Resorbable Calcium Phosphate Research Articles

Hydrothermal synthesis of calcium-deficient hydroxyapatite whiskers and their thermal transformation to polycrystalline β-tricalcium phosphate short fibers

Resorbable calcium phosphate fibers are of particular interest to reinforce biodegradable bone substitutes for load-bearing applications. The aim of the present study was to prepare calcium-deficient hydroxyapatite (dHAp) whiskers with a molar Ca/P ratio of 1·5 by a hydrothermal synthesis and to transform them to β-tricalcium phosphate (β-TCP) by a subsequent thermal treatment. For the hydrothermal synthesis, calcium tripolyphosphate was used and different amounts of 2-propanol were added to adjust the pH. Average whisker lengths of 160 µm were obtained at a 2-propanol content of 27 volume-percent. However, 33% dicalcium phosphate anhydrate (DCPA) were present as a secondary phase. The amount of DCPA could be reduced by increasing the amount of 2-propanol. Whisker samples consisting of 88% dHAp and 12% DCPA were selected to investigate microstructural changes and phase transformations during thermal treatment. Polycrystalline single phase -TCP short fibers were obtained after a thermal treatment at 1125°C.

Brushite cement additives inhibit attachment to cell culture beads

Brushite-forming calcium phosphate cements are of great interest as bone replacement materials because they are resorbable in physiological conditions. Cell-attached culture beads formed from this material could be of great use for cell therapy. Despite a significant amount of work on optimizing the physicochemical properties of these materials, there are very few studies that have evaluated the capacity of the materials to facilitate cell adhesion. In this study, we have formed resorbable calcium phosphate (brushite) culture beads and for the first time we showed that cell attachment to the surface of the brushite cement (BC) could be inhibited by the presence of an intermediate dicalcium phosphate-citrate complex, formed in the cement as a result of using citric acid, a retardant and viscosity modifier used in many cement formulations. The BC beads formed from the mixture of β-TCP/orthophosphoric acid using citric acid did not allow cell attachment without further treatment. Ageing of BC beads in serum-free Dulbecco's Modified Eagle's Medium (DMEM) solution at 37°C for 1 week greatly enhanced the cell adhesion capacity of the material. Scanning electron microscopy, X-ray diffraction (XRD), and confocal Raman microspectrometry indicated the increased capacity for cell adhesion was due to the changes in phase composition of BC. XRD patterns collected before and after ageing in aqueous solution and a high initial mass loss, suggest the formation of a dicalcium phosphate-citrate complex within the matrix. Since compacts formed from brushite powder supported cell attachment, it was hypothesized that the dicalcium phosphate-citrate complex prevented attachment to the cement surface.

Variability of the paracrine‐induced osteoclastogenesis by human breast cancer cell lines

Breast cancer frequently metastasizes to the bone, often leading to the formation of osteolytic lesions. This work compares the paracrine-induced osteoclastogenesis mediated by four human breast cancer cell lines, the estrogen-receptor positive T47D and MCF-7 and the estrogen-negative SK-BR-3 and Hs-578T cell lines. Human osteoclast precursor cells were cultured in the presence of conditioned media from the breast cancer cell lines (10% and 20%), collected at different culture periods (48 h, 7 days, and 14 days). Cultures performed in the absence or the presence of M-CSF and RANKL served as negative and positive control, respectively. Results showed that the cell lines differentially expressed several osteoclastogenic genes. All cell lines exhibited a significant osteoclastogenic potential, evidenced by a high TRAP activity and number of osteoclastic cells, expression of several osteoclast-related genes, and, particularly, a high calcium phosphate resorption activity. Differences among the osteoclastogenic potential of the cell lines were noted. T47D and MCF-7 cell lines displayed the highest and the lowest osteoclastogenic response, respectively. Despite the variability observed, MEK and NF-κB signaling pathways, and, at a lesser extent, PGE2 production, seemed to have a central role on the observed osteoclastogenic response. In conclusion, the tested breast cancer cell lines exhibited a high osteoclastogenic potential, although with some variability on the cell response profile, a factor to be considered in the development of new therapeutic approaches for breast cancer-induced bone metastasis.

