Tumor-supportive and osteoclastogenic changes induced by breast cancer-derived factors are reversed by inhibition of {gamma}-secretase.

During breast cancer metastasis to bone, tumor cells home to bone marrow, likely targeting the stem cell niche, and stimulate osteoclasts, which mediate osteolysis required for tumor expansion. Although osteoblasts contribute to the regulation of the hematopoietic stem cell niche and control osteoclastogenesis through production of proresorptive cytokine RANKL (receptor activator of NF-κB ligand), their role in cancer metastases to bone is not fully understood. C57BL/6J mouse bone marrow cells were treated for 3-12 days with ascorbic acid (50 µg/ml) in the presence or absence of 10% medium conditioned by breast carcinoma cells MDA-MB-231, 4T1, or MCF7. Treatment with cancer-derived factors resulted in a sustained 40-60% decrease in osteoblast differentiation markers, compared with treatment with ascorbic acid alone, and induced an osteoclastogenic change in the RANKL/osteoprotegerin ratio. Importantly, exposure of bone cells to breast cancer-derived factors stimulated the subsequent attachment of cancer cells to immature osteoblasts. Inhibition of γ-secretase using pharmacological inhibitors DAPT and Compound E completely reversed cancer-induced osteoclastogenesis as well as cancer-induced enhancement of cancer cell attachment, identifying γ-secretase activity as a key mediator of these effects. Thus, we have uncovered osteoblasts as critical intermediary of premetastatic signaling by breast cancer cells and pinpointed γ-secretase as a robust target for developing therapeutics potentially capable of reducing both homing and progression of cancer metastases to bone.

Development and characterization of a PLGA-HA composite material to fabricate 3D-printed scaffolds for bone tissue engineering.

Additive manufacturing is a rising field in bone tissue engineering. Additive fabrication offers reproducibility, high precision and rapid manufacture of custom patient-specific scaffolds. The development of appropriate composite materials for biomedical applications is critical to reach clinical application of these novel biomaterials. In this work, medical grade poly(lactic-co-glycolic) acid (PLGA) was mixed with hydroxyapatite nanoparticles (nHA) to fabricate 3D porous scaffolds by Fused Deposition Modeling. We have first confirmed that the composite material could be printed in a reproductive manner. Physical characterization demonstrated a low degradation of the material during manufacturing steps and an expected loading and homogeneous distribution of nHA. In vitro biodegradation of the scaffolds showed modifications of morphological and physicochemical properties over time. The composite scaffolds were biocompatible and high cell viability was observed in vitro, as well as a maintain of cell proliferation. As expected, the addition of nHA displayed a positive impact on osteodifferentiation in vitro. Furthermore, a limited inflammatory reaction was observed after subcutaneous implantation of the materials in the rat. Overall, this study suggests that this composite material is suitable for bone tissue engineering applications.

3D printed polymer-mineral composite biomaterials for bone tissue engineering: Fabrication and characterization.

Applications in additive manufacturing technologies for bone tissue engineering applications requires the development of new biomaterials formulations. Different three-dimensional (3D) printing technologies can be used and polymers are commonly employed to fabricate 3D printed bone scaffolds. However, these materials used alone do not possess an effective osteopromotive potential for bone regeneration. A growing number of studies report the combination of polymers with minerals in order to improve their bioactivity. This review exposes the state-of-the-art of existing 3D printed composite biomaterials combining polymers and minerals for bone tissue engineering. Characterization techniques to assess scaffold properties are also discussed. Several parameters must be considered to fabricate a 3D printed material for bone repair (3D printing method, type of polymer/mineral combination and ratio) because all of them affect final properties of the material. Each polymer and mineral has its own advantages and drawbacks and numerous composites are described in the literature. Each component of these composite materials brings specific properties and their combination can improve the biological integration of the 3D printed scaffold. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2579-2595, 2019.

A Unique Triculture Model to Study Osteoblasts, Osteoclasts, and Endothelial Cells.

