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International audience; Immediately upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to the biomaterial surface and the protein layer affects both blood cell functions and biomaterial bioactivity. Previously, we reported that 80-200 µm biphasic calcium phosphate (BCP) microparticles embedded in a blood clot, induce ectopic woven bone formation in mice, when 200-500 µm BCP particles induce mainly fibrous tissue. Here, in a LC-MS/MS proteomic study we compared the differentially expressed blood proteins (plasma and blood cell proteins) and the deregulated signaling pathways of these osteogenic and fibrogenic blood composites. We showed that blood/BCP-induced osteogenesis is associated with a higher expression of fibrinogen (FGN) and an upregulation of the Myd88- and NF-κB-dependent TLR4 signaling cascade. We also highlighted the key role of the LBP/CD14 proteins in the TLR4 activation of blood cells by BCP particles. As FGN is an endogenous ligand of TLR4, able to modulate blood composite stiffness, we propose that different FGN concentrations modify the blood clot mechanical properties, which in turn modulate BCP/blood composite osteoactivity through TLR4 signaling. The present findings provide an insight at the protein level, into the mechanisms leading to an efficient bone reconstruction by blood/BCP composites. STATEMENT OF SIGNIFICANCE: Upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to bone substitute surface and this protein layer affects both biomaterial bioactivity and bone healing. Therefore, for the best outcome for patients, it is crucial to understand the molecular interactions between blood and bone scaffolds. Biphasic calcium phosphate (BCP) ceramics are considered as the gold standard in bone reconstruction surgery. Here, using proteomic analyses we showed that the osteogenic properties of 80-200 µm BCP particles embedded in a blood clot is associated with a higher expression of fibrinogen. Fibrinogen upregulates the Myd88- and NF-κB-dependent TLR4 pathway in blood cells and, BCP-induced TLR4 activation is mediated by the LBP and CD14 proteins.

We previously reported that biphasic calcium phosphate (BCP) microparticles embedded in a blood clot induces ectopic bone formation in mice and repairs a critical femoral defect in rat. The present pilot study aimed to evaluate in dog and in two models of large defects the efficacy of this composite named "blood for reconstruction of bone" (BRB). We show here that BRB is a cohesive biomaterial easy to prepare from dog autologous blood and to mold to fill large bone defects. First in a model of cylindrical femoral condyle defect, the BRB was compared with BCP particles alone. After 8 weeks, this revealed that the amount of mature bone was slightly and significantly higher with BRB than with BCP particles. Second, in a model consisting in a 2 cm-long critical interruptive defect of the ulna, the BRB was compared with autologous bone. After 6 months, we observed that implantation of BRB can induce the complete reconstruction of the defect and that newly formed bone exhibits high regenerative potential. Comparison with the results obtained with autologous bone grafting strongly suggests that the BRB might be an efficient biomaterial to repair large bone defects, as an alternative or in addition to autologous bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1842-1850, 2018.

An injectable purely apatitic calcium phosphate cement (CPC) was successfully combined to a water-soluble radiopaque agent (i.e., Xenetix® ), to result in an optimized composition that was found to be as satisfactory as poly(methyl methacrylate) (PMMA) formulations used for vertebroplasty, in terms of radiopacity, texture and injectability. For that purpose, the Xenetix dosage in the cement paste was optimized by injection of the radiopaque CPC in human cadaveric vertebrae under classical PMMA vertebroplasty conditions, performed by interventional radiologists familiar with this surgical procedure. When present in the cement paste up to 70 mg I mL-1 , Xenetix did not influence the injectability, cohesion, and setting time of the resulting composite. After hardening of the material, the same observation was made regarding the microstructure, mechanical strength and alpha-tricalcium phosphate to calcium deficient apatite transformation rate. Upon implantation in bone in a small animal model (rat), the biocompatibility of the Xenetix-containing CPC was evidenced. Moreover, an almost quantitative release of the contrast agent was found to occur rapidly, on the basis of in vitro static and dynamic quantitative studies simulating in vivo implantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2786-2795, 2018.

Background There are different mouthwashes containing chlorhexidine in different concentrations, as well as various excipients. Chlorhexidine induce stains or discoloration in teeth and mucous membranes. The aim of this work was to design a model to reproduce in vitro staining associated with the use of different mouthwashes containing chlorhexidine. Material and methods We used as substrates of natural teeth and elephant ivory slices. Different incubation baths were conducted over 21 days in culture dishes at 37°C. At the beginning of experiment before incubation (D0) and after 21 days (D21) of incubation with different mouthwashes, pictures of substrates were taken in a standardized manner and an image analysis software was used to analyse and quantify the staining under the various conditions by using the 3 main colours (Red, Green, Blue, RGB). Results The results of this work demonstrate a very good reproducibility of the protocol, and secondly, a different expression statistically significant of the primary blue colour. We suggest that for a given concentration of chlorhexidine, the staining effects may vary depending on the excipients used. Conclusions This replicable model, easy to implement over a relatively short duration, can be used for evaluation of existing mouthwashes, and to test the excipients anti discoloration proposed by manufacturers. Key words:In vitro, chlorhexidine, mouthwashes, dental stain, tooth discoloration.

