acid-phosphatase and tricalcium-phosphate

It has been reported that surface microstructural dimensions can influence the osteoinductivity of calcium phosphates (CaPs), and osteoclasts may play a role in this process. We hypothesised that surface structural dimensions of ≤ 1 μm trigger osteoinduction and osteoclast formation irrespective of macrostructure (e.g., concavities, interconnected macropores, interparticle space) or surface chemistry. To test this, planar discs made of biphasic calcium phosphate (BCP: 80% hydroxyapatite, 20% tricalcium phosphate) were prepared with different surface structural dimensions - either ~ 1 μm (BCP1150) or ~ 2-4 μm (BCP1300) - and no macropores or concavities. A third material was made by sputter coating BCP1150 with titanium (BCP1150Ti), thereby changing its surface chemistry but preserving its surface structure and chemical reactivity. After intramuscular implantation in 5 dogs for 12 weeks, BCP1150 formed ectopic bone in 4 out of 5 samples, BCP1150Ti formed ectopic bone in 3 out of 5 samples, and BCP1300 formed no ectopic bone in any of the 5 samples. In vivo, large multinucleated osteoclast-like cells densely colonised BCP1150, smaller osteoclast-like cells formed on BCP1150Ti, and osteoclast-like cells scarcely formed on BCP1300. In vitro, RAW264.7 cells cultured on the surface of BCP1150 and BCP1150Ti in the presence of osteoclast differentiation factor RANKL (receptor activator for NF-κB ligand) proliferated then differentiated into multinucleated osteoclast-like cells with positive tartrate resistant acid phosphatase (TRAP) activity. However, cell proliferation, fusion, and TRAP activity were all significantly inhibited on BCP1300. These results indicate that of the material parameters tested - namely, surface microstructure, macrostructure, and surface chemistry - microstructural dimensions are critical in promoting osteoclastogenesis and triggering ectopic bone formation.

Topics: Acid Phosphatase; Animals; Calcium Phosphates; Cell Differentiation; Cell Line; Cell Proliferation; Cell Survival; Coated Materials, Biocompatible; Dogs; Hydroxyapatites; Isoenzymes; Male; Mice; Microscopy, Electron, Scanning; Osteoclasts; Osteogenesis; Porosity; Prostheses and Implants; Surface Properties; Tartrate-Resistant Acid Phosphatase; Time Factors; Titanium; X-Ray Diffraction

A potential standard method for measuring the relative dissolution rate to estimate the resorbability of calcium-phosphate-based ceramics is proposed. Tricalcium phosphate (TCP), magnesium-substituted TCP (MgTCP) and zinc-substituted TCP (ZnTCP) were dissolved in a buffer solution free of calcium and phosphate ions at pH 4.0, 5.5 or 7.3 at nine research centers. Relative values of the initial dissolution rate (relative dissolution rates) were in good agreement among the centers. The relative dissolution rate coincided with the relative volume of resorption pits of ZnTCP in vitro. The relative dissolution rate coincided with the relative resorbed volume in vivo in the case of comparison between microporous MgTCPs with different Mg contents and similar porosity. However, the relative dissolution rate was in poor agreement with the relative resorbed volume in vivo in the case of comparison between microporous TCP and MgTCP due to the superimposition of the Mg-mediated decrease in TCP solubility on the Mg-mediated increase in the amount of resorption. An unambiguous conclusion could not be made as to whether the relative dissolution rate is predictive of the relative resorbed volume in vivo in the case of comparison between TCPs with different porosity. The relative dissolution rate may be useful for predicting the relative amount of resorption for calcium-phosphate-based ceramics having different solubility under the condition that the differences in the materials compared have little impact on the resorption process such as the number and activity of resorbing cells.. The evaluation and subsequent optimization of the resorbability of calcium phosphate are crucial in the use of resorbable calcium phosphates. Although the resorbability of calcium phosphates has usually been evaluated in vivo, establishment of a standard in vitro method that can predict in vivo resorption is beneficial for accelerating development and commercialization of new resorbable calcium phosphate materials as well as reducing use of animals. However, there are only a few studies to propose such an in vitro method within which direct comparison was carried out between in vitro and in vivo resorption. We propose here an in vitro method based on measuring dissolution rate. The efficacy and limitations of the method were evaluated by international round-robin tests as well as comparison with in vivo resorption studies for future standardization. This study was carried out as one of Versailles Projects on Advanced Materials and Standards (VAMAS).