Simple Modifications to Standard TRIzol® Protocol Allow High-Yield RNA Extraction from Cells on Resorbable Materials

Resorbable bioceramics are attractive for medical applications such as bone substitution. Biochemical analysis on cells cultured on these biomaterials is vital to predict the impact of the materials in vivo and RNA extraction is an essential step in gene expression study using RT-qPCR. In this study, we describe simple modifications to the TRIzol® RNA extraction protocol widely used in biology and these allow high-yield extraction of RNA from cells on resorbable calcium phosphates. Without the modifications, RNA is trapped in the co-precipitated calcium compounds, rendering TRIzol® extraction method infeasible. Among the modifications, the use of extra TRIzol® to dilute the lysate before the RNA precipitation step is critical for extraction of RNA from porous ?-tricalcium phosphate (?-TCP) discs. We also investigate the rationale behind the undesirable precipitation so as to provide clues about the modifications required for other resorbable materials with high application potential in bone tissue engineering.

Cellular compatibility of highly degradable bioactive ceramics for coating of metal implants

Resorbable ceramics can promote the bony integration of implants. Their rate of degradation should ideally be synchronized with bone regeneration. This study examined the effect of rapidly resorbable calcium phosphate ceramics 602020, GB14, 305020 on adherence, proliferation and morphology of human bone-derived cells (HBDC) in comparison to β-TCP. The in vitro cytotoxicity was determined by the microculture tetrazolium (MTT) assay. HBDC were grown on the materials for 3, 7, 11, 15 and 19 days and counted. Cell morphology, cell attachment, cell spreading and the cytoskeletal organization of HBDC cultivated on the substrates were investigated using laser scanning microscopy and environmental scanning electron microscopy. All substrates supported sufficient cellular growth for 19 days and showed no cytotoxicity. On each material an identical cell colonisation of well communicating, polygonal, vital cells with strong focal contacts was verified. HBDC showed numerous well defined stress fibres which give proof of well spread and strongly anchored cells. Porous surfaces encouraged the attachment and spreading of HBDC. Further investigations regarding long term biomaterial/cell interactions in vitro and in vivo are required to confirm the utility of the new biomaterials.

Direct and indirect effects of functionalised fluorescence-labelled nanoparticles on human osteoclast formation and activity

Recently, it was demonstrated that phosphonate-functionalised nanoparticles were successfully taken up by mesenchymal stem cells without influencing their viability and differentiation capacity, suggesting that they may provide a promising basis for the development of nanoparticles for drug delivery or cell labelling. The present study aimed to investigate the effects of these nanoparticles on osteoclast formation and activity as well as on the inflammatory response of osteoclasts and osteoblasts. The intracellular uptake of the particles by human osteoclasts and osteoblasts was demonstrated by confocal laser scanning microscopy, transmission electron microscopy and fluorescence microscopy. The expression of tartrate-resistant acid phosphatase, carboanhydrase II, cathepsin K, calcitonin receptor and osteoclast-specific vacuolar proton pump subunit TCIRG1 as well as actin ring formation were not significantly altered in osteoclasts by particle treatment, as demonstrated by cytochemical staining and immunostaining. Active calcium phosphate resorption by osteoclasts was also not significantly influenced by the particles. The expression and secretion of pro-inflammatory cytokines (IL-6, IL-1β) by osteoclasts and osteoblasts and the expression of osteoclast-regulating genes (M-CSF, OPG, RANKL) in osteoblasts were similarly not significantly affected. In conclusion, phosphonate-functionalised nanoparticles did not affect osteoclast formation and activity either directly or indirectly, suggesting that they could provide a promising tool for the development of particle-based treatments for anti-resorptive therapies.

Oral Malodorous Compound Induces Osteoclast Differentiation Without Receptor Activator of Nuclear Factor κB Ligand

Hydrogen sulfide (H(2)S), the main component of halitosis, is one of the etiologic factors for periodontitis. We recently reported that H(2)S may induce pathologic changes in rat alveolar bone. The objective of this study is to determine the effect of H(2)S on osteoclast differentiation. Murine macrophage cells RAW264 were cultured in medium lacking nuclear factor κB ligand (receptor activator of nuclear factor κB ligand) in 5% CO(2) with air at 37°C for 24 hours; then 0.05, 0.5, or 5 ng/ml H(2)S was added to the CO(2)-air mix for 4 days. The controls received the CO(2)-air mix with no H(2)S. Cell differentiation was evaluated by counting the tartrate-resistant acid-phosphatase (TRAP)-positive cells. Extracellular signaling-regulated kinase1/2 (ERK1/2) and mitogen-activated protein kinase p38 phosphorylation were examined by Western blotting. The bone-resorption activity was determined with the resorption assay of calcium phosphate. There were significantly more TRAP-positive cells at a concentration of 0.05 ng/ml H(2)S than at the other concentrations (P <0.001). Cathepsin K protein, a specific marker for osteoclasts, was expressed in the H(2)S-induced multinuclear cells. Resorption of calcium phosphate significantly increased in the H(2)S-induced TRAP-positive cells cultured on plates coated with calcium phosphate apatite (P <0.01). The phosphorylation of ERK1/2 and p38 were accelerated by H(2)S, and increased with time. PD98059 and SB203580, specific inhibitors of ERK1/2 and p38, suppressed the activation of these enzymes and osteoclast differentiation by H(2)S. Results demonstrate that H(2)S at physiologic concentrations in mouth air induces osteoclasts from RAW264 cells.