International audience; Study design: An animal model of anterior lumbar arthrodesis was designed to investigate whether a calcium phosphate cement (CPC) mixed with autologous whole blood might favourably compete with autograft for enhanced spine fusion.Objective: The objective of this study was to use a large animal model to investigate a new approach for spine fusion which consists in the injection of a highly resorbable CPC / blood composite to fill the interbody fusion cage, once placed in the intervertebral space.Summary of background data: Spinal fusion via intervertebral arthrodesis is a common surgical procedure for which the gold standard consists in filling the internal hollow part of the cage with bone autograft. However, this approach suffers from some drawbacks, including a second surgical site which requires additional time spent in the surgical room, and the occurrence of additional new pain on the bone harvesting site along with risks of long-term morbidity.Methods: An animal model of anterior lumbar arthrodesis involved 14 sheep that received a fusion cage in two non-adjacent lumbar levels. For one-half of the animals, the cages were filled after impaction by injection of a CPC/blood composite in one level and the blood-free CPC reference in the other. For the other seven animals, the CPC/blood composite was compared to bone autograft.Results: SEM and histological examination of the explants gave evidence of a good osteointegration for the three types of implanted materials. For the first group, no spine fusion was observed for the blood-free CPC reference while a highly successful spine fusion (6/7 cages) was present for the CPC/blood composite, three months after surgery. For the second group, successful arthrodesis was observed for 57% (4/7) of cages filled with iliac autograft, while the fusion rate was 71% (5/7) in the case of cages filled with the CPC/blood composite.Conclusion: This study reports that the observed fusion rate when filling the cage with a calcium phosphate cement mixed with autologous whole blood was at least as good as results obtained with autograft. Once placed in the intervertebral space, the cage was easily and homogeneously filled with the CPC/blood composite, and this new strategy might thus offer a less invasive option for intervertebral spine fusion.

The in vivo resorption rate of two injectable apatitic calcium phosphate cements used in clinics (Graftys® HBS and NORIAN®) was compared, using a good laboratory practice (GLP) study based on an animal model of critical-sized bone defect. To rationalize the markedly different biological properties observed for both cements, key physical features were investigated, including permeability and water-accessible porosity, total porosity measured by mercury intrusion and gravimetry, and microstructure. Due to a different concept for creating porosity between the two cements investigated in this study, a markedly different microstructural arrangement of apatite crystals was observed in the intergranular space, which was found to significantly influence both the mechanical strength and in vivo degradation of the two calcium phosphate cements.

Polarized Raman measurements were recorded on a monoclinic brushite (CaHPO4.2H2O) crystal in the 800–1200 cm−1 spectral range, corresponding to the PO stretching modes. This study is a continuation of the investigation of the phosphate stretching modes observed in polarized infrared reflectance spectra of brushite crystal. In such ionic non-centrosymmetric crystals, the splitting between the transverse and longitudinal components of the optic vibrations was observed in the polarized Raman spectra recorded at several scattering geometries. A″ symmetry modes of the brushite crystal were measured. Using a simple model based on the symmetry of the PO4 group, the Raman intensities of the stretching modes are calculated and compared with experimental bands. Copyright © 2016 John Wiley & Sons, Ltd.

Mesoporous bioactive glasses (MBGs) in the system SiO2-CaO-P2O5-Ga2O3 have been synthesized by the evaporation induced self-assembly method and subsequent impregnation with Ga cations. Two different compositions have been prepared and the local environment of Ga(III) has been characterized using 29Si, 71Ga and 31P NMR analysis, demonstrating that Ga(III) is efficiently incorporated as both, network former (GaO4 units) and network modifier (GaO6 units). In vitro bioactivity tests evidenced that Ga-containing MBGs retain their capability for nucleation and growth of an apatite-like layer in contact with a simulated body fluid with ion concentrations nearly equal to those of human blood plasma. Finally, in vitro cell culture tests evidenced that Ga incorporation results in a selective effect on osteoblasts and osteoclasts. Indeed, the presence of this element enhances the early differentiation towards osteoblast phenotype while disturbing osteoclastogenesis. Considering these results, Ga-doped MBGs might be proposed as bone substitutes, especially in osteoporosis scenarios., 35 pages, 8 figures

International audience; Abstract: Postoperative pain after bone reconstruction is a serious complication that could jeopardize the global success of a surgery. This pain must be controlled and minimized during the first 3 to 4 postoperative days to prevent it from becoming chronic. In this study, a critical-size bone defect was created at the femoral distal end of rats and filled by an injectable calcium phosphate cement (CPC) loaded or not with local anesthetics (bupivacaine or ropivacaine). A functional evaluation of the gait was performed using the CatWalk system to compare the postoperative pain relief enhanced by the different CPCs after such a bone filling surgery. The results demonstrated significant pain relief during the short-term postoperative period, as shown by the print area and intensity parameters of the operated paw. At 24 hours, the print area decreased by 65%, 42%, and 24%, and the intensity decreased by 25%, 9%, and 1% for unloaded, ropivacaine-loaded, and bupivacaine-loaded CPCs, respectively, compared with the preoperative values. Bupivacaine-loaded CPC provided an earlier return to full functional recovery than ropivacaine-loaded CPC. Moreover, the CPCs retained their biologic and mechanical properties. For all these reasons, anesthetic-loaded CPCs could be part of the global pain management protocol after bone reconstruction surgery such as iliac crest bone grafting procedures.Perspective: Bupivacaine-loaded CPC provided an earlier return to full gait function than ropiva-caine-loaded CPC, with preserved bone filling properties. Such analgesic CPCs deserve further in vivo investigation and may be part of the global pain management protocol after bone reconstruction or bone augmentation surgery such as iliac crest bone grafting.

International audience; Management of postoperative pain following bone surgery includes administration of local anesthetics (LAs). Smart delivery systems, including triggered systems, have been designed to provide a continuous release of LA in situ. However, these systems can provide a high level of LA locally. This review will examine the state-of-the-art regarding the LA delivery systems optimized for management of postoperative pain in bone surgery and will discuss the potential adverse effects of LAs on the overall pathways of bone healing, including the inflammation response phase, hemostasis phase, tissue repair phase and remodeling phase. There is a clinical need to document these effects and the potential impacts on the clinical outcome of the patient.

