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International audience; Particulate forms of biphasic calcium phosphate (BCP) biomaterials below 500 mum are promising bone substitutes that provide with interconnected open porosity allowing free circulation of fluids and cells. Dispersion of the particles in the surrounding tissues at the time of implantation is a major drawback preventing from an easy use. We have asked whether blood clot could be a convenient natural hydrogel for handling BCP microparticles, and we hypothesized that blood clot might also confer osteoinductive properties to these particles. We show here that blood clotted around BCP microparticles constitutes a cohesive, moldable, and adaptable biomaterial that can be easily implanted in subcutaneous sites but also inserted and maintained in segmental bone defects, conversely to BCP microparticles alone. Moreover, implantation in bony and ectopic sites revealed that this composite biomaterial has osteogenic properties. It is able to repair a 6-mm critical femoral defect in rat and induced woven bone formation after subcutaneous implantation. Parameters such as particle size and loading into the clot are critical for its osteogenic properties. In conclusion, this blood/BCP microparticle composite is a moldable and osteoinductive biomaterial that could be used for bone defect filling in dental and orthopedic surgery.

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. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2007

A new way of optimizing osteoconduction of biomaterials is to bring to them biological properties. In this work, we associated a novel release system with an electrodeposited calcium phosphate (CaP) coated titanium alloy Ti6Al4V. The characterization of this material was performed by means of light microscopy, scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and X-ray energy dispersive spectroscopy (EDXS). The electrodeposited CaP coating was a tricalcium phosphate, and the release system was composed of microcapsules entrapped in an alginate film. We observed that the alginate matrix had a close contact with the coating. An intermediate layer containing calcium and phosphorus appeared at the interface between the alginate matrix and the CaP coating. These results allowed us to conclude that the association of two techniques, i.e. electrodeposition followed by deposition of a calcium alginate matrix, led to the elaboration of a new biomaterial.

International audience; A combination of autologous bone marrow stromal cells (BMSCs) and biomaterials is a strategy largely developed in bone tissue engineering, and subcutaneous implantation in rodents or large animals is often a first step to evaluate the potential of new biomaterials. This study aimed at investigating the influence of the immune status of the recipient animal on BMSCs-induced bone formation. BMSCs prepared from C57BL/6 mice, composed of a mixture of mesenchymal stromal and monocytic cells, were combined with a biomaterial that consisted of biphasic calcium phosphate (BCP) particles and plasma clot. This composite was implanted subcutaneously either in syngenic C57BL/6 immune-competent mice or in T-lymphocyte-deficient Nude (Nude) mice. Using histology, immunohistochemistry, and histomorphometry, we show here that this BMSC/BCP/plasma clot composite implanted in Nude mice induces the formation of mature lamellar bone associated to hematopoietic areas and numerous vessels. Comparatively, implantation in C57BL/6 results in the formation of woven bone without hematopoietic tissue, a lower number of new vessels, and numerous multinucleated giant cells (MNGCs). In situ hybridization, which enabled to follow the fate of the BMSCs, revealed that BMSCs implanted in Nude mice survived longer than BMSCs implanted in C57BL/6 mice. Quantitative expression analysis of 280 genes in the implants indicated that the differences between C57BL/6 and Nude implants corresponded almost exclusively to genes related to the immune response. Gene expression profile in C57BL/6 implants was consistent with a mild chronic inflammation reaction characterized by Th1, Th2, and cytotoxic T-lymphocyte activation. In the implants retrieved from T-deficient Nude mice, Mmp14, Il6st, and Tgfbr3 genes were over-expressed, suggesting their putative role in bone regeneration and hematopoiesis. In conclusion, we show here that the T-mediated inflammatory microenvironment is detrimental to BMSCs-induced bone formation and shortens the survival of implanted cells. Conversely, the lack of T-lymphocyte reaction in T-deficient animals is beneficial to BMSCs-induced mature bone formation. This should be taken into account when evaluating cell/biomaterial composites in these models.

