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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.

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.

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.

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.

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.

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.

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.

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...

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.

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.

International audience; Different possible options were investigated to combine an apatitic calcium phosphate cement with gal-lium ions, known as bone resorption inhibitors. Gallium can be either chemisorbed onto calcium-deficient apatite or inserted in the structure of b-tricalcium phosphate, and addition of these gallium-doped components into the cement formulation did not significantly affect the main properties of the biomaterial, in terms of injectability and setting time. Under in vitro conditions, the amount of gal-lium released from the resulting cement pellets was found to be low, but increased in the presence of osteoclastic cells. When implanted in rabbit bone critical defects, a remodeling process of the gallium-doped implant started and an excellent bone interface was observed. Statement of Significance The integration of drugs and materials is a growing force in the medical industry. The incorporation of pharmaceutical products not only promises to expand the therapeutic scope of biomaterials technology but to design a new generation of true combination products whose therapeutic value stem equally from both the structural attributes of the material and the intrinsic therapy of the drug. In this context, for the first time an injectable calcium phosphate cement containing gallium was designed with properties suitable for practical application as a local delivery system, implantable by minimally invasive surgery. This important and original paper reports the design and in-depth chemical and physical characterization of this groundbreaking technology.

International audience; This study evaluated the in vivo behavior of an injectable calcium phosphate bone cement implanted in bone defects at the distal end of rabbit femora. After 3 weeks, samples were harvested and processed for undecalcified sectioning. Scanning electron microscopy, transmission electron microscopy, and Fourier transform infrared microspectroscopy showed direct contact of bone and cement without soft tissue interposition, biocompatibility, and bioactivity with osteoconductive properties.

Despite the increasing number of techniques for the preservation of bone ridges after dental avulsion, no precise evaluation of alveolar filling has been performed to date. The criteria of available measurement techniques (probes, retroalveolar or panoramic radiography, and lateral teleradiography) are not sufficiently reliable and precise. This study investigated the reliability of evaluation based on CT images in comparison with retroalveolar radiography (the most precise radiographic technique, providing standardised images), direct measurements, and images obtained in scanning electron microscopy. After a preliminary investigation ex vivo, a study was performed in vivo on three beagles. Mandibular premolars were extracted, and the corresponding alveoli were filled with an injectable bone substitute composed of a calcium phosphate mineral load associated with hydroxypropyl methylcellulose. Measurements performed on CT images relative to visual and automatic detection of density changes and studies of density curves provided better precision than those obtained by retroalveolar radiography.

A biphasic calcium phosphate (BCP) powder is synthesized, mixed with various amounts of naphthalene particles, pressed and sintered at different temperatures to obtain ceramics with isolated macropores (from 3% to 52% of the specimen volume) and residual microporosity resulting from an incomplete sintering (from 2% to 45% of the ceramic matrix volume). Young’s modulus is measured on a classical three-point bending setup. A good agreement is found between an existing analytical model and the experimental Young’s modulus measurements, on the overall ranges of macro- and microporosity. The Young’s modulus variation as a function of macroporosity is also calculated by a finite element method, by simulating the mechanical response of a periodic tri-dimensional repetition of elemental cubes containing various dispersions of macropores. The contribution of the shape and dispersion (in size and location) of the macropores on the decrease of Young’s modulus as a function of porosity is simulated. Calculated trends are confirmed by experimental results.

This study investigated the in vivo performance of two composite injectable bone substitutes (IBS), each with different calcium-phosphate particles granulometries [40-80 (IBS 40-80) and 200-500 microm (IBS 200-500)]. These biomaterials were obtained by associating a biphasic calcium-phosphate (BCP) ceramic mineral phase with a 3% aqueous solution of a cellulosic polymer (hydroxy-propyl-methyl-cellulose). Both materials were injected for periods of 2, 3, 8, or 12 weeks into bone defects at the distal end of rabbit femurs. Quantitative results on new bone formation, BCP resorption, and staining for tartrate-resistant acid phosphatase (TRAP) activity were studied for statistical purposes. Measurements with scanning electron microscopy and image analysis showed that the final rates of newly formed bone were similar for both tested IBS after 12 weeks of implantation. Bone colonization occurred more extensively during early implantation times for IBS 40-80 than for IBS 200-500. For the latter, BCP degradation occurred regularly throughout the implantation period, whereas it was very intensive during the first 2 weeks for IBS 40-80. Positive TRAP-stained degradation cells were significantly more numerous for IBS 40-80 than for IBS 200-500 regardless of implantation time. With the granulometry of either mineral phase, both tested IBS supported extensive bone colonization, which was greater than that previously reported for an equivalent block of macroporous BCP. The resorption-bone substitution process seemed to occur earlier and faster for IBS 40-80 than for IBS 200-500. Both tested IBS expressed similar biological efficiency, with conserved in vivo bioactivity and bone-filling ability.