International audience; Two commercial formulations of apatitic calcium phosphate cements (CPCs), Graftys Ò Quickset (QS) and Graftys Ò HBS (HBS), similar in composition but with different initial setting time (7 and 15 min, respectively), were combined to ovine whole blood. Surprisingly, although a very cohesive paste was obtained after a few minutes, the setting time of the HBS/blood composite dramatically delayed when compared to its QS analogue and the two blood-free references. Using solid state NMR, scanning electron microscopy and high frequency impedance measurements, it was shown that, in the particular case of the HBS/blood composite, formation of a reticulated and porous organic network occurred in the intergranular space, prior to the precipitation of apatite crystals driven by the cement setting process. The resulting microstructure conferred unique biological properties to this material upon implantation in bone defects, since its degradation rate after 4 and 12 weeks was more than twice that for the three other CPCs, with a significant replacement by newly formed bone. Statement of Significance A major challenge in the design of bone graft substitutes is the development of injectable, cohesive, resorbable and self-setting calcium phosphate cement (CPC) that enables rapid cell invasion with initial mechanical properties as close as bone ones. Thus, we describe specific conditions in CPC-blood composites where the formation of a 3D clot-like network can interact with the precipitated apatite crystals formed during the cement setting process. The resulting microstructure appears more ductile at short-term and more sensitive to biological degradation which finally promotes new bone formation. This important and original paper reports the design and in-depth chemical and physical characterization of this groundbreaking technology.

An injectable purely apatitic calcium phosphate cement (CPC) was successfully combined to a water-soluble radiopaque agent (i.e., Xenetix

We previously reported that biphasic calcium phosphate (BCP) microparticles embedded in a blood clot induces ectopic bone formation in mice and repairs a critical femoral defect in rat. The present pilot study aimed to evaluate in dog and in two models of large defects the efficacy of this composite named "blood for reconstruction of bone" (BRB). We show here that BRB is a cohesive biomaterial easy to prepare from dog autologous blood and to mold to fill large bone defects. First in a model of cylindrical femoral condyle defect, the BRB was compared with BCP particles alone. After 8 weeks, this revealed that the amount of mature bone was slightly and significantly higher with BRB than with BCP particles. Second, in a model consisting in a 2 cm-long critical interruptive defect of the ulna, the BRB was compared with autologous bone. After 6 months, we observed that implantation of BRB can induce the complete reconstruction of the defect and that newly formed bone exhibits high regenerative potential. Comparison with the results obtained with autologous bone grafting strongly suggests that the BRB might be an efficient biomaterial to repair large bone defects, as an alternative or in addition to autologous bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1842-1850, 2018.

International audience; Calcium phosphate (CaP)-based biomaterials are commonly used in bone reconstructive surgery to replace the damaged tissue, and can also serve as vectors for local drug delivery. Due to its inhibitory action on osteoclasts, the semi-metallic element gallium (Ga) is used for the systemic treatment of disorders associated with accelerated bone resorption. As it was demonstrated that Ga could be incorporated in the structure of CaP biomaterials, we investigated the biological properties of Ga-loaded CaP biomaterials. Culturing bone cells on Ga-CaP, we observed a decrease in osteoclast number and a downregulation of late osteoclastic markers expression, while Ga-CaP upregulated the expression of osteoblastic marker genes involved in the maturation of bone matrix. We next investigated in vivo bone reconstructive properties of different Ga-loaded biomaterials using a murine bone defect healing model. All implanted biomaterials showed a good osseointegration into the surrounding host tissue, accompanied by a successful bone ingrowth and bone marrow reconstruction, as evidenced by histological analysis. Moreover, quantitative micro-computed tomography analysis of implants revealed that Ga enhanced total defect filling. Lastly, we took advantage for the first time of a particular mode of non-linear microscopy (second harmonic generation) to quantify in vivo bone tissue reconstruction within a CaP bone substitute. By doing so, we showed that Ga exerted a positive impact on mature organized collagen synthesis. As a whole, our data support the hypothesis that Ga represents an attractive additive to CaP biomaterials for bone reconstructive surgery. Copyright © 2017 John Wiley & Sons, Ltd.

In this work, we show that it is possible to overcome the limitations of solid-state MRI for rigid tissues due to large line broadening and short dephasing times by combining Magic Angle Spinning (MAS) with rotating pulsed field gradients. This allows recording ex vivo 31P 3D and 2D slice-selected images of rigid tissues and related biomaterials at very high magnetic field, with greatly improved signal to noise ratio and spatial resolution when compared to static conditions. Cross-polarization is employed to enhance contrast and to further depict spatially localized chemical variations in reduced experimental time. In these materials, very high magnetic field and moderate MAS spinning rate directly provide high spectral resolution and enable the use of frequency selective excitation schemes for chemically selective imaging. These new possibilities are exemplified with experiments probing selectively the 3D spatial distribution of apatitic hydroxyl protons inside a mouse tooth with attached jaw bone with a nominal isotropic resolution nearing 100 µm.

International audience; The recent progress in oncologic management of patients with localized cancer or metastatic disease has permitted a significant improvement in life expectancy. Nevertheless, bone metastases and their consequent skeletal-related events (SREs) are still associated with unfavorable prognosis and greatly affect quality of life. Global management of these bone metastases includes traditional local approaches (surgery, radiotherapy, etc.) and systemic administration of chemotherapeutic agents. This review focuses on treatments specific for bone metastases and, in particular, on inhibitors of bone resorption that are effective for preventing and delaying the development of SREs.