IMPACT STATEMENT: In this article, we first developed a new medium to culture together primary human osteoblastic, osteoclastic, and endothelial cells (ECs) chosen to represent the three major bone cell tissues. Indeed, no study has been conducted on primary human cells and on the phenotype/activity retention of these three primary human cell types. Thus, we established an original triculture model with osteoblastic, osteoclastic, and ECs, where not only both cell phenotype and cell activity were maintained but also cell culture homeostasis. These promising results will permit further investigations to create in vitro conditions to mimic the bone microenvironment and analyze cell interactions in ex vivo studies.

Layer-by-layer bioassembly of poly(lactic) acid membranes loaded with coculture of HBMSCs and EPCs improves vascularization in vivo.

Layer-by-layer (LBL) BioAssembly method was developed to enhance the control of cell distribution within 3D scaffolds for tissue engineering applications. The objective of this study was to evaluate in vivo the development of blood vessels within LBL bioassembled membranes seeded with human primary cells, and to compare it to cellularized massive scaffolds. Poly(lactic) acid (PLA) membranes fabricated by fused deposition modeling were seeded with monocultures of human bone marrow stromal cells or with cocultures of these cells and endothelial progenitor cells. Then, four cellularized membranes were assembled in LBL constructs. Early osteoblastic and endothelial cell differentiation markers, alkaline phosphatase, and von Willebrand's factor, were expressed in all layers of assemblies in homogenous manner. The same kind of LBL assemblies as well as cellularized massive scaffolds was implanted subcutaneously in mice. Human cells were observed in all scaffolds seeded with cells, but not in the inner parts of massive scaffolds. There were significantly more blood vessels observed in LBL bioassemblies seeded with cocultures compared to all other samples. LBL bioassembly of PLA membranes seeded with a coculture of human cells is an efficient method to obtain homogenous cell distribution and blood vessel formation within the entire volume of a 3D composite scaffold.

The use of RANKL-coated brushite cement to stimulate bone remodelling.

Calcium phosphate cements were first proposed as synthetic bone substitutes over two decades ago, however, they are characterised by slow chemical or cellular resorption and a slow osteointegration. In contrast, bone autograft has been shown to stimulate osteoclastogenesis and angiogenesis resulting in active bone remodelling and rapid graft incorporation. Therefore, we aimed to develop a biomaterial able to release a key stimulator of the bone remodelling process, cytokine RANKL. Cylinders of brushite cement, hydroxyapatite cement and sodium alginate were loaded with RANKL either by incorporation into the cement or by coating the material with soluble RANKL. To test the biological activity of these formulations, we assessed their effectiveness in inducing osteoclast formation from RAW 264.7 monocytic cell line. Only brushite and hydroxyapatite cements coated with RANKL allowed for retaining sufficient biological activity to induce osteoclast formation. Most efficient was coating 40 mg cylinder of brushite cement with 800 ng RANKL. We have found that RANKL-coated brushite cement exhibits osteoclastogenic activity for at least 1 month at 37 degrees C. Thus, we developed a formulation of brushite cement with RANKL - a synthetic bone graft that is similar to autografts in its ability to actively induce osteoclastogenesis.

Bioactivity of bone resorptive factor loaded on osteoconductive matrices: stability post-dehydration.

Since calcium phosphate cements were proposed two decades ago, extensive research has been realized to develop and improve their properties. They have proved their efficiency as bone graft substitutes and their ability to incorporate and release drugs. However, to date, all 'resorbable' osteoconductive synthetic biomaterials are in fact simply soluble. In order to investigate a synthetic material capable of inducing osteoclast remodelling post-implantation, a formulation of calcium phosphate cement loaded with a pro-resorptive cytokine (RANKL) was studied. Many prior release studies on calcium phosphates did not confirm that the matrix had no detrimental effect on the molecule to be released during storage prior to use or that bioactivity was maintained during storage. In this report, the stability of our protein was tested after loading onto the cement, and various regimens to improve stability were compared. The presence of trehalose was shown to stabilize the bioactivity of RANKL adsorbed to brushite cement. The reduction of both moisture and oxygen in the storage vessel improved osteoclastogenic potential of the matrix compared with that stored in ambient atmosphere and temperature. No loss in activity was observed over the study period for the loaded matrix stored in dry nitrogen.