Topics: Acid Phosphatase; Animals; Bone Resorption; Calcium Phosphates; Ceramics; Hydrogen-Ion Concentration; Isoenzymes; Materials Testing; Porosity; Rabbits; Tartrate-Resistant Acid Phosphatase

Zinc-containing tricalcium phosphate (Zn-TCP) was synthesized to investigate the role of zinc in osteoblastogenesis, osteoclastogenesis and in vivo bone induction in an ectopic implantation model. Zinc ions were readily released in the culture medium. Zn-TCP with the highest zinc content enhanced the alkaline phosphatase activity of human bone marrow stromal cells and tartrate-resistant acid phosphatase activity, as well as multinuclear giant cell formation of RAW264.7 monocyte/macrophages. RAW264.7 cultured with different dosages of zinc supplements in medium with or without zinc-free TCP showed that zinc could influence both the activity and the formation of multinuclear giant cells. After a 12-week implantation in the paraspinal muscle of canines, de novo bone formation and bone incidence increased with increasing zinc content in Zn-TCP - up to 52% bone in the free space. However, TCP without zinc induced no bone formation. Although the observed bone induction cannot be attributed to zinc release alone, these results indicate that zinc incorporated in TCP can modulate bone metabolism and render TCP osteoinductive, indicating to a novel way to enhance the functionality of this synthetic bone graft material.

Topics: Acid Phosphatase; Animals; Calcium Phosphates; Cell Differentiation; Cell Line; Cell Proliferation; Cell Shape; Cell Size; Dogs; Giant Cells; Humans; Ions; Isoenzymes; Macrophages; Male; Mesenchymal Stem Cells; Mice; Models, Biological; Osteogenesis; Plastics; Tartrate-Resistant Acid Phosphatase; X-Ray Diffraction; Zinc

This study investigated the influence of the surface microporosity of beta-tri-calcium phosphate (β-TCP) ceramics on the resorption capacity of osteoclasts. This was achieved by first compacting commercially available β-TCP powder into disks that were sintered at various temperatures, thereby yielding different surface microporosities. Scanning electron microscopy (SEM) and subsequent image processing verified different degrees of surface microporosity on the disks. Rabbit osteoclasts in a bone marrow derived cell suspension were then seeded onto these disks and incubated for 48 h. Tartrate resistant acid phosphatase (TRAP) staining confirmed the presence of osteoclasts on all disks. Actin ring staining that detected actively resorbing OCs showed an inverse linear correlation between the number of actively resorbing osteoclasts (percentage of total OCs on the surfaces) with surface microporosity. These findings should be taken into consideration for the design and/or production of new β-TCP bone graft substitutes.

Topics: Acid Phosphatase; Actins; Animals; Biocompatible Materials; Bone Resorption; Calcium Phosphates; Cell Count; Cell Nucleus; Ceramics; Isoenzymes; Osteoclasts; Porosity; Rabbits; Staining and Labeling; Tartrate-Resistant Acid Phosphatase; X-Ray Diffraction

Calcium phosphate (CaP) scaffolds with three-dimensionally-interconnected pores play an important role in mechanical interlocking and biological fixation in bone implant applications. CaPs alone, however, are only osteoconductive (able to guide bone growth). Much attention has been given to the incorporation of biologics and pharmacologics to add osteoinductive (able to cause new bone growth) properties to CaP materials. Because biologics and pharmacologics are generally delicate compounds and also subject to increased regulatory scrutiny, there is a need to investigate alternative methods to introduce osteoinductivity to CaP materials. In this study silica (SiO2) and zinc oxide (ZnO) have been incorporated into three-dimensional printed β-tricalcium phosphate (β-TCP) scaffolds to investigate their potential to trigger osteoinduction in vivo. Silicon and zinc are trace elements that are common in bone and have also been shown to have many beneficial properties, from increased bone regeneration to angiogenesis. Implants were placed in bicortical femur defects introduced to a murine model for up to 16 weeks. The addition of dopants into TCP increased the capacity for new early bone formation by modulating collagen I production and osteocalcin production. Neovascularization was found to be up to three times more than the pure TCP control group. The findings from this study indicate that the combination of SiO2 and ZnO dopants in TCP may be a viable alternative to introducing osteoinductive properties to CaPs.

Topics: Acid Phosphatase; Animals; Blood Vessels; Bone and Bones; Calcium Phosphates; Collagen Type I; Implants, Experimental; Ions; Isoenzymes; Mice; Neovascularization, Physiologic; Osteocalcin; Osteoclasts; Osteogenesis; Printing; Rats; Rats, Sprague-Dawley; Silicon Dioxide; Tartrate-Resistant Acid Phosphatase; Tissue Engineering; Tissue Scaffolds; Zinc Oxide