We studied a new injectable biomaterial for bone and dental surgery consisting of a hydrophilic polymer as matrix and bioactive calcium phosphate (CaP) ceramics as fillers. This material is composed of complex fluids whose flow is determined by the laws of rheology. We investigated the macromolecular effects on this composite in a tube. The stability of the polymer and the mixture is essential to the production of a ready-to-use injectable biomaterial. These flow properties are necessary to obtain CaP bioactivity in a dental canal or bone defect during percutaneous surgery. Macromolecules provide spaces between CaP ceramic granules and facilitate the role of the biological agents of bone substitution.

The purpose of this study was to develop an injectable bone substitute (IBS) for percutaneous orthopedic surgery. The multiphasic material used was composed of a 2% aqueous solution of methylhydroxypropylcellulose (MHPC) and biphasic calcium phosphate (BCP, 60% hydroxyapatite and 40% beta-tricalcium phosphate) in which MHPC served as the carrier for 80-200 microm of BCP granules. The best BCP/polymer ratio was determined by the rheological properties and higher BCP content of the material. Steam sterilization was more effective than gamma irradiation in maintaining the stability of the mixture and conserving its physiochemical and mechanical properties. The in vitro biocompatibility of the composite was checked by direct-contact cytotoxicity and cell-proliferation assays. A preliminary in vivo test was performed in the rabbit using intraosseous implantations in the femoral epiphysis. Histological analysis was done after 1, 2, 4, and 10 weeks. Bone ingrowth into the IBS, in close association with BCP granules, was observed after 1 week and increased regularly from the surface inward at 2, 4, and 10 weeks. At the same time, smaller BCP granules (less than 80 microns in diameter) were degraded and resorbed. This injectable biomaterial proved suitable for cavity filling. The water solubility and viscosity of the polymer allow cells to recolonize, with in situ bonding of the mineral phase.

International audience; Calcium phosphate ceramics are widely used as bone substitutes in dentistry and orthopedic applications. For minimally invasive surgery an injectable calcium phosphate ceramic suspension (ICPCS) was developed. It consists in a biopolymer (hydroxypropylmethylcellulose: HPMC) as matrix and bioactive calcium phosphate ceramics (biphasic calcium phosphate: BCP) as fillers. The stability of the suspension is essential to this generation of "ready to use" injectable biomaterial. But, during storage, the particles settle down. The engineering sciences have long been interested in models describing the settling (or sedimentation) of particles in viscous fluids. Our work is dedicated to the comprehension of the effect of the formulation on the stability of calcium phosphate suspension before and after steam sterilization. The rheological characterization revealed the macromolecular behavior of the suspending medium. The investigations of settling kinetics showed the influence of the BCP particle size and the HPMC concentration on the settling velocity and sediment compactness before and after sterilization. To decrease the sedimentation process, the granule size has to be smaller and the polymer concentration has to increase. A much lower sedimentation velocity, as compared to Stokes law, is observed and interpreted in terms of interactions between the polymer network in solution and the particles. This experimentation highlights the granules spacer property of hydrophilic macromolecules that is a key issue for interconnection control, one of the better ways to improve osteoconduction and bioactivity.

Several studies have shown that macro micro porous bioceramics ectopically implanted promote bone tissue formation. This study aims at investigating the inflammatory response towards biphasic calcium phosphate (BCP) ceramic micro particles. BCP composed of hydroxyapatite (HA) and beta-tricalcium phosphate, HA/beta -TCP ratio of 50/50, were prepared by sintering at 1200 degrees C for 5 h. After crushing, 3 fractions of BCP micro particles < 20, 40-80 and 80-200 micro m were sieved. The micro particles were carefully characterized by using X-ray diffraction (XRD), scanning electron microscopy (SEM) and laser scattering. The inflammatory reactions induced by BCP micro particles implanted in quadriceps muscles of rats for 7, 14 and 21 days were studied by histology (n = 8/group). A fibrous tissue encapsulation of the BCP micro particles implanted in muscle tissue was observed and fibrosis was similar for the 3 groups of micro particles. The comparison of the cellular response indicated that the total number of cells was significantly higher for BCP < 20 micro m than for 40-80 and 80-200 micro m (p < 0.0001). The number of macrophages was relatively higher for the smallest than for the intermediate and largest fractions (p < 0.0001). The relative percentage of giant cells was higher for the intermediate and largest size of particles than for the smallest. The number of lymphocytes was comparable for the 3 fractions and after the 3 delays. Therefore, the BCP micro particles < 20 micro m initiated an inflammatory response which might play an important role in osteogenesis.