Brushite-collagen composites for bone regeneration.

Brushite-based biomaterials are of special interest in bone regeneration due to their biocompatibility and biodegradability; on the other hand, collagen is a well-known osteoconductive biomaterial. In the present study a new brushite-collagen composite biomaterial is reported. This new biomaterial was prepared by combining citric acid/collagen type I solutions with a brushite cement powder. The obtained biomaterial was a cement paste, with improved handling properties. The effect of collagen on the setting reaction of brushite cement was studied, and was found to speed up the cement setting reaction. The cement paste set into a hard ceramic material within 18.5+/-2.1min and had compressive strength similar to that of spongeous bone (48.9+/-5.9MPa in dry conditions and 12.7+/-1.5MPa in humid conditions). The combination of collagen with citric acid revealed an interesting synergistic effect on the compressive strength of the composite material. Moreover, this new biomaterial had excellent cohesion properties (ninefold better than brushite cement), and high cellular adhesion capacity (threefold higher than brushite cement). The composite biomaterial described in this study combines good handling properties, compressive strength, cohesion and cell adhesion capacity, along with the osteoconductive and biodegradable properties inherent in brushite and in collagen-based biomaterials.

Characterization of printed PLA scaffolds for bone tissue engineering.

Autografts remain the gold standard for orthopedic transplantations. However, to overcome its limitations, bone tissue engineering proposes new strategies. This includes the development of new biomaterials such as synthetic polymers, to serve as scaffold for tissue production. The objective of this present study was to produce poly(lactic) acid (PLA) scaffolds of different pore size using fused deposition modeling (FDM) technique and to evaluate their physicochemical and biological properties. Structural, chemical, mechanical, and biological characterizations were performed. We successfully fabricated scaffolds of three different pore sizes. However, the pore dimensions were slightly smaller than expected. We found that the 3D printing process induced decreases in both, PLA molecular weight and degradation temperatures, but did not change the semicrystalline structure of the polymer. We did not observe any effect of pore size on the mechanical properties of produced scaffolds. After the sterilization by γ irradiation, scaffolds did not exhibit any cytotoxicity towards human bone marrow stromal cells (HBMSC). Finally, after three and seven days of culture, HBMSC showed high viability and homogenous distribution irrespective of pore size. Thus, these results suggest that FDM technology is a fast and reproducible technique that can be used to fabricate tridimensional custom-made scaffolds for tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 887-894, 2018.

Anti-osteoclastic effects of C-glucosidic ellagitannins mediated by actin perturbation.

Actin subunits assemble into actin filaments whose dynamics and three-dimensional architectures are further regulated by a variety of cellular factors to establish the functional actin cytoskeleton. The C-glucosidic ellagitannin vescalagin and its simpler analogue vescalin, affect both the dynamics and the ultrastructure of the actin cytoskeleton by directly binding to F-actin. Herein, we show that in vitro, the two compounds induce the formation of distinct F-actin networks characterized by different superstructures and dynamics. In living mature osteoclasts, highly specialized bone-degrading cells that constantly remodel their cytoskeleton, vescalagin and vescalin alter actin dynamics at podosomes and compromise the integrity of the podosome belt that forms the bone-degrading apparatus. Both compounds target the bone-resorbing activity at concentrations that preserve osteoclastic maturation and survival and with no detectable impact on the behaviour of bone-forming osteoblastic cells. This anti-osteoclastic activity of vescalagin and vescalin reveals the potential of targeting actin dynamics as a new therapeutic opportunity and, in this case, as a plausible approach for the local treatment of osteoporosis.

Hybrid composites of calcium phosphate granules, fibrin glue, and bone marrow for skeletal repair.