International audience; Autologous bone graft is considered as the gold standard in bone reconstructive surgery. However, the quantity of bone available is limited and the harvesting procedure requires a second surgical site resulting in severe complications. Due to these limits, scientists and clinicians have considered alternatives to autologous bone graft. Calcium phosphates (CaPs) biomaterials including biphasic calcium phosphate (BCP) ceramics have proven efficacy in numerous clinical indications. Their specific physico-chemical properties (HA/TCP ratio, dual porosity and subsequent interconnected architecture) control (regulate/condition) the progressive resorption and the bone substitution process. By describing the most significant biological responses reported in the last 30 years, we review the main events that made their clinical success. We also discuss about their exciting future applications as osteoconductive scaffold for delivering various bioactive molecules or bone cells in bone tissue engineering and regenerative medicine. Statement of Significance Nowadays, BCPs are definitely considered as the gold standard of bone substitutes in bone reconstructive surgery. Among the numerous clinical studies in literature demonstrating the performance of BCP, Passuti et al. and Randsford et al. studies largely contributed to the emergence of the BCPs. It could be interesting to come back to the main events that made their success and could explain their large adhesion from scientists to clinicians. This paper aims to review the most significant biological responses reported in the last 30 years, of these BCP-based materials. We also discuss about their exciting future applications as osteoconductive scaffold for delivering various bioactive molecules or bone cells in bone tissue engineering and regenerative medicine.

International audience; Gallium (Ga) is a semi-metallic element that displays antitumor, antiresorptive, anti-inflammatory and immunosuppressive properties. Among all these properties, antitumor properties were the most extensively applied and have shown efficacy in treatment of Paget's disease, myeloma and hypercalcemia in cases of malignancy. By contrast, no clinical trials have been conducted in prevention and/or treatment of osteoporosis. In this article I focus on Ga effects on bone tissue and cells, as well as on molecular mechanisms governing Ga internalization into cells. Eventually, the potential of Ga as an antiosteoporotic agent is discussed.

International audience; Staphylococcusaureus, a major responsible microorganism of osteomyelitis, represents a challenge to treat because of the poor penetration of antibiotics in bone and increasing minimum inhibitory concentrations (MICs) to glycopeptides. The calcium-deficient apatites (CDA), closer to the biological components found in bone and other calcified tissues, have osteoconductive properties. So, to process severe osseous infections, CDA can be used to deliver in the infectious site antibiotics like linezolid. The acute experimental osteomyelitis due to methicillin-resistant Staphylococcusaureus (MRSA) was induced in rabbit’s femurs and surgery mimicking human procedures was performed at day three after inoculation. Animals were randomly assigned to treatment groups: L(IV)[4-day linezolid IV infusion, human-equivalent dose of 10 mg/kg/12 h], L(CDA50%) (100 mg CDA with linezolid 500 μg/mg) and L (CDA50%) +L (IV). Surviving bacteria were counted in bonemarrow (BM) and bone (Bo) at day 3 (before treatment), day 7 (4-day treatment) or day 17 (14-day treatment). L(iv)was effective after a 4-day treatment with a log[10]CFU/g decrease of −2.63±1.92 and −2.17±1.58 in bone marrowand bone, respectively. CDA loaded with linezolid enhance the efficacy of the IV linezolid regimen by more than one log[10]CFU/g.

A novel gallium-doped -tricalcium phosphate (-TCP) ceramics with rhombohedral structure (R3c space group) can be obtained by the addition of a Ga precursor in the typical reaction protocols used for the preparation of -TCP. It was found, from the refinement of their XRD patterns, that the incorporation of Ga in the -TCP network occurs by substitution of one of the five calcium sites, while occupation of another Ca site decreases in inverse proportion to the gallium content in the structure. A decrease of the unit cell volume is observed with increasing gallium content, together with improved mechanical properties. Indeed, the compressive strength of these new bioceramics is enhanced in direct proportion of the Ga content, up to a 2.6-fold increase as compared to pure -TCP.

Several substituted â-tricalcium phosphates have been prepared with different cations (monovalent, divalent and trivalent) and at various levels of substitution. Structural investigations have proved that fewer than ~10% wt substitution, the substituted compounds are isostructural to â- TCP, leading to solid solutions. These samples have been characterized by infrared and Raman spectroscopies. The vibrational spectra show mainly the bands related to the vibrations of PO43- tetrahedrons present in the structure. As Raman scattering and infrared absorption are local probes, the bands are sensitive to the local environment of the distinct tetrahedrons, related to the site of substitution and to the nature of the cations.

Calcium phosphate cements have been widely used in medical and dental applications for decades. However, their intrinsic high brittleness and low strength prohibit their use in many stress-bearing locations, which would require an improvement in mechanical properties. The influence of microstructural parameters on the latter have nevertheless barely been investigated in a systematic manner. Furthermore, due to their inferior reproducibility which is sensitive to the variations introduced during the preparation and the way they are measured, mechanical properties of CPC cannot simply be characterized using mean values, but request a more reliable method. In this aim, apatite cements have been fabricated by mixing liquid and powders based on α-TCP (α-tricalcium phosphate), and their mechanical properties have been measured (Young’s modulus, fracture toughness, compressive strength and flexural strength) in wet environment as a function of various parameters (liquid-to-powder ratio; amount and morphology of porosity, including macropores created by porogens). The reliability of compressive strength of CPC is analysed using Weibull statistics. The above results indicate that fabrication and microstructural features of CPC significantly influence their mechanical properties.