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.

Calcium-phosphate ceramics used in surgery, as bone-bonding materials, are currently available in different forms (blocks, granules, etc.). However, progress in noninvasive surgery has favored the development of injectable composite materials associating a polymeric and a dusty mineral phase. The purpose of this study was the in vivo evaluation of biphasic calcium phosphate of various grains sizes, to elucidate the role of granulometries in ceramic degradation/resorption, bone ingrowth, and inflammatory reactions. Three particle sizes were compared: 10-20, 80-100, and 200-400 microm. The 10-20-microm powders provided the best bone ingrowth, with a higher resorption/degradation rate in conjunction with stronger early inflammatory reactions. The 200-400-microm powders showed higher bone ingrowth than 80-100-microm ones, indicating that properties of cell recruitment for osseous apposition and mechanical support for bone bonding may both play a role in both ingrowth mechanisms. Our results suggest that the strong inflammatory reaction in 10-20-microm granulated powders was due to a faster reversal of the resorption/apposition sequence in bone. This may have resulted from massive release of bone ingrowth factors, which implies that the brief inflammatory process observed in the early stages of implantation was favorable to the osteoconduction process.

International audience; This study reports on the incorporation of the self-setting polysaccharide derivative hydrogel (silanized-hydroxypropyl methylcellulose, Si-HPMC) into the formulation of calcium phosphate cements (CPCs) to develop a novel injectable material for bone substitution. The effects of Si-HPMC on the handling properties (injectability, cohesion and setting time) and mechanical properties (Young's modulus, fracture toughness, flexural and compressive strength) of CPCs were systematically studied. It was found that Si-HPMC could endow composite CPC pastes with an appealing rheological behavior at the early stage of setting, promoting its application in open bone cavities. Moreover, Si-HPMC gave the composite CPC good injectability and cohesion, and reduced the setting time. Si-HPMC increased the porosity of CPCs after hardening, especially the macroporosity as a result of entrapped air bubbles; however, it improved, rather than compromised, the mechanical properties of composite CPCs, which demonstrates a strong toughening and strengthening effect. In view of the above, the Si-HPMC composite CPC may be particularly promising as bone substitute material for clinic application. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

International audience; In the context of bone regeneration in an osteoporotic environment, the present study describes the development of an approach based on the use of calcium phosphate (CaP) bone substitutes that can promote new bone formation and locally deliver in situ bisphosphonate (BP) directly at the implantation site. The formulation of a CaP material has been optimized by designing an injectable apatitic cement that (i) hardens in situ despite the presence of BP and (ii) provides immediate mechanical properties adapted to clinical applications in an osteoporotic environment. We developed a large animal model for simulating lumbar vertebroplasty through a two-level lateral corpectomy on L3 and L4 vertebrae presenting a standardized osteopenic bone defect that was filled with cements. Both 2-D and 3-D analysis of microarchitec-tural parameters demonstrated that implantation of BP-loaded cement in such vertebral defects positively influenced the microarchitecture of the adjacent trabecular bone. This biological effect was dependent on the distance from the implant, emphasizing the in situ effect of the BP and its release from the cement. As a drug device combination, this BP-containing apatitic cement shows good promise as a local approach for the prevention of osteoporotic vertebral fractures through percutaneous vertebroplasty procedures.

This work reports the development of a relevant and general method based on high frequency impedance measurements, for the in situ monitoring of the alpha-tricalcium phosphate (α-TCP) to calcium-deficient hydroxyapatite (CDA) transformation which is the driving force of the hardening processes of some calcium phosphate cements (CPC) used as bone substitutes. The three main steps of the setting reaction are identified in a non invasive way through the variation of dielectric permittivity and dielectric losses. The method is also likely to characterize the effect of the incorporation of additives (i.e, antiosteoporotic bisphosphonate drugs such as Alendronate) in the CPC formulation on the hydration process. It allows not only to confirm the retarding effect of bisphosphonate by an accurate determination of setting times, but also to assess the phenomena taking place whether alendronate is added in the liquid phase or combined to the solid phase of the cement composition. Compared to the conventional Gillmore needle test, the present method offers the advantage of accurate, user-independent, in situ and real-time determination of the initial and final times of the chemical hardening process, which are important parameters when considering surgical applications.