Calcium phosphate cements (CPCs) are successfully used as bone substitutes in dentistry and orthopaedic applications. This study investigated the physico-chemical-mechanical properties of and in vitro biological properties (cell response) of CPCs prepared with amorphous calcium carbonate phosphate (ACCP) doped with magnesium (ACCP-Mg), zinc (ACCp-Zn) or fluoride (ACCP-F) ions. The experimental CPC consisted of alpha-TCP, doped ACCP, and MPCM powders as matrix and biphasic calcium phosphate (BCP) granules. X-ray diffraction analysis showed that the matrix converted to apatite with poor crystallinity (reflecting small crystal size) after setting for 24 h, while BCP remained apparently unchanged. Cements with ACCP-F (F-CPC) had shorter setting times and greater compressive strength compared to cements with ACCP-Mg (Mg-CPC) or ACCP-Zn (Zn-CPC). Scanning electron microscopy (SEM) showed that crystals set on Mg-CPC and Zn-CPC were smaller compared to those on F-CPC. The total porosity of Mg-CPC was greater compared to Zn-CPC or F-CPC. Osteoblast-like cells, MC3T3-E1, remained viable and maintained their ability to express alkaline phosphatase in contact with the CPCs with doped ACCPs.

Calcium phosphates coatings were deposited onto titanium alloy discs via en electrodeposition method. Titanium alloy discs were blasted with calcium phosphate particles, then etched in a mixture of nitric and fluoric acids and rinsed in demineralized water. The titanium alloy disc (cathode) and platinum mesh (anode) were immersed in a supersaturated calcium phosphate electrolyte buffered at pH 7.4 and connected to a current generator. The microstructure, chemical composition and crystallinity of the electrodeposited coatings were studied as function of time 10-120 min, temperature 25-80 degrees C, current density 8-120 mA/cm(2), magnesium and hydrogen carbonate amounts (0.1-1 mM). Uniform calcium phosphate coatings were obtained in 30 min but coating thickness increased with deposition time. Raising the temperature of electrolyte resulted in more uniform coatings as ionic mobility increased. Low current density was preferable due to hydrogen gas evolving at the cathode, which disturbed the deposition of calcium phosphate crystals on titanium. The amounts of magnesium and hydrogen carbonate ions affected both the homogeneity and morphology of the coatings. This study showed that the electrodeposition method is efficient for coating titanium with osteoconductive calcium phosphate layers.

International audience; This study is the first report of a clinical evaluation of an injectable bone substitute (IBS). This IBS was prepared by suspending biphasic calcium phosphate (BCP) particles with diameters ranging between 80 and 200 microm in a water-soluble cellulose polymer carrier phase. It was used for filling bone defects after tooth extractions in 11 patients. The first objective of the study was to investigate the safety of the filler material. The second objective was to investigate the efficacy of the material for filling human tooth sockets and preventing alveolar bone loss. Radiographic density measurements of the surgical sites gradually increased to those of the surrounding host bone. Three years after surgery, small biopsies of the implanted areas were harvested and analyzed by using micro-computed tomography, non-decalcified histology and histomorphometry. The BCP granules appeared in direct contact with mineralized bone tissue, thereby supporting bone growth. A gradual substitution of the filler by bone tissue was observed thus preserving the height of the alveolar bone crest.