The dynamic, fluid-filled RCCS! bioreactor, based on the operational principle of relative micro-gravity condition, is a new efficient system devised to perform long-term three-dimensional (3D) culture of cells and tissue explants. In the present study, we evaluated the impact of the RCCS!-produced hydrodynamic condition on an innovative 3D in vitro model of osteocytes’ differentiation and bone matrix formation, obtained by coupling primary human osteoblasts with biphasic calcium phosphate (BCP) ceramic particles.

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.

The use of bone grafts is constantly increasing, their employ is principally linked to bone trauma, prosthesis revision surgery, and arthrodesis applications. In the case of biological bone grafts and depending on the origin of the graft, these grafts are classified as autografts, allografts, or xenografts. The autograft is the most commonly used and corresponds to a fresh bone graft harvesting taken from a second operating site, i.e. iliac crest, parietal bone, tibial plateaux or the fibula. The autograft has many advantages in terms of biotolerance and osteogenic potential, which justify its widespread utilization in reconstructive surgery[1]. From a practical point of view, sampling and grafting take place during the same surgical session. However, the longer exposure to the anesthetic and the surgical operation per se increases the risk of complications. For example, this procedure results in sever post-operation pain, iliac hernias, or even haemorrhages[2]. Furthermore, the volume of the bone graft taken is generally limited to 20 cm3. In the case of allografts, it generally leads to an acute inflammatory reaction which participates to the resorption/substitution process. Xenografts are less used since it involves a donor and a recipient from different species.

Background and purpose: Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer-related hypercalcemia and Paget's disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro. Experimental approach: In different osteoclastic models [osteoclasts isolated from long bones of neonatal rabbits (RBC), murine RAW 264.7 cells and human CD14-positive cells], we have performed resorption activity tests, staining for tartrate resistant acid phosphatase (TRAP), real-time polymerase chain reaction analysis, viability and apoptotic assays. We also evaluated the effect of Ga on osteoblasts in terms of proliferation, viability and activity by using an osteoblastic cell line (MC3T3-E1) and primary mouse osteoblasts. Key results: Gallium dose-dependently (0–100 µM) inhibited the in vitro resorption activity of RBC and induced a significant decrease in the expression level of transcripts coding for osteoclastic markers in RAW 264.7 cells. Ga also dramatically reduced the formation of TRAP-positive multinucleated cells. Ga down-regulated in a dose-dependant manner the expression of the transcription factor NFATc1. However, Ga did not affect the viability or activity of primary and MC3T3-E1 osteoblasts. Conclusions and implications: Gallium exhibits a dose-dependent anti-osteoclastic effect by reducing in vitro osteoclastic resorption, differentiation and formation without negatively affecting osteoblasts. We provide evidence that this inhibitory mechanism involves down-regulation of NFATc1 expression, a master regulator of RANK-induced osteoclastic differentiation.

Macroporous biphasic calcium phosphate (BCP) bioceramics, for bone substitution applications, have been synthesized, cold isostatically pressed and pressureless sintered, using naphthalene particles as a porogen to produce macropores. The resulting materials are mixtures of P-tricalcium phosphate and hydroxyapatite with various microporosities and macroporosities. Mechanical properties (Young's modulus, compressive strength and fracture toughness) were measured on specimens over the widest attainable ranges of porosities, and compared to previously proposed analytical models and hypotheses. These models describe the evolution of the mechanical properties as functions of macroporosity and microporosity separately, the strength model considering macropores as critical flaws in the ceramic. Results show that the presence of macropores strongly influences the critical flaw size, but the latter appears to increase with macroporosity. This phenomenon can be explained by the presence of clusters of macropores, acting as critical flaws, becoming larger as macroporosity increases. (C) 2009 Elsevier Ltd. All rights reserved.

In the domain of implantable materials, the porosity and pore size distribution of a material in contact with bone is decisive for bone ingrowth and thus the control of the porosity is of great interest. The use of a new porogen agent, i.e. sucrose is proposed to create a porosity in biphasic calcium phosphate blocks. The technological procedure is as follows: sucrose and mineral powder are mixed, then compressed by isostatic compression and sintering finally eliminates sucrose. Blocks obtained were compared to a manufactured product: Triosite (R) (Zimmer, Etupes, France) which porosity is created through a naphthalene sublimation process. Results have shown that the incorporation of sucrose allows the preparation of porous blocks with controlled porosity varying from 40 to 80% and with macro-, meso- and microporosity characteristics depending on the percentage of sucrose added as well as on the granulometry of both sucrose and mineral powder. (C) 2009 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

The fixation of an orthopedic implant depends strongly upon its initial stability. Peri-implant bone may resorb shortly after the surgery. This resorption is directly followed by new bone formation and implants fixation strengthening, the so-called secondary fixation. If the initial stability is not reached, the resorption continues and the implant fixation weakens, which leads to implant loosening. Studies with rats and dogs have shown that a solution to prevent peri-implant resorption is to deliver bisphosphonate from the implant surface. The aims of the study were, first, to develop a model of bone remodeling around an implant delivering bisphosphonate, second, to predict the bisphosphonate dose that would induce the maximal peri-implant bone density, and third to verify in vivo that peri-implant bone density is maximal with the calculated dose. The model consists of a bone remodeling equation and a drug diffusion equation. The change in bone density is driven by a mechanical stimulus and a drug stimulus. The drug stimulus function and the other numerical parameters were identified from experimental data. The model predicted that a dose of 0.3 microg of zoledronate on the implant would induce a maximal bone density. Implants with 0.3 microg of zoledronate were then implanted in rat femurs for 3, 6 and 9 weeks. We measured that peri-implant bone density was 4% greater with the calculated dose compared to the dose empirically described as best. The approach presented in this paper could be used in the design and analysis processes of experiments in local delivery of drug such as bisphosphonate.