International audience; Since their initial formulation in the 1980s, calcium phosphate cements (CPCs) have been increasingly used as bone substitutes. This article provides an overview on the chemistry, kinetics of setting and handling properties (setting time, cohesion and injectability) of CPCs for bone substitution, with a focus on their mechanical properties. Many processing parameters, such as particle size, composition of cement reactants and additives, can be adjusted to control the setting process of CPCs, concomitantly influencing their handling and mechanical performance. Moreover, this review shows that, although the mechanical strength of CPCs is generally low, it is not a critical issue for their application for bone repair - an observation not often realized by researchers and clinicians. CPCs with compressive strengths comparable to those of cortical bones can be produced through densification and/or homogenization of the cement matrix. The real limitation for CPCs appears to be their low fracture toughness and poor mechanical reliability (Weibull modulus), which have so far been only rarely studied. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Neonatal rabbit bone cells were cultured for 1 and 4 days on biphasic calcium phosphate ceramic specimens to study the osteoclastic resorption of the ceramic. Scanning electron microscopic studies after removal of stromal cells with pronase E and EDTA revealed many osteoclast-associated lacunae on the ceramic surface. The degraded crystals inside the lacunae appeared to have been dissolved by acids. Cellular degradation of the ceramic was clearly due to the extracellular process characteristic of osteoclastic resorption. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 37, 346-352, 1997.

A new injectable biomaterial for bone and dental surgery is a composite consisting of a polymer as a matrix and bioactive calcium phosphate (CaP) ceramics as fillers. The stability of the polymer is essential in the production of a ready-to-use injectable sterilized biomaterial. The purpose of this study was to detect possible polymer degradation which may have been caused by the interaction with the fillers using Fourier transform infrared spectroscopy. Composites containing CaP fillers (biphasic calcium phosphate, hydroxyapatite and peroxidized hydroxyapatite) and polymer (hydroxypropyl methyl cellulose) were prepared. To investigate the properties of the polymer, the inorganic and organic phases of the composite were separated using several extraction methods. The difficulty in separating the organic (polymer) from the mineral (CaP fillers) phases in the composite investigated in this study suggested the presence of strong interactions between the two phases. Spectra of extracted polymers showed new absorption bands of low intensities and indications that some chemical modifications of the original polymers have occurred. Results also indicated that the filler composition has an effect on the integrity of the polymer.

Biodegradation processes (both intra- and extracellular) occur immediately after implantation of calcium phosphate (CaP) ceramics. Monocytes and macrophages, among the first cells to appear in wound healing, are largely implicated in phagocytosis and may be involved in CaP degradation because of their sensitivity to secreted cytokines. We tested the behaviour of human monocytes placed on the surface of hydroxyapatite (HA) and biphasic calcium phosphate (BCP) tablets in the presence of vitamin D-3 (VD3) and interferon gamma (INF gamma). After short-term culture (6 days), morphological events were observed in histological and scanning electron microscopy studies, and degradation lacunae were characterized. There were cell prints but no pits on the HA surface, but pits appeared near cells on the BCP surface. Preincubation of biomaterial in culture medium was essential. Variations in cell morphology were observed in different culture types. In the presence of VD3, degradation was greater than in the control, and cells were more polarized and rounded. With INF gamma, cells were extensively spread out on the sample surface, and the biomaterial seemed to be extracted from the surface by cells. Thus, monocytes are clearly influenced by soluble factors (vitamins, cytokines) and could be key cells in initiating the degradation of biomaterial. (C) 1996 Elsevier Science Limited

International audience; Porous structures are becoming more and more important in biology and material science because it helps in reducing the density of the grafted material. For biomaterials, porosity also increases the accessibility of cells and vessels inside the grafted area. However, descriptors of porosity are scanty. We have used a series of biomaterials with different types of porosity (created by various porogens: fibers, beads ...). Blocks were studied by microcomputed tomography for the measurement of 3D porosity. 2D sections were re-sliced to analyze the microarchitecture of the pores and were transferred to image analysis programs: star volumes, interconnectivity index, Minkowski-Bouligand and Kolmogorov fractal dimensions were determined. Lacunarity and succolarity, two recently described fractal dimensions, were also computed. These parameters provided a precise description of porosity and pores' characteristics. Non-linear relationships were found between several descriptors e.g. succolarity and star volume of the material. A linear correlation was found between lacunarity and succolarity. These techniques appear suitable in the study of biomaterials usable as bone substitutes.