Synthetic bone substitutes, such as calcium phosphate ceramics, give good results in clinical applications. In order to adapt to surgical sites, bioceramics come in the form of blocks or granules, and are either dense or porous. Combining these bioceramics with fibrin glue provides a mouldable and self-hardening composite biomaterial with the biochemical properties of each component. Critical-sized defects in the femoral condyle of rabbits were filled with TricOs/fibrin glue/bone marrow hybrid/composite material. The TricOs granules (1-2 mm) were composed of hydroxyapatite and beta tricalcium phosphate (60/40 in weight). The fibrin glue was composed of fibrinogen, thrombin and other biological factors and mixed with MBCP granules either simultaneously or sequentially. Bone marrow was also added to the MBCP/fibrin composite prior to filling the defects. After 3, 6, 12, and 24 weeks of implantation, the newly-formed bone was analysed with histology, histomorphometry and mechanical tests. The newly-formed bone had grown centripetally. Simultaneous application of fibrin glue showed better results for mechanical properties than sequential application after 6 weeks. Around 40% of bone had formed after 24 weeks in the three groups. Although the addition of bone marrow did not improve bone formation, the MBCP/fibrin material could be used in clinical bone filling applications.

Regulation of Osteoclast Growth and Fusion by mTOR/raptor and mTOR/rictor/Akt.

Osteoclasts are giant bone cells formed by fusion from monocytes and uniquely capable of a complete destruction of mineralized tissues. Previously, we have demonstrated that in energy-rich environment not only osteoclast fusion index (the number of nuclei each osteoclast contains), but also cytoplasm volume per single nucleus was increased. The goal of this study was to investigate the regulation of metabolic sensor mTOR during osteoclast differentiation in energy-rich environment simulated by addition of pyruvate. We have found that in the presence of pyruvate, the proportion of mTOR associated with raptor increased, while mTOR-rictor-mediated Akt phosphorylation decreased. Inhibition of mTOR with rapamycin (10 nM) significantly interfered with all aspects of osteoclastogenesis. However, rapamycin at 1 nM, which preferentially targets mTOR-raptor complex, was only effective in control cultures, while in the presence of pyruvate osteoclast fusion index was successfully increased. Inhibition of Akt drastically reduced osteoclast fusion, however in energy-rich environment, osteoclasts of comparable size were formed through increased cytoplasm growth. These data suggest that mTOR-rictor mediated Akt signaling regulates osteoclast fusion, while mTOR-raptor regulation of protein translation contributes to fusion-independent cytoplasm growth. We demonstrate that depending on the bioenergetics microenvironment osteoclastogenesis can adjust to occur through preferential multinucleation or through cell growth, implying that attaining large cell size is part of the osteoclast differentiation program.

Micro-architecture of calcium phosphate granules and fibrin glue composites for bone tissue engineering.

Calcium phosphate ceramics are currently used as bone graft substitutes in various types of clinical applications. Fibrin glue is also used in surgery due to its haemostatic, chemotactic and mitogenic properties. By combining these two biomaterials, new composite scaffolds were prepared. In this study, we attempt to analyse whether thrombin concentration in the fibrin glue could influence the properties of the composite. The association between fibrin glue and calcium phosphate ceramic granules was characterized at the ultra structural level. Micro and macroporous biphasic calcium phosphate ceramic granules with a diameter of 1-2mm composed of hydroxyapatite and beta-tricalcium phosphate (60/40) were associated to fibrin glue. The composites were observed by scanning and transmission electron microscopy and microcomputed tomography. Fibre thickness, porosity and homogeneity of the fibrin clot were modified by increased the thrombin concentration. Mixing fibrin glue with calcium phosphate granules (1:2) did not modify the microstructure of the fibrin clot in the composite. Nevertheless, thrombin interacted with the bioceramic by inducing the nucleation of crystalline precipitate at the ceramic/fibrin glue interface. Combining fibrin sealant and calcium phosphate ceramics could lead to new scaffolds for bone tissue engineering with the synergy of the properties of the two biomaterials.

Ectopic bone formation by microporous calcium phosphate ceramic particles in sheep muscles.