International audience; In this work, calcium-deficient apatites (CDA) were synthesized by ammonia hydrolysis reaction of dicalcium phosphate dihydrate (DCPD; CaHPO4 x 2 H2O) to obtain biphasic calcium phosphates (BCP) without any extraionic substitution. The influence of three parameters was studied: temperature of the reaction (70 and 100 degrees C), time of the reaction (4 and 18 h), and the pressure (open and closed system). Experiments were made according to a factorial design method (FDM) allowing optimization of the number of samples as well as statistical analysis of results. Moreover, the influence of temperature (until 200 degrees C) was investigated. The crystal size of CDA was determined according to the Scherrer's formula and from Rietveld refinements taking the CDA anisotropy into account. The last method seems to be a reliable method to determine crystallite sizes of CDA, since crystallite sizes of CDA along and directions were accessible. The results describe the hydroxyapatite % (HA%) in BCP by a first-order polynomial equation in the experimental area studied and the HA content was found to increase by raising time and temperature of the reaction. Moreover, the type of reaction system (open/closed vessel) appeared to have little influence on HA%.

In France, despite the ban of meat-and-bone meal (MBM) in cattle feed, bovine spongiform encephalopathy (BSE) was detected in hundreds of cattle born after the ban. To study the role of MBM, animal fat, and dicalcium phosphate on the risk for BSE after the feed ban, we conducted a spatial analysis of the feed industry. We used data from 629 BSE cases as well as data on use of each byproduct and market area of the feed factories. We mapped risk for BSE in 951 areas supplied by the same factories and connection with use of byproducts. A disease map of BSE with covariates was built with the hierarchical Bayesian modeling methods, based on Poisson distribution with spatial smoothing. Only use of MBM was spatially linked to risk for BSE, which highlights cross-contamination as the most probable source of infection after the feed ban.

International audience; We have used a new synthetic injectable composite constituted of hydroxyapatite/tricalcium phosphate (HA/TCP) particles in suspension in a self-hardening Si-hydroxypropylmethylcellulose (HPMC) hydrogel. The aim of this study was to evaluate in vivo the biocompatibility and the new bone formation efficacy of this scaffold loaded with undifferentiated bone marrow stromal cells (BMSCs). This biomaterial was mixed extemporaneously with BMSCs prepared from C57BL/6 mice, injected in subcutaneous and intramuscular sites and retrieved 4 and 8 weeks after implantation. Dissection of the implants revealed a hard consistency and the absence of a fibrous capsule reflecting a good integration into the host tissues. Histological analysis showed mineralized woven bone in the granule inter-space with numerous active osteoclasts attached to the particles as assessed by the presence of multinucleated cells positively stained for TRAP activity and for the a3 subunit of the V-ATPase. Small vessels were homogenously distributed in the whole implants. Similar results were obtained in SC and IM sites and no bone formation was observed in the control groups when cell-free and particle-free transplants were injected. These results indicate that this injectable biphasic calcium phosphate-hydrogel composite mixed with undifferentiated BMSCs is a new promising osteoinductive bone substitute. It also provides with an original in vivo model of osteoclast differentiation and function.

International audience; A new injectable and self-crosslinkable bone substitute (IBS2) was developed for filling bone defects. The IBS2 consisted of a chemically modified polymer solution mixed with biphasic calcium phosphate (BCP) ceramic particles. The polymer hydroxypropylmethyl cellulose was functionalized with silanol groups (Si-HPMC) and formed a viscous solution (3 wt %) in alkaline medium. With a decrease in pH, self-hardening occurred due to the formation of intermolecular -Si-O- bonds. During setting, BCP particles, 40 to 80 microm in diameter, were added to the polymer solution at a weight ratio of 50/50. The resulting injectable material was bilaterally implanted into critically sized bone defects at the distal femoral epiphyses of nine New Zealand White rabbits. The IBS2 filled the bone defects entirely and remained in place. After 8 weeks, bone had grown centripetally and progressed towards the center of the defects. Newly formed bone, ceramic, and nonmineralized tissue ratios were 24.6% +/- 5.6%, 21.6% +/- 5.8%, and 53.7% +/- 0.1%, respectively. Mineralized and mature bone was observed between and in contact with the BCP particles. The bone/ceramic apposition was 73.4% +/- 10.6%. The yield strength for the IBS2-filled defects was 16.4 +/- 7.2 MPa, significantly higher than for the host trabecular bone tissue (2.7 +/- 0.4 MPa). This study showed that BCP particles supported the bone healing process by osteoconduction while the Si-HPMC hydrogel created intergranular space for bone ingrowth. This new injectable and self-crosslinkable bone substitute could be used conveniently in orthopedic surgery for filling critical-size bone defects.