The integration of drugs and devices is a growing force in the medical industry. The incorporation of pharmaceutical products not only promises to expand the therapeutic scope of device technology but to access combination products whose therapeutic value stem equally from both the structural attributes of the device and the intrinsic therapy of the drug. For example, the orthopedic industry is exploring drug-coated hip, knee and bone reconstruction implants capable of promoting healing as an added therapeutic benefit for device recipients. In this context, the drug is eluted locally, being targeted in a specific site of interest, thus offering a convenient strategy to avoid adverse effects commonly observed for systemic treatments of some diseases, as an additional benefit. In addition, these new technologies are generally well adapted to the development of minimally invasive surgery for their implantation. In this context, given the wide use of calcium phosphates (CaPs) and bisphosphonates (BPs) for the therapy of bone-related affections, there was great interest to investigate the chemistry taking place when combining the two systems since: (i) it could provide better insight in the mechanism of BP fixation on bones (ii) such combination could act as efficient BP delivery systems when implanted in bone defects.

In this study, we propose a simple and effective strategy to prepare injectable macroporous calcium phosphate cements (CPCs) by syringe-foaming via hydrophilic viscous polymeric solution, such as using silanized-hydroxypropyl methylcellulose (Si-HPMC) as a foaming agent. The Si-HPMC foamed CPCs demonstrate excellent handling properties such as injectability and cohesion. After hardening the foamed CPCs possess hierarchical macropores and their mechanical properties (Young's modulus and compressive strength) are comparable to those of cancellous bone. Moreover, a preliminary in vivo study in the distal femoral sites of rabbits was conducted to evaluate the biofunctionality of this injectable macroporous CPC. The evidence of newly formed bone in the central zone of implantation site indicates the feasibility and effectiveness of this foaming strategy that will have to be optimized by further extensive animal experiments.A major challenge in the design of biomaterial-based injectable bone substitutes is the development of cohesive, macroporous and self-setting calcium phosphate cement (CPC) that enables rapid cell invasion with adequate initial mechanical properties without the use of complex processing and additives. Thus, we propose a simple and effective strategy to prepare injectable macroporous CPCs through syringe-foaming using a hydrophilic viscous polymeric solution (silanized-hydroxypropyl methylcellulose, Si-HPMC) as a foaming agent, that simultaneously meets all the aforementioned aims. Evidence from our in vivo studies shows the existence of newly formed bone within the implantation site, indicating the feasibility and effectiveness of this foaming strategy, which could be used in various CPC systems using other hydrophilic viscous polymeric solutions.

The reaction of aqueous Zoledronate solutions with β-tricalcium phosphate resulted in the precipitation of a crystalline Zoledronate complex on the surface of the calcium phosphate. This complex (C...

The infrared and Raman spectra of Na substituted β-TCP are presented for various levels of substitution. The influence of Na content on main vibrational modes appears significant and among them the ν1 symmetric stretching band is very sensitive to PO4 3- tetrahedrons environment. Thus, this mode was particularly investigated and has been decomposed using Lorentzian shapes components. We assigned the calculated components to the different types of PO4 3- tetrahedrons present in the crystalline structure.

Macroporous biphasic calcium phosphate bioceramics, for use as bone substitutes, have been fabricated by cold isostatic pressing and conventional sintering, using naphthalene particles as a porogen to produce macropores. The resulting ceramics, composite materials made of hydroxyapatite and β-tricalcium phosphate containing various macroporosities and microporosities, have been submitted to compression and three-point bending tests. The mechanical tests performed on the sintered ceramics tend to validate the modelling approach and its hypothesis, i.e. the material can be considered as a microporous matrix containing isolated macropores, and the critical flaw is a macropore.

Macroporous biphasic calcium phosphate bioceramics, for use as bone substitutes, have been fabricated by cold isostatically pressing and conventional sintering, using naphtalen or saccharose particles to produce macropores. The resulting ceramics, composite materials made of hydroxyapatite and b-tricalcium phosphate containing ~ 45% macropores and ~ 25% micropores, have been submitted to compression and three-point bending tests, toughness tests by single-edge-notched-bending, and spherical indentation tests. A new model is established to describe mechanical properties as a function of the amount and morphology of porosity, and propositions are made to optimise the fabrication procedure. Finally, those highly porous ceramics, although very brittle, exhibit a damage-tolerant contact behaviour, due to the compaction of the porous body under the indenter.

An injectable bone substitute (IBS) made of a suspension of calcium phosphate ceramic was used to filled dental root canal after removing of canal pulp. Compared with current filling materials, which are toxic to periapical tissues, calcium phosphate materials, due to their biocompatibility and bioactive properties, may be viewed as possible alternatives. The aim of this study was first to determine if an injectable bone substitute could be used to obtain further healing of apical tissue by the neoformation of a mineralized barrier. In the next step, the paper will focus on rheological measurements as a tool for physical characterisation and on the improvement of the injection technique. Rheology concerns the flow and deformation of the suspension and, in particular, its behaviour in the transient area between solids and fluids. The results showed that injection is possible with a good level of BCP granules at the end of the root dental canal with extracted tooth. Other experiments with other animal models closer to a Human model have to be performed before human trials.