Calcium phosphate ceramics are widely used in bone reconstructive surgery because of their osteconductive properties. However, these materials generally lack osteoinductive properties required to support bone healing in large defects. In this article, we study the osteoinductive potential of calcium phosphate ceramic particles implanted for 6 months into the dorsal muscles of eight adult female sheep. Microporous biphasic calcium phosphate (MBCP) granules of 1-2 mm composed of hydroxyapatite and beta-tricalcium phosphate (60/40) had macropores of 450 microm, micropores of 0.43 microm, and a specific surface area of 1.8 m(2)/g. After 6 months in the back muscles of sheep, the explants composed of MBCP granules were hard and encapsulated by normal muscle tissue. Ectopic bone formation with Haversian structures was observed in close contact with the MBCP granules in histological sections. Back-scattered electron microscopy and micro-computed tomography indicated that approximately 10% of well-mineralized bone with mature osteocytes had formed between or upon the granules. The ectopic bone showed trabeculae bridging the MBCP granules. Both the number and thickness of the trabeculae formed between the MBCP particles were comparable to those measured in spongious bone. The overall results therefore confirmed the presence of mature bone after intramuscular implantation of MBCP granules. The different hypotheses explaining ectopic bone formation induced by MBCP granules are discussed. Synthetic bone substitutes with osteoinductive properties could be used in bone reconstructive surgery.

Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery.

Moderate excess of pyruvate augments osteoclastogenesis.

Cell differentiation leads to adaptive changes in energy metabolism. Conversely, hyperglycemia induces malfunction of many body systems, including bone, suggesting that energy metabolism reciprocally affects cell differentiation. We investigated how the differentiation of bone-resorbing osteoclasts, large polykaryons formed through fusion and growth of cells of monocytic origin, is affected by excess of energy substrate pyruvate and how energy metabolism changes during osteoclast differentiation. Surprisingly, small increases in pyruvate (1-2 mM above basal levels) augmented osteoclastogenesis in vitro and in vivo, while larger increases were not effective in vitro. Osteoclast differentiation increased cell mitochondrial activity and ATP levels, which were further augmented in energy-rich conditions. Conversely, the inhibition of respiration significantly reduced osteoclast number and size. AMP-activated protein kinase (AMPK) acts as a metabolic sensor, which is inhibited in energy-rich conditions. We found that osteoclast differentiation was associated with an increase in AMPK levels and a change in AMPK isoform composition. Increased osteoclast size induced by pyruvate (1 mM above basal levels) was prevented in the presence of AMPK activator 5-amino-4-imidazole carboxamide ribonucleotide (AICAR). In keeping, inhibition of AMPK using dorsomorphin or siRNA to AMPKγ increased osteoclast size in control cultures to the level observed in the presence of pyruvate. Thus, we have found that a moderate excess of pyruvate enhances osteoclastogenesis, and that AMPK acts to tailor osteoclastogenesis to a cell's bioenergetics capacity.

The effect of autoclaving on the physical and biological properties of dicalcium phosphate dihydrate bioceramics: brushite vs. monetite.

Dicalcium phosphate dihydrate (brushite) is an osteoconductive biomaterial with great potential as a bioresorbable cement for bone regeneration. Preset brushite cement can be dehydrated into dicalcium phosphate anhydrous (monetite) bioceramics by autoclaving. This heat treatment results in changes in the physical characteristics of the material, improving in vivo bioresorption. This property is a great advantage in bone regeneration; however, it is not known how autoclaving brushite preset cement might improve its capacity to regenerate bone. This study was designed to compare brushite bioceramics with monetite bioceramics in terms of physical characteristics in vitro, and in vivo performance upon bone implantation. In this study we observed that monetite bioceramics prepared by autoclaving preset brushite cements had higher porosity, interconnected porosity and specific surface area than their brushite precursors. In vitro cell culture experiments revealed that bone marrow cells expressed higher levels of osteogenic genes Runx2, Opn, and Alp when the cells were cultured on monetite ceramics rather than on brushite ones. In vivo experiments revealed that monetite bioceramics resorbed faster than brushite ones and were more infiltrated with newly formed bone. In summary, autoclaving preset brushite cements results in a material with improved properties for bone regeneration procedures.

Biocompatibility of magnesium phosphate minerals and their stability under physiological conditions.