International audience; The influence of cellulose ether additives (CEAs) on the performance of final calcium phosphate cement (CPC) products is thoroughly investigated. Four CEAs were added into the liquid phase of apatitic CPCs based on the hydrolysis of α-tricalcium phosphate, to investigate the influence of both molecular weight and degree of substitution on the CPCs' properties, including handling (e.g. injectability, cohesion, washout resistance and setting time), microstructure (e.g. porosity and micromorphology) and mechanical properties (e.g. fracture toughness and compressive strength). The results showed that even a small amount of CEAs modified most of these CPCs' features, depending on the structural parameters of the CEAs. The CEAs dramatically improved the injectability, cohesion and washout resistance of the pastes, prolonged the final setting time and increased the porosity of CPCs. Moreover, the CEAs had an evident toughening effect on CPCs, and this effect become more significant with increasing molecular weight and mass fraction of CEAs, inducing a significant tolerance to damage. Overall, the molecular weight of CEAs played a major role compared to their degree of substitution in CPCs' performances.

There is a growing interest in bone tissue engineering for bone repair after traumatic, surgical or pathological injury, such as osteolytic tumor or osteoporosis. In this regard, calcium phosphate (Cap) bone substitutes have been used extensively as bone-targeting drug-delivery systems. This localized approach improves the osteogenic potential of bone substitutes by delivering bone growth factors, thus extending their biofunctionality to any pathological context, including infection, irradiation, tumor and osteoporosis. This review briefly describes the physical and chemical processes implicated in the preparation of drug-delivering CaPs. It also describes the impact of these processes on the intrinsic properties of CaPs, especially in terms of the drug-release profile. In addition, this review focuses on the potential influence of drugs on the resorption rate of CaPs. Interestingly, by modulating the resorption parameters of CaP biomaterials, it should be possible to control the release of bone-stimulating ions, such as inorganic phosphate, in the vicinity of bone cells. Finally, recent in vitro and in vivo evaluations are extensively reported. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Combining Bisphophonates (BPs) and Calcium Phosphate Cement (CPC) to form a new medical device for the local treatment of Osteoporosis is a promising challenge. Our formulation was optimized from an apatitic-type CPC and we have shown that the best solution consists in introducing the bisphosphonate (Alendronate) in the calcium deficient apatite (CDA), a solid component of the cement, through a chemical exchange reaction. The cement obtained was characterized by 31P NMR and high frequency impedance for monitoring the CPC setting. The presence of Alendronate in the cement was also demonstrated by 31P NMR which has been also used to characterize the chemical transformation of α-TCP (main component of the apatitic cement) during the setting process. BP absorption/desorption experiments have been realized on cement blocks, under continuous flow condition, to model the release profile of the Alendronate. In vivo experiments showed promising results in terms of resorbability of the Alendronate–loaded cement while promoting new bone formation. The same methodology is considered to introduce gallium, a potential inhibitor of osteoclastic resorption, in a CPC formulation. First experiments have shown that gallium can be incorporated in calcium phosphate ceramics (i.e. β-TCP) where gallium is part of the network.

Resorption of synthetic bone substitute materials is essential for the integration of these materials into the natural bone remodeling process. Osteoclast behavior in the presence of calcium phosphate bioceramics (CaPB) is partially understood, and a better understanding of the underlying mechanisms is expected to facilitate the development of new synthetic bone substitutes to improve bone regeneration. In the present study, our aim was to investigate osteoclastic resorption of various synthetic CaPB. We used neonatal total rabbit bone cells to generate osteoclasts. Osteoclast-generated resorption on dentine and multiple CaPB was investigated by quantifying the surface resorbed and measuring tartrate resistant acid phosphatase (TRAP) enzyme activity. In this study, we observed that osteoclastic cells responded in a different way to each substrate. Both dentine and CaPB were resorbed but the quantitative results for the surface resorbed and TRAP activity showed a specific response to each substrate and that increased mineral density seemed to inhibit osteoclast activity.

International audience; Osteomyelitis is a severe bone infection frequently caused by Staphylococcus aureus, which shows significant resistance to methicillin. One therapeutic treatment would be to insert a bone substitute loaded to an antibiotic, which would enable the bone to be filled while the illness is being treated. Linezolid is an oxazolidinone antibiotic with a large spectrum of action. It is effective against most Gram-positive bacteria and displays a specific mode of action. The aim of this work was to study the association of linezolid with a calcium phosphate-deficient apatite matrix. Granules containing 10% and 50% linezolid were prepared by wet granulation and characterized. Porosity analyses performed by mercury porosimetry and scanning electron microscopy revealed that grain porosity with 50% linezolid was higher than that of the grains containing 10% linezolid. NMR analyses showed no change in structure of linezolid when linked to calcium-deficient apatite. These results were confirmed by studying the antibacterial activity of linezolid, which remained proportional to the quantity of loaded linezolid, proving that the antibiotic released was active. The in vitro release time varied from 9 days for granules containing 10% linezolid to 26 days for granules containing 50% linezolid.