In vivo bone augmentation in an osteoporotic environment using bisphosphonate-loaded calcium deficient apatite

Resorbable calcium phosphate (CaP) biomaterials have demonstrated considerable efficacy in bone reconstructive surgery. Furthermore, bisphosphonates (BPs) are well known anti-resorptive agents largely used in clinical treatments for osteoporosis. An injectable BP-combined CaP matrix has been developed in order to biologically reinforce osteoporotic bone by increasing the bone fraction and improving bone micro-architecture. Our previous in vitro studies have shown that CaP is effective for loading and releasing BPs at doses that can inhibit excessive bone resorption without affecting osteoblasts. In vivo studies in relevant animal models are necessary to explore the effect of our injectable BP-combined biomaterial on femur bone structure by performing three-dimensional microtomography analysis, histological studies and SEM observations. Firstly, in rat model, our BP-combined CaP matrix significantly improved the bone micro-architecture as compared to CaP alone. The implantation of the BP-loaded biomaterial within proximal femurs of osteoporotic ewes led to a significant increase in relative bone content and an improvement of its micro-architecture. These modifications were confirmed by histological and SEM observations, which revealed CaP granule resorption and new bone trabeculae formation. This approach could be considered in the future for preventing osteoporotic fractures that are preferentially localized in the proximal femur, vertebral bodies or wrist.

Biphasic Resorbable Calcium Phosphate Ceramic for Bone Implants and Local Alendronate Delivery

AbstractA novel biphasic calcium phosphate ceramic composed of tricalcium phosphate (TCP) and calcium pyrophosphate (CP) is synthesized in order to tailor the biodegradation behavior of the ceramic. The results show that biphasic TCP/CP ceramic has a strength of 62.2 ± 2.1 MPa, which is superior to single‐phase TCP and CP ceramics, which show strengths of 44.3 ± 3.0 and 53.0 ± 4.8 MPa, respectively. In addition, biphasic TCP/CP ceramic displays a controlled strength degradation from 62.2 ± 2.1 to 40.5 ± 1.0 MPa in stimulated body fluid over a period of 28 d. An in vitro cell materials interaction study using human fetal osteoblast cells indicates that TCP/CP ceramic is cytocompatible. TCP/CP ceramic also show a good loading capacity for alendronate. Adsorption of alendronate (AD) on the TCP/CP surface is found to proceed via ligand exchange mechanism and the in vitro release profile of AD from TCP/CP surface is characterized by an initial fast release followed by a slow and sustained release. Strong electrostatic interactions between AD groups and surface Ca2+ ions enable the slow and sustained release of AD. These results demonstrate that the newly developed biphasic ceramic, with its controlled strength degradation and drug release, shows promise for use in orthopedic and tissue engineering applications.

Injectable and fast resorbable calcium phosphate cement for body-setting bone grafts

In this work a calcium phosphate (CPC)/polymer blend was developed with the advantage of being moldable and capable of in situ setting to form calcium deficient hydroxyapatite under physiological conditions in an aqueous environment at body temperature. The CPC paste consists in a mix of R cement, glycerol as a liquid phase carrier and a biodegradable hydrogel such as Polyvinyl alcohol, which acts as a binder. Microstructure and mechanical analysis shows that the CPC blend can be used as an injectable implant for low loaded applications and fast adsorption requirements. The storage for commercial distribution was also evaluated and the properties of the materials obtained do not significantly change during storage at -18 degrees C.