Magnesium phosphates such as newberyite (MgHPO(4)·3H(2)O) are formed in vivo and are known to be biodegradable and nontoxic after implantation. Indeed, magnesium apatites have been shown to support osteoblast differentiation and function, and bone formation can occur around metallic magnesium implants. However, very little is known regarding the precipitation and stability of magnesium phosphates in physiological environments. In order to address this, the aqueous formation of magnesium phosphate as a function of pH, temperature and ion concentration is reported. Physicochemical characterization of the precipitates was carried out; additionally, biocompatibility and gene expression of osteoblast differentiation markers for bone formation via an in vitro cell culture assay were determined. Precipitation conditions for newberyite, tribasic magnesium phosphate pentahydrate, holtedahlite, bobierrite and cattiite were determined. Under physiological conditions of pH, temperature and magnesium phosphate concentration, no precipitates were formed. However, at concentrations 10-100 times higher than physiological, magnesium phosphate precipitates of cattiite and newberyite were formed. These two minerals demonstrated biocompatibility with osteoblast cultures and induced osteoblast adhesion and differentiation. The pattern of expression of OCN and CollA1 genes in the presence of newberyite crystals was comparable to that of calcium phosphate bioceramics. In our experiments, we have shown that certain magnesium phosphate phases such as newberyite and cattiite are able to promote in vivo osteogenic activity in a similar way to calcium phosphates such as hydroxyapatite and brushite. This confirms the great potential of magnesium phosphate ceramics in the development of new biomaterials for bone regeneration.

Ascorbic acid accelerates osteoclast formation and death.

Ascorbic acid (AA) plays a key role in bone formation. However controversy remains about the effect of AA on cells responsible for bone destruction, osteoclasts. We investigated the effect of AA on osteoclastogenesis using primary mouse bone marrow cultures and monocytic RAW 264.7 cells treated with osteoclastogenic factors RANKL and MCSF. Treatment with AA resulted in significant increase in osteoclast number, size and nucleation. To assess osteoclast oxidative stress level, a ratio of reduced (GSH) to oxidized (GSSG) glutathione and the total glutathione content (GSH(t)) were evaluated. Osteoclast differentiation was associated with a decrease in GSH/GSSG and GSH(t). AA induced further decrease in both parameters, and resulted in significant production of H(2)O(2), indicating its pro-oxidant action. At low concentration, H(2)O(2) induced similar effects to AA, although less potently, and catalase partially inhibited AA-induced osteoclastogenesis. To assess the modification in osteoclast metabolism, the mitochondrial activity was evaluated using JC-1 and the ATP levels were assessed. Osteoclast formation was associated with the increase in mitochondrial activity and ATP concentration, which were further increased in the presence of AA. Importantly, the stimulatory effect of AA was only evident at early phase of osteoclastogenesis, whereas at the late stage AA significantly accelerated osteoclast death. Thus, during osteoclastogenesis AA acts as an oxidant, first stimulating osteoclast formation, but later limiting osteoclast lifespan. This duality of AA action allows reconciling the stimulatory action of AA on osteoclastogenesis observed in vitro with an overall attenuation of bone resorption in the presence of AA observed in vivo.

Bone tissue formation in sheep muscles induced by a biphasic calcium phosphate ceramic and fibrin glue composite.

Some biomaterials are able to induce ectopic bone formation in muscles of large animals. The osteoinductive potential of macro- micro-porous biphasic calcium phosphate (MBCP) ceramic granules with fibrin glue was evaluated by intramuscular implantation for 6 months in six adult female sheep. The MBCP granules were 1-2 mm in size and were composed of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) in a 60/40 ratio. The fibrin glue was composed of fibrinogen, thrombin and other biological factors. After 6 months of implantation in the dorsal muscles of sheep, the explants were rigid. Histology, back-scattered electron microscopy and micro-computed tomography of the implants indicated that approximately 12% of mineralized bone had formed in between the MBCP granules. The ectopic bone appeared well-mineralized with mature osteocytes and Haversian structures. In addition, the number and thickness of bone trabeculae formed in between the MBCP particles were similar to those measured in trabecular bone in sheep. The overall results therefore confirmed the formation of well-mineralized ectopic bone tissue after intramuscular implantation of MBCP/fibrin glue composites. These bone substitutes exhibiting osteoinductive properties could be used for the reconstruction of large bone defects.