Calcium phosphate cements have been used in medical and dental applications for many years. However, their low strength and their high brittleness prohibit their use in many stress-bearing locations, which would require an improvement in mechanical properties. The influence of microstructural parameters on the latter has nevertheless barely been investigated in a systematic manner. To this aim, apatite cements have been fabricated through alpha-TCP (alpha-tricalcium phosphate) hydrolysis, and their mechanical properties have been measured (Young's modulus, fracture toughness and compressive strength), as a function of various parameters (particle size, liquid-to-powder ratio, amount and morphology of porosity, including macropores created by mannitol particles used as porogen). Five days following the mixing of phases, identification and microstructural observation indicated the presence of unreacted alpha-TCP particles, exhibiting very weak links with the apatite matrix and often surrounded by microcracks. The latter provoke a decrease in Young's modulus. The coarser the microstructure, the larger the critical flaw size causing fracture. In the case of macroporous materials, the critical flaw size increases with macroporosity. The knowledge gained should allow, in the end, to improve mechanical properties by controlling the microstructure, and to find a better compromise between strength and biological behaviour. (C) 2010 Elsevier B.V. All rights reserved.

International audience; Combination of a bisphosphonate (BP) anti-osteoporotic drug, alendronate, with an apatitic calcium phosphate cement does not significantly affect the main properties of the biomaterial, in terms of injectability and setting time, provided that the BP is introduced chemisorbed onto calcium-deficient apatite, one of the components of the cement. In contrast to other modes of introducing the BP into the cement formulation, this mode allows to minimize alendronate release in the cement paste, thus limiting the setting retardant effect of the BP. An original approach based on high frequency impedance measurements is found to be a convenient method for in situ monitoring of the cement setting reaction. The release profile of the drug from a cement block under continuous flow conditions can be well described using a coupled chemistry/transport model, under simulated in vivo conditions. The results show that the released alendronate concentration is expected to be much lower than the cytotoxic concentration.

Resorbable calcium phosphate (CaP) biomaterials have demonstrated considerable efficacy in bone reconstructive surgery. Furthermore, bisphosphonates (BPs) are well known anti-resorptive agents largely used in clinical treatments for osteoporosis. An injectable BP-combined CaP matrix has been developed in order to biologically reinforce osteoporotic bone by increasing the bone fraction and improving bone micro-architecture. Our previous in vitro studies have shown that CaP is effective for loading and releasing BPs at doses that can inhibit excessive bone resorption without affecting osteoblasts. In vivo studies in relevant animal models are necessary to explore the effect of our injectable BP-combined biomaterial on femur bone structure by performing three-dimensional microtomography analysis, histological studies and SEM observations. Firstly, in rat model, our BP-combined CaP matrix significantly improved the bone micro-architecture as compared to CaP alone. The implantation of the BP-loaded biomaterial within proximal femurs of osteoporotic ewes led to a significant increase in relative bone content and an improvement of its micro-architecture. These modifications were confirmed by histological and SEM observations, which revealed CaP granule resorption and new bone trabeculae formation. This approach could be considered in the future for preventing osteoporotic fractures that are preferentially localized in the proximal femur, vertebral bodies or wrist. (C) 2010 Elsevier Ltd. All rights reserved.

The present study investigates the development of controlled drug delivery devices by association of bisphosphonates (BPs) with calcium-deficient apatite (CDA) to obtain a prolonged drug delivery. In a first part, we studied the microencapsulation of methylene bisphosphonic acid, our model of BPs, in biodegradable PLGA by the double emulsion (w/o/w) solvent evaporation/extraction process. Secondly, we associated BPs, either in a free form or microencapsulated, with calcium phosphate biomaterials. The association of free BPs with CDA was performed by isostatic compression at 80 MPa and we tested the interest of adding a binder, HPMC, in the formulation to reinforce the association. In parallel, microparticles were associated with calcium-deficient apatite, either by simple mixture or by isostatic compression. To compare the different formulations, in vitro dissolution studies were performed. All the formulations tested appear to be efficient to produce BPs loaded biomaterials able to deliver the drug slowly and at a constant rate. The slowest release rate (2.7% in 14 days) was obtained with the blend of microencapsulated BPs with CDA.