Novel chemoembolization using calcium‐phosphate ceramic microsphere incorporating TNP‐470, an anti‐angiogenic agent

The purpose of the present study was to develop a new method of chemoembolization to improve the therapeutic effectiveness and safety profile of cancer treatment. A chemoembolization approach was designed for human solid tumors using resorbable calcium-phosphate ceramic microspheres loaded with an agent anti-angiogenic to tumor vasculature in vivo. The human uterine sarcoma cell line FU-MMT-3 was used in this study because this tumor is aggressive and also exhibits a poor response to radiotherapy or any chemotherapy currently used. The calcium-phosphate ceramic microspheres loaded with TNP-470, an anti-angiogenic agent, were injected into FU-MMT-3 xenografts in nude mice three times per week for 8 weeks. The treatment using TNP-470-loaded microspheres suppressed tumor growth, compared to treatment with TNP-470 alone, microspheres alone, and the control. The mean tumor weight after treatment using TNP-470-loaded microspheres was significantly lower than that after treatment with microspheres alone. These ceramic microspheres were remarkably embolized in tumor microvessels as well as in the feeding arteries and a significant reduction of intratumoral vascularity was also demonstrated following treatment with TNP-470-loaded microspheres. Severe loss of body weight was not observed in any mice treated with the TNP-470-loaded microspheres, compared to treatment with TNP-470 alone. These results suggest that targeting tumor vasculature in human uterine sarcoma using calcium-phosphate microspheres might be more effective and safer than the treatment that employs anti-angiogenic agent alone. This new chemoembolization method incorporating an anti-angiogenic agent may contribute to the effective treatment of locally advanced or recurrent solid tumors.

The Sol–Gel Production of Bioceramics

Sol–gel synthesis is used for the fabrication of new materials with technological applications including ceramics for implants manufacturing, usually termed bioceramics. Many bioactive and resorbable bioceramics, that is, calcium phosphates, glasses and glass–ceramics, have been improved by using the sol–gel synthesis. In addition, the soft thermal conditions of sol–gel methods made possible to synthesize more reactive materials than those synthesized by traditional methods. Moreover, new families of bioactive materials such as organic–inorganic hybrids and inorganic compounds with ordered mesostructure can be produced. In hybrid materials, the inorganic component ensures the bioactive response whereas the organic polymeric component allows modulating other properties of the resulting biomaterial such as mechanical properties, degradation, etc. On the other hand, the sol–gel processes also allow the synthesis of silica ordered mesoporous materials, which are bioactive and exhibit – as an added value – a possible application as matrices for the controlled release of biologically active molecules (drugs, peptides, hormones, etc.). Finally, by combining the bioactive glasses composition with synthesis strategies of mesoporous materials, template glasses with ordered mesoporosity can be obtained. In this chapter, the advances that sol–gel technology has brought to the silica-based bioactive bioceramics are presented.

Effect of surface roughness, porosity, and a resorbable calcium phosphate coating on osseointegration of titanium in a minipig model

The aim of this study was to compare the osseointegration of four different implant surfaces in the Göttingen minipig femur model. They were prepared by glasspearlblasting (A), sandblasting (B) and titaniumplasma spraying (C and D). Surface D received additionally an electrochemically deposited layer of a resorbable calcium phosphate (CaP) layer, made mainly of brushite. Sample size was n = 20 per group. Implants were placed in the intertrochanteric and intercondylar sites of both femora. After 12 weeks, implant anchorage was measured by the pull-out test and histomorphometry measurements were carried out at the bone-implant interface. Implant anchorage was 0.7 +/- 0.3 MPa for surface A, 3.2 +/- 0.6 MPa for surface B, 6.5 +/- 1.5 MPa for surface C and 7.3 +/- 1.9 MPa for surface D. The differences between surfaces were statistically significant, with exception of C and D. The stiffness of the bone-implant interface showed no statistically significant difference between surfaces. After pull-out, surface A and B showed nearly no bone spots, while on surfaces C and D bone remains were found. Bone-implant contact was 1.9 +/- 1.1% for surface A, 10.5 +/- 3.6% for surface B, 22.4 +/- 4.5% for surface C and 48.8 +/- 4.5% for surface D. The differences were statistically significant. Implant location, intertrochanteric and intercondylar, did not affect the data. In this minipig model, rougher surfaces showed better osseointegration. After 12 weeks of healing, the resorbable CaP layer enhanced significantly the bone-implant contact but not the level of anchorage. The findings also suggest that the pull-out test should be critically evaluated to determine the shear strength between bone and porous surfaces.