International audience; 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.

International audience; Basic calcium phosphate (BCP) crystals, including hydroxyapatite, octacalcium phosphate (OCP) and carbonate-apatite, have been associated with severe osteoarthritis and several degenerative arthropathies. Most studies have considered the chondrocyte to be a bystander in the pathogenesis of calcium crystal deposition disease, assuming that synovial cell cytokines were the only triggers of chondrocyte activation. In the present study we identified direct activation of articular chondrocytes by OCP crystals, which are the BCP crystals ith the greatest potential for inducing inflammation. OCP crystals induced nitric oxide (NO) production and inducible nitric oxide synthase (NOS) mRNA expression by isolated articular chondrocytes and cartilage fragments, in a dose-dependent manner and with variations over time. OCP crystals also induced IL-1β mRNA expression. Using pharmacological and cytokine inhibitors, we observed that OCP crystals induced NO production and inducible NOS mRNA activation were regulated at both the transcriptional and the translational levels; were independent from IL-1β gene activation; and involved p38 and c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, as further confirmed by OCP crystal-induced p38 and JNK MAPK phosphorylation. Taken together, our data suggest that the transcriptional inducible NOS response to OCP crystals involved both the p38 and the JNK MAPK pathways, probably under the control of activator protein-1. NO, a major mediator of cartilage degradation, can be directly produced by BCP crystals in chondrocytes. Together with synovial activation, this direct mechanism may be important in the pathogenesis of destructive arthropathies triggered by microcrystals.

International audience; BACKGROUND: The aim of the present study was to assess the efficacy of a ready-to-use injectable bone substitute for bone regeneration around dental implants placed into fresh extraction sockets. METHODS: Third and fourth mandibular premolars were extracted from three beagle dogs and the interradicular septa were surgically reduced to induce a mesial bone defect. Thereafter, titanium implants were immediately placed. On the left side of the jaw, mesial bone defects were filled with an injectable bone substitute (IBS), obtained by combining a polymer and biphasic calcium phosphate ceramic granules. The right defects were left unfilled as controls. After 3 months of healing, specimens were prepared for histological and histomorphometric evaluations. RESULTS: No post-surgical complications were observed during the healing period. In all experimental conditions, histological observations revealed a lamellar bone formation in contact with the implant. Histomorphometric analysis showed that IBS triggers a significant (P

International audience; Synthesis of grafting silane on a hydro soluble cellulose ether (HPMC) was described. In alkaline medium, this derivate is under gel form. With a decrease of the pH, a self-hardening occurs due to the silanol condensation. For potential biomedical use, we described the silated-HPMC synthesis, the gel behavior after steam sterilization and the parameters of the silanol condensation i.e. pH, silane percentage and temperature. Minimum kinetic of the condensation was observed for pH between 5.5 and 6.5. So temperature catalyzed the reaction and the self-hardening speed was increased by silane percentage.

International audience; The purpose of this study was to check the chemical stability of an injectable bone substitute (IBS) composed of a 50/50 w/w mixture of 2.92% hydroxypropyl methylcellulose (HPMC) solution in deionized water containing biphasic calcium phosphate (BCP) granules (60% hydroxyapatite/40% beta-tricalcium phosphate w/w). After separation of the organic and mineral phases, capillary gas chromatography (GC) was used to study the possible modification of HPMC due to the contact with BCP granules following steam sterilisation and 32 days storage at room temperature. HPMC was extracted from IBS in aqueous medium, and a dialytic method was then used to extract calcium phosphate salts from the HPMC. The percentage of HPMC extracted from BCP was 98.5%+/-0.5%, as measured by UV. GC showed no chemical modifications after steam sterilisation and storage.