Interconnection, one of the main structural features of macroporous calcium-phosphate ceramics, contributes to the biological and physicochemical properties of bone substitutes. As no satisfactory method exists for evaluating this feature, analysis was performed to determine the permeability, tortuosity, and equivalent diameter of interconnecting channels, that is the parameters that appear to be representative of the way pores are linked. The testing of two ceramics with similar porosity levels revealed important differences in all three interconnection parameters. One ceramic showed poor permeability, corresponding to a small equivalent diameter for interconnecting channels in conjunction with a high tortuosity factor, while the other displayed high permeability, a large diameter for interconnecting channels, and a low tortuosity factor. The methodology used, which can be applied to the quantification of interconnection in all calcium-phosphate ceramics, constitutes the first step in a complete study of the role of this feature in cellular colonization of the ceramic, matrix dissolution, and drug release from the calcium-phosphate matrix.

International audience; The aim of the present study was to assess the efficacy of a ready-to-use injectable bone substitute on the prevention of alveolar ridge resorption after tooth extraction. Maxillary and mandibular premolars were extracted from 3 Beagle dogs with preservation of alveolar bone. Thereafter, distal sockets were filled with an injectable bone substitute (IBS), obtained by combining a polymer solution and granules of a biphasic calcium phosphate (BCP) ceramic. As a control, the mesial sockets were left unfilled. After a 3 months healing period, specimens were removed and prepared for histomorphometric evaluation with image analysis. Histomorphometric study allowed to measure the mean and the maximal heights of alveolar crest modifications. Results always showed an alveolar bone resorption in unfilled sockets. Resorption in filled maxillary sites was significantly lower than in control sites. Interestingly, an alveolar ridge augmentation was measured in mandibular filled sockets including 30% of newly-formed bone. It was concluded that an injectable bone substitute composed of a polymeric carrier and calcium phosphate can significantly increase alveolar ridge preservation after tooth extraction.

Biphasic calcium phosphate (BCP) bioceramics, for use as resorbable bone substitutes, containing both isolated macropores and interconnected micropores, have been fabricated by sintering, using naphtalen particles as a porogen to produce macropores. The resulting ceramics contain ~ 45% macropores and various amounts of microporosity. Mechanical properties (compression and bending strength, toughness and hardness) have been measured and modeled by combining two approaches, at two different scales: the one describes the mechanical properties of a partly sintered stacking of grains, supposed to account for the interconnected microporosity, the other one holds in the case of closed and isolated macropores within a continuous matrix. The material is then represented as a quasi-continuous matrix containing macropores, the matrix being itself microporous. The model also considers that fracture always initiates on a macropore, which allows to set a correspondence between fracture toughness and fracture stress equations. The mechanical tests performed on the sintered ceramics tend to validate the modeling approach.

Macroporous biphasic calcium phosphate bioceramics, for use as bone substitutes, have been fabricated by cold isostatic pressing and conventional sintering, using naphtalen particles as a porogen to produce macropores. The resulting ceramics, composite materials made of hydroxyapatite and β-tricalcium phosphate (TCP) containing ∼45% macropores and with various microporosities, have been submitted to compression and three-point bending tests, toughness tests by single-edge-notched-bending (SENB), and spherical indentation tests. By combining two approaches at two different scales, one for closed porosity and one for open porosity, a model is established to describe mechanical properties as a function of the amount and morphology of porosity. The model assumes a quasi-continuous matrix containing macropores, the matrix being itself microporous, and considers that fracture always initiates on a macropore. The preliminary mechanical tests performed on the sintered ceramics tend to validate the modelling approach.

The role of adrenoceptor subtypes was studied in rat brown adipose tissue (BAT). The type II 5'-deiodinase (5'DII) was activated in response to simultaneous stimulation by beta3- and alpha1-adrenergic agonists, BRL 37344 or CGP 12177, and cirazoline, in brown adipocytes. Inhibition of the alpha1- and beta-adrenergic phenylephrine-stimulated 5'DII activity was obtained by the alpha1-adrenergic antagonists in the order of prazosin/= wb 41015-methylurapidil. In comparison, the binding of [3H]prazosin to rat BAT plasma membranes was inhibited by alpha1-adrenergic antagonists in the order of prazosinWB 4101 = benoxathian5-methylurapidil. Although the order of the alpha1-adrenergic competition seemed to be rather typical for the alpha1B-adrenergic receptors, a molecular analysis on adrenoceptor mRNAs should be made to confirm the exact alpha1-adrenergic subtypes at the level of brown adipocytes, since the possibility of a mixture of different receptor subtypes in brown fat cells and/or tissue may interact with the pharmacological characterization. Thus, specific alpha1- and beta-adrenoceptor subtypes participate in the regulation of 5'DII activity in the rat brown adipocytes, and therefore, an impaired alpha1- and beta-adrenergic co-work may be involved in a defective BAT function, e.g., in obese Zucker rats, too. An interesting possibility is that the decreased number of alpha1-adrenoceptors in the BAT of obese Zucker rats is due to the decrease in the alpha1B-adrenoceptor subtype which would further be involved especially in the regulation of BAT 5'DII activity.