Calcium Phosphate Ceramic Blasting on Titanium Surface Improve Bone Ingrowth

Surface roughness modulates the osseointegration of orthopaedic and dental titanium implants. High surface roughness is currently obtained by blasting of titanium implants with silica or aluminium abrasive particles. This process includes into the surface abrasive particles and may cause the release of cytotoxic silica or aluminium ions in the peri implant tissue. To overcome this drawback, we currently develop an innovative gridblasting process using Biphasic Calcium Phosphate (BCP) particles (RBBM Resorbable and Biocompatible Blast Media) to generate biocompatible roughened titanium surface. This work present the technique of blasting using RBBM particles to provide a roughened surface which does not release cytotoxic elements and (ii) to assess the effects of such a roughened surface for bone osteointegration in critical size rabbit defect. Our results demonstrate that resorbable biphasic calcium phosphate abrasive particles can be used to create titanium surface roughness. This grid blasting process increases surface roughness of titanium implants and offers a non cytotoxic surface for rapid and efficient osteointegration.

Repairing Segmental Defect with a Composite Associating Collagen Membrane and MBCP® Combined with Total Bone Marrow Graft in Irradiated Bone Defect: an Experimental Study in Rabbit

The aim of this study was to study bone marrow quality from various location and species for reconstruction of segmental critical size defect in irradiated weigh bearing bone. Sample of bone marrow aspirates from rabbits and Beagle dog were analyzed. Rabbits were implanted with a composite associating resorbable collagen membrane plus micro macroporous biphasic calcium phosphate (MBCP®) and autologous bone marrow (BM) injected after irradiation. Bone marrow samples were found to be significantly less rich in tibia than in humerus and ilium in Dog and less rich in Dog than in Rabbit (p&lt;0,05). Successful osseous colonization bridging of the defect were obtain at 16 weeks in all animals. Identical repartition of bone ingrowth and residual ceramic at the different levels of the implant suggest an osteoinduction role of the bone marrow graft in the center of the defect. This model succeeded in reconstruct a large segmental defect in weight bearing and irradiated bone in rabbit.

Histological and histomorphometric investigations on bone integration of rapidly resorbable calcium phosphate ceramics

Resorbable ceramics can promote the bony integration of implants. Their rate of degradation should ideally be synchronized with bone regeneration. We report here the results of a histological study of implants with two resorbable calcium phosphate ceramic coatings: Ca(2)KNa(PO(4))(2)-(GB14) and Ca(10)[K/Na](PO(4))(7)-(602020). The results attained with these ceramic-coated implants show the benefits of these materials with regard to bioactive bone-healing stimulation, compared with uncoated implants. The GB14 ceramic coating exhibited greater bone regeneration and differentiation on its surface than the conventional hydroxyapatite coating and helped bone tissue achieve more extensive contact free of connective tissue. Not until the coating disintegrated did the histological features of GB14- and 602020-coated implants converge-both implant types were integrated into bone. Rapid disintegration of the coating material, as with 602020, supports osteoblast proliferation but has negative effects on bone mineralization. Both resorbable ceramics tested, GB14 and 602020, demonstrated bioactivity; even metal surfaces coated with these materials were populated by mature bone tissue without connective tissue after disintegration of their ceramic coating. The less rapidly degrading material, GB14, achieved better results. Degradable calcium phosphate coatings have the potential to stimulate bone regeneration. From the histological viewpoint, the resorbable ceramics examined here can be recommended as coating materials for clinical use.

Influence of Platelet-Rich Plasma on Osteogenic Differentiation of Mesenchymal Stem Cells and Ectopic Bone Formation in Calcium Phosphate Ceramics

Platelet-rich plasma (PRP) contains a mixture of growth factors that play an important role in wound and fracture healing. While PRP enhanced bone formation by autogenous cancellous bone grafts, its influence in combination with different bone substitutes remained unknown. This study evaluated the effect of PRP on osteogenic differentiation and ectopic bone formation of human mesenchymal stem cells (MSC) in distinct resorbable calcium phosphate ceramics. Calcium-deficient hydroxyapatite (CDHA) blocks with a large specific surface area (48 m²/g) and β-tricalcium phosphate (β-TCP) with a low specific surface area (<0.5 m²/g) were loaded with 2 × 10⁵ bone marrow-derived MSC. Half of the specimens were treated with 5-fold concentrated PRP. Biocomposites were implanted subcutaneously into SCID mice or kept under osteogenic culture conditions for 2 weeks before implantation. The addition of PRP increased the specific alkaline phosphatase (ALP) activity (p = 0.012) in undifferentiated MSC/CDHA composites but not in MSC/β-TCP composites. Osteogenic preinduction was ineffective for CDHA and reduced ALP activity of β-TCP composites significantly at explantation. Ectopic bone formation was stronger in MSC/CDHA (7/32) compared to MSC/β-TCP (2/30) composites, but no influence of PRP was evident. In conclusion, the effect of PRP depended on the type of ceramic and the differentiation status of the MSC, and enhanced ALP activity of MSC on the high surface scaffold CDHA only, but PRP did not improve osteogenesis in our setting.