This study investigates the biological effects of traditional Chinese medicines on the activities of bone cells using rat bone cells. Then, a mixture of a GGT composite, that is, a novel biodegradable composite containing genipin crosslinked gelatin and tricalcium phosphate, and traditional Chinese medicine (TCM) was prepared as a GGT-TCM composite. A cultured neonatal rat calvarias organ was used to measure the potential of GGT-TCM composite for use in promoting the regeneration of defective bone tissue. The mitochondria activity of the bone cells following exposure to various concentrations of crude extracts of five herbal Chinese medicines was measured by colorimetric assay. Biochemical markers, such as alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) titers were analyzed to evaluate the activities of bone cells. Finally, we examined the organ culture units, which were maintained in cultured medium for 5 weeks. Morphology of tissue was observed, and the quantitative evaluation of the regenerated bone was determined. In a bone cells culture experiment, adding Cuscuta chinensis Lam. (TCM-5) to the bone cells culture clearly promoted the proliferation and differentiation of the osteoblasts from their precursor cells; but the reduced amount of TRAP indicated that the medicine significantly inhibited the osteoclasts activities. Opposite bone cell responses were observed when Loranthus parasiticus Merr. (TCM-3) and Achyranthes bidentata Bl. (TCM-4) were added to the bone cells culture. Eucommia ulmoides Oliv. (TCM-1) and Dipsacus asper Wall. (TCM-2) potentially influence the proliferation and differentiation of the osteoblasts from their precursor cells, but they did not affect the osteoclasts activities. The finding from the organ culture indicated that Chinese medicine effectively increased the rate of tissue regeneration of damaged bones.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Bone Substitutes; Calcium Phosphates; Cell Differentiation; Cells, Cultured; Coculture Techniques; Gelatin; Isoenzymes; Medicine, Chinese Traditional; Organ Culture Techniques; Osteoblasts; Osteoclasts; Rats; Rats, Sprague-Dawley; Skull; Tartrate-Resistant Acid Phosphatase

The interactive effects of phosphate solubilizing bacteria, N2 fixing bacteria and arbuscular mycorrhizal fungi (AMF) were studied in a low phosphate alkaline soil amended with tricalcium insoluble source of inorganic phosphate on the growth of an aromatic grass palmarosa (Cymbopogon martinii). The microbial inocula consisted of the AM fungus Glomus aggregatum, phosphate solubilizing rhizobacteria Bacillus polymyxa and N2 fixing bacteria Azospirillum brasilense. These rhizobacteria behaved as "mycorrhiza helper" and enhanced root colonization by G. aggregatum in presence of tricalcium phosphate at the rate of 200 mg kg(-1) soil (P1 level). Dual inoculation of G. aggregatum and B. polymyxa yielded 21.5 g plant dry weight (biomass), while it was 21.7 g in B. polymyxa and A. brasilense inoculated plants as compared to 14.9 g of control at the same level. Phosphate content was maximum (0.167%) in the combined treatment of G. aggregatum, B. polymyxa and A. brasilense at P1 level, however acid phosphatase activity was recorded to be 4.75 pmol mg(-1) min(-1) in G. aggregatum, B. polymyxa and A. brasilense treatment at P0 level. This study indicates that all microbes inoculated together help in the uptake of tricalcium phosphate which is otherwise not used by the plants and their addition at 200 mg kg(-1) of soil gave higher productivity to palmarosa plants.

Topics: Acid Phosphatase; Acyclic Monoterpenes; Alkaline Phosphatase; Azospirillum brasilense; Bacillus; Calcium Phosphates; Fungi; Plant Roots; Poaceae; Soil Microbiology; Terpenes

TGF-beta 1-coated beta-TCP pellets were grafted in experimental defects of rat calvariae to study the effects on new bone formation.. The grafted sites were evaluated by light microscopy using hematoxylin-eosin (H-E) staining for histology and detection of alkaline phosphatase (ALPase) and tartrate-resistant acid phosphatase (TRACPase) activities to demonstrate osteoblastic and osteoclastic cells. Confocal laser scanning microscopy (CLSM) was performed for morphometry of newly formed bone.. H-E sections showed more new bone formation in sites grafted with TGF-beta 1-coated beta-TCP pellets (experimental sites) than those with beta-TCP pellets only (control sites). TRACPase-positive and ALPase-positive cells at experimental sites were more frequent than at control sites. The bone formation rate calculated by computerized CLSM pixel image analysis showed more new bone formation at the experimental sites than at control sites (3.4 +/- 0.8% vs 9.3 +/- 1.7% on week 2 and 11.8 +/- 2.1% vs 39.8 +/- 10.9% on week 4).. TGF-beta 1-coated beta-TCP pellets promote new bone formation and may be a useful modality in synthetic bone grafting.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Bone Regeneration; Bone Substitutes; Calcium Phosphates; Drug Combinations; Drug Implants; Humans; Image Processing, Computer-Assisted; Isoenzymes; Male; Microscopy, Confocal; Osteoblasts; Osteoclasts; Rats; Rats, Wistar; Recombinant Proteins; Skull; Tartrate-Resistant Acid Phosphatase; Transforming Growth Factor beta; Wound Healing

beta-TCP was implanted in surgically prepared alveolar bone defects on the mesial side of the upper canine. The dogs that we used were sacrificed after 5 weeks, fixed by perfusion, and the beta-TCP resorbing cells were examined ultrastructurally and histochemically, with the following results: (1) beta-TCP was resorbed by macrophages and multinucleated giant cells. (2) Mitochondria, vacuoles and Golgi apparatus were abundant in beta-TCP-resorbing multinucleated giant cells that possessed neither ruffled borders nor clear zones. (3) The addition of tartric acid inhibited acid phosphatase activity in the cytoplasm of the multinucleated giant cells and macrophages.

Topics: Acid Phosphatase; Animals; Calcium Phosphates; Dental Implants; Dogs; Giant Cells; Histocytochemistry; Macrophages; Microscopy, Electron; Osteoblasts; Periodontium; Tartrates