This in vivo study investigated the efficiency of an injectable calcium phosphate bone substitute (IBS) for bone regenerative procedures through non-destructive three-dimensional (3D) micro-tomographic (microCT) imaging, biomechanical testing with a non-destructive micro-indentation technique and 2D scanning electron microscopy (SEM) analysis. The injectable biomaterial was obtained by mixing a biphasic calcium phosphate (BCP) ceramic mineral phase and a cellulosic polymer. The BCP particles were 200-500 microm or 80-200 microm in diameter. The injectable material was implanted for 6 weeks into critical-sized bone defects at the distal end of rabbit femurs. Extensive new bone apposition was noted with both 2D and 3D techniques. Micro-CT showed that newly formed bone was in perfect continuity with the trabecular host bone structure and demonstrated the high interconnectivity of the restored bone network. For both IBS formulations, SEM and microCT gave very close measurements. The only detected significant difference concerned the amount of newly formed bone obtained with IBS 80-200 that appeared significantly higher with microCT analysis than with SEM (p=0.00007). Student t-tests did not show any significant difference in the amount of newly formed bone and remaining ceramic obtained from microCT analysis or SEM. Regression analysis showed satisfactory correlation between both the amount of newly formed bone and remaining ceramic obtained from microCT or SEM. For IBS 200-500, the newly formed bone rate inside the defect was 28.0+/-5.2% with SEM and yield strength of the samples was 18.8+/-5.4 MPa. For IBS 80-200, the newly formed bone rate inside the defect was 31.7+/-5.1% with SEM and yield strength of the samples was 26.8+/-4.5 MPa. Yield strength appeared well correlated with the amount of newly formed bone, specially observed with microCT. This study showed the ability of non-destructive techniques to investigate biological and mechanical aspects of bone replacement with injectable biomaterials.

In this study a biodegradable polymer microspheres were used to make an alpha-TCP calcium phosphate bone cement macroporous. The biodegradable polymer microspheres were synthesised at the laboratory and a sieve fraction of this microspheres ranging between 100 and 300 microns was incorporated in a calcium phosphate cement, where the powder contained a-Ca3(PO4)2 (a-TCP). The microspheres content of the cement was 10 %. The cement pastes were prepared at liquid-to-powder ratios from 0.32 to 0.40 ml/g. X-ray diffraction and Infra-red spectroscopy was performed to analyse the reaction on powdered samples that were let to set at different times in a Ringer’s solution at body temperature. Mercury porosimetry has been used at the main method for porosity measurements. Scanning electron microscopy (SEM) was performed on fractured samples fro microstructural analysis. The microspheres seemed to retard slightly the cement setting and to reduce progressively the compressive strength after aging in aqueous solutions which were continuously refreshed. SEM pictures and XRD patterns of the samples after 1 and 4 weeks of aging showed that the microspheres were dissolved progressively. Mercury porosimetry measurements assessed at different times of aging showed an improvement in the total porosity and the pore diameter of the set material and confirmed SEM and XRD results.

Bone invasion is common in case of Squamous Cell Carcinomas (SCC) of the upper aero-digestive tract. Radiotherapy is required in addition to large surgical tumor removal. This treatment usually generates irreversible injuries on the reparation properties of the tissues, especially on bone. The quality of life of patients undergoing major surgery and radiotherapy in maxillary and mandible areas is reduced, but could be improved by bone reconstruction. The aim of this study was to evaluate the bone reconstruction possibilities by Macroporous Biphasic Calcium-Phosphate (MBCPÔ). The MBCP substitute was evaluated as granules and associated to autologous bone marrow (BM) graft in irradiated areas, in an inbreeding rodent model. Radiation sequels were created on inferior members of half of the rats. 3 weeks later, 3-mm osseous defects were created on each animal. The inbreeding model allows BM to be grafted without graft-versus-host reaction. Defects were filled either with MBCP alone, BM alone or a mixture of MBCP and BM. Six weeks after implantation, animals were sacrificed: bone repair and ceramic degradation were evaluated by qualitative and quantitative study. Results showed that bioceramics were well osteointegrated. Filling the defects with BM alone showed a significant increased of newly-formed bone formation but only after irradiation, whereas filling defects with MBCP alone increased new-bone formation only without previous irradiation. Associating MBCP to BM provided the best new-bone formation rates after irradiation. Degradation of the ceramic was the most important in case of BM grafting. This study demonstrated that BM added to MBCP constitute an appropriate material to be considered in case of bone defect occurring in irradiated tissue, and could be foreseen for use after bone removal for oncologic obligations.

International audience; One type of potent aminobisphosphonate (Zoledronate) has been chemically associated onto b-tricalcium phosphate [b-TCP] and calcium deficients apatite [CDA]. Two different association modes have been observed, according to the nature of the Calcium Phosphate [CaP] support and/or the initial concentration of the Zoledronate solution. b-TCP appears to promote Zoledronate-containing crystals formation. On the other hand, at concentrations < 0.05 mol.L-1 CDA seems to undergo chemisorption of the drug through a surface adsorption process, due to PO3 for PO4 exchange, which is well described by Freundlich equations. At concentrations > 0.05 mol.L-1, crystalline needles of a Zoledronate complex form onto the CDA surface. The ability of CDA to release Zoledronate, resulting in the inhibition of osteoclastic activity, was shown using a specific in vitro bone resorption model.

The present study investigates vancomycin microencapsulation in biodegradable PLAGA microparticles. To optimize encapsulation efficiency by the double emulsion (w/o/w) solvent evaporation/extraction process, two parameters were studied: surfactant (Span 80) rate and external aqueous phase saturation. In vitro dissolution studies, laser granulometry and scanning electron microscopy were performed to characterize the microparticles. The best results were obtained by stabilizing the first emulsion with 0.5% Span 80 and saturating the external phase with sodium chloride. Such parameters allowed a 95% drug encapsulation efficiency. This process yielded round microparticles with a mean diameter of approximately 170 microm and presenting a smooth surface without any pores. Moreover, this formulation induces a sustained drug release at a constant rate over a period of 10 days. Such materials could be associated with biphasic calcium phosphate granules to form an antibiotic-loaded injectable bone substitute offering a long-term activity in situ.

This paper is aimed at describing the evolution of newly formed bone dens ity and micro- architecture after implantation of an Injectable Bone Substitute ( IBS). Cancellous bone growth (rabbit) is described as dependent of IBS formulation parameters. Pos sible improvements to the IBS formulation are finally discussed.