Calcium Phosphate‐Based Resorbable Ceramics: Influence of MgO, ZnO, and SiO2 Dopants

Resorbable calcium phosphate (CaP)‐based biomaterials are important because they can significantly improve health care by shortening the time necessary for restoration of functional loading of grafted bones. Although synthetic CaPs show exceptional similarities to natural bone, however, they are deficient in one major area, in that they do not have the same mineral content of bone. The focus of our work is to understand the influence of dopants on the physical, mechanical, and biological properties of tricalcium phosphate (TCP) resorbable ceramics with special emphasis toward in vitro strength degradation and cell–materials interactions as a function of time. For this purpose, β‐TCP was doped with magnesia (MgO), zinc oxide (ZnO), and silica (SiO2). Those dopants were added as individual dopants, and their binary and ternary compositions. It was found that these dopants significantly influenced densification behavior and as sintered microstructures of TCP. In vitro mineralization studies in simulated body fluids (SBF) for 12 weeks showed apatite growth on the highly porous compositions either on the surface or inside. From scanning electron microscopic analysis it was evident that surface degradation occurred on all compositions in SBF. Compression strengths for samples up to 12 weeks in SBF showed that it is possible to tailor strength loss behavior through compositional modifications. The highest compression strength was found for binary MgO–ZnO doped TCP. Overall, samples showed either a similar strength level during the 12 weeks test period, or a continuous decrease or a continuous increase in strength depending on dopant chemistry or amount. In vitro human osteoblast cell culture was used to determine influence of dopants on cell‐materials interactions. All samples were non‐toxic and biocompatible. Dopant chemistry also influenced adhesion, proliferation, and differentiation of osteoblastic precursor cell line 1 (OPC1) cells on these matrices.

Composition and crystal structure of resorbable calcium phosphate thin films

Silicon stabilized tricalcium phosphate (Si-TCP) is formed, among other phases, as a result of sintering hydroxyapatite (HA) in the presence of silica (SiO2) at >800°C. Calcium phosphate films sintered at 1000°C on quartz substrates are examined with and without additional SiO2 added to the starting precipitate. Data from transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) separate the undoped film morphology into a surface layer with a monoclinic crystal structure P21/a characteristic of α or Si-tricalcium phosphate and grain size in the range 100–1000 nm and a substrate layer with a crystal structure which is predominantly apatitic P63/m and grain size in the range 30–100 nm. The silicon content is greatest in the substrate layer. The addition of SiO2 to the film material during fabrication induces a more uniform grain size of 10–110 nm and a higher Si content. The structural and phase evolution of these films suggests the nucleation of α-TCP by the local formation of Si-TCP at a SiO2-hydroxyapatite interface. The results are consistent with X-ray diffraction studies and are explained by a model of nucleation and growth developed for bulk powders.

Elevated extracellular calcium stimulates secretion of bone morphogenetic protein 2 by a macrophage cell line

It has been suggested that macrophages and multinucleated giant cells are responsible for phagocytosis of resorbable calcium phosphate (CaP) compounds implanted in bone defects. However, function of macrophages around the CaP, if continuously exposed to various concentration of extracellular calcium ions ([Ca 2+] o), is still unknown. The present study showed that when resorbable octacalcium phosphate was implanted in mouse calvaria, macrophage-like cells were observed around the implant during bone formation. Then, experiments were designed to investigate whether secretion of bone morphogenetic protein 2 (BMP-2) is enhanced by extracellular calcium ions in a macrophage cell line (J774A.1) in vitro. The mRNA expression and the secretion of BMP-2 in J774A.1 cells were significantly increased when incubated in the medium with [Ca 2+] o up to 14 mM. The promotion of mRNA expression was maintained even when incubated with a small amount of minute CaP crystals. The present results suggest that [Ca 2+] o above physiological concentration may stimulate macrophages to induce osteogenic cytokine, such as BMP-2, for bone formation by osteoblasts.