. This reparative dentine was not completely mineralized after 6 weeks healing. Experiments were now made to determine the capacity of hOP-1 to preserve the vitality of and induce reparative dentine in vital radicular pulps. The extent of tissue mineralization present after 1, 2, 4 and 6 months' healing time in permanent monkey teeth was assessed. Radicular pulp vitality was maintained, reparative dentine formed, and mineralization was nearly 75% complete after 1 month and more than 95% after 4 months. The effects of irrigating the exposed pulps with EDTA, sodium hypochlorite or saline were also compared. Significantly more sodium hypochlorite-treated pulps became non-vital and the root canals of all the non-vital teeth contained bacteria at the time of sacrifice.

Topics: Animals; Bacteria; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Dental Pulp; Dental Pulp Cavity; Dentin; Dentin, Secondary; Edetic Acid; Humans; Macaca fascicularis; Proteins; Pulpectomy; Recombinant Proteins; Root Canal Irrigants; Sodium Chloride; Sodium Hypochlorite; Time Factors; Tooth Calcification; Transforming Growth Factor beta; Wound Healing

Dental pulp cells have the potential to differentiate into odontoblasts. The molecular mechanisms underlying differentiation are not clear. Demineralized dentin matrix is osteoinductive and contains bone morphogenetic protein (BMP) activity. BMPs have been implicated in embryonic odontogenic differentiation and hence may play a role in the differentiation of adult pulp cells into odontoblasts during pulpal healing. This study examined the hypothesis that BMPs induce dentin formation on amputated canine pulp. Recombinant human BMP-2 and BMP-4 were capped with inactivated dentin matrix on amputated pulp. At two months, the amputated pulp was filled with tubular dentin in the lower part and osteodentin in the upper part. The amount of dentin formed was markedly diminished when dentin matrix alone was implanted. These findings imply that recombinant human BMP-2 and BMP-4 induce differentiation of adult pulp cells into odontoblasts. Thus, BMPs may have a role in dentistry as a bioactive pulp-capping agent to induce dentin formation.

Topics: Animals; Bone Morphogenetic Proteins; Dental Pulp; Dental Pulp Capping; Dentin; Dentin, Secondary; Dogs; Extracellular Matrix; Growth Substances; Humans; Odontoblasts; Proteins; Pulpectomy; Recombinant Proteins; Tooth Calcification; Transforming Growth Factor beta

Osteogenic protein-1 (OP-1, BMP-7), a member of the transforming growth factor-beta supergene family, induces cartilage and bone formation when implanted in intra- and extraskeletal sites in vivo. The human OP-1 gene has been cloned and biologically active recombinant OP-1 homodimers (hOP-1) produced. The amount of bone induced by hOP-1 in vivo is related to the amount of protein implanted. Dentine possesses bone morphogenetic protein (BMP) activity. Impure material from allogenic bone with BMP activity induced reparative dentine formation in dogs. The objective of this study was to determine if the amount of reparative dentine stimulated by hOP-1 is related to the amount of protein utilized in direct pulp-capping experiments. Freshly exposed molar and premolar pulps were treated with varying amounts of a complex comprising hOP-1 and a carrier matrix of purified bovine type-1 collagen powder (CM) moistened with sterile saline. Reparative dentine was present in all hOP-1/CM treated teeth (12 of 15) that remained sealed for the 6 weeks' healing. Substantially more new dentine was present in teeth treated with hOP-1/CM than in those treated with Ca(OH)2 paste and the amount of reparative dentine formed was proportional to the amount of hOP-1/CM (P < 0.05). No reparative dentine formed in collagen carrier or untreated teeth. The appearances of the new tissue suggested that much of the mass of the hOP-1/CM was replaced first by a pulp-like connective tissue, which mineralized to form reparative dentine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Calcium Hydroxide; Collagen; Dental Pulp; Dental Pulp Capping; Dentin, Secondary; Female; Humans; Macaca fascicularis; Pharmaceutical Vehicles; Proteins; Recombinant Proteins; Transforming Growth Factor beta; Wound Healing