David Marchat

Accurate characterization of pure silicon-substituted hydroxyapatite powders synthesized by a new precipitation route

This paper presents a new aqueous precipitation method to prepare silicon-substituted hydroxyapatites Ca10(PO4)6-y(SiO4)y(OH)2-y(VOH)y (SiHAs) and details the characterization of powders with varying Si content up to y=1.25molmolSiHA(-1). X-ray diffraction, transmission electron microscopy, solid-state nuclear magnetic resonance and Fourier transform infrared spectroscopy were used to accurately characterize samples calcined at 400°C for 2h and 1000°C for 15h. This method allows the synthesis of monophasic SiHAs with controlled stoichiometry. The theoretical maximum limit of incorporation of Si into the hexagonal apatitic structure is y<1.5. This limit depends on the OH content in the channel, which is a function of the Si content, temperature and atmosphere of calcination. These results, particularly those from infrared spectroscopy, raise serious reservations about the phase purity of previously prepared and biologically evaluated SiHA powders, pellets and scaffolds in the literature.

The pH in the microenvironment of human mesenchymal stem cells is a critical factor for optimal osteogenesis in tissue-engineered constructs.

The present study aimed at elucidating the effect of local pH in the extracellular microenvironment of tissue-engineered (TE) constructs on bone cell functions pertinent to new tissue formation. To this aim, we evaluated the osteogenicity process associated with bone constructs prepared from human Bone marrow-derived mesenchymal stem cells (hBMSC) combined with 45S5 bioactive glass (BG), a material that induces alkalinization of the external medium. The pH measured in cell-containing BG constructs was around 8.0, that is, 0.5 U more alkaline than that in two other cell-containing materials (hydroxyapatite/tricalcium phosphate [HA/TCP] and coral) constructs tested. When implanted ectopically in mice, there was no de novo bone tissue in the BG cell-containing constructs, in contrast to results obtained with either HA/TCP or coral ceramics, which consistently promoted the formation of ectopic bone. In addition, the implanted 50:50 composites of both HA/TCP:BG and coral:BG constructs, which displayed a pH of around 7.8, promoted 20-30-fold less amount of bone tissue. Interestingly, hBMSC viability in BG constructs was not affected compared with the other two types of material constructs tested both in vitro and in vivo. Osteogenic differentiation (specifically, the alkaline phosphatase [ALP] activity and gene expression of RUNX2, ALP, and BSP) was not affected when hBMSC were maintained in moderate alkaline pH (≤7.90) external milieu in vitro, but was dramatically inhibited at higher pH values. The formation of mineralized nodules in the extracellular matrix of hBMSC was fully inhibited at alkaline (>7.54) pH values. Most importantly, there is a pH range (specifically, 7.9-8.27) at which hBMSC proliferation was not affected, but the osteogenic differentiation of these cells was inhibited. Altogether, these findings provided evidence that excessive alkalinization in the microenvironment of TE constructs (resulting, for example, from material degradation) affects adversely the osteogenic differentiation of osteoprogenitor cells.

Comparative study of the osteogenic ability of four different ceramic constructs in an ectopic large animal model.

Tissue‐engineered constructs combining bone marrow mesenchymal stem cells with biodegradable osteoconductive scaffolds are very promising for repairing large segmental bone defects. Synchronizing and controlling the balance between scaffold‐material resorption and new bone tissue formation are crucial aspects for the success of bone tissue engineering. The purpose of the present study was to determine, and compare, the osteogenic potential of ceramic scaffolds with different resorbability. Four clinically relevant granular biomaterial scaffolds (specifically, Porites coral, Acropora coral, beta‐tricalcium phosphate and banked bone) with or without autologous bone marrow stromal cells were implanted in the ectopic, subcutaneous‐pouch sheep model. Scaffold material resorption and new bone formation were assessed eight weeks after implantation. New bone formation was only detected when the biomaterial constructs tested contained MSCs. New bone formation was higher in the Porites coral and Acropora coral than in either the beta‐tricalcium phosphate or the banked bone constructs; furthermore, there was a direct correlation between scaffold resorption and bone formation. The results of the present study provide evidence that, among the biomaterials tested, coral scaffolds containing MSCs promoted the best new bone formation in the present study. Copyright

In vitro three-dimensional bone tissue models: from cells to controlled and dynamic environment.

Most of our knowledge of bone cell physiology is derived from experiments carried out in vitro on polystyrene substrates. However, these traditional monolayer cell cultures do not reproduce the complex and dynamic three-dimensional (3D) environment experienced by cells in vivo. Thus, there is a growing interest in the use of 3D culture systems as tools for understanding bone biology. These in-vitro-engineered systems, less complex than in vivo models, should ultimately recapitulate and control the main biophysical, biochemical, and biomechanical cues that define the in vivo bone environment, while allowing their monitoring. This review focuses on state-of-the-art and the current advances in the development of 3D culture systems for bone biology research. It describes more specifically advantages related to the use of such systems, and details main characteristics and challenges associated with its three main components, that is, scaffold, cells, and perfusion bioreactor systems. Finally, future challenges for noninvasive imaging technologies are addressed.

Physico-Chemical Characterization and In Vitro Biological Evaluation of Pure SiHA for Bone Tissue Engineering Application

Studies about silicon-substituted hydroxyapatites exhibit several shortcomings that leave unanswered questions regarding the properties and subsequent biological outcomes generated by this biomaterial. Firstly, samples characterization is often incomplete, meaning that phase purity on the pellet surface is not assured. In fact, ceramic materials used in literature that are claimed to be pure are actually polluted through second phase as superficial polymerized silicate. In this study, we have successfully synthesized a phase pure silicon hydroxyapatite powder Ca10(PO4)5.5(SiO4)0.5(OH)1.5 (Si0.5HA) compressed this powder into pellets, sintered them, and evaluated the biological response of osteoblast cells (C3H10 line) seeded on the pellet surface. Besides, the solubility in aqueous media of HA and Si0.5HA pellets were determined through static experiments. These tests attempt to provide a comprehensive picture of the cellular response to the SiHA material, in order to determine the mechanism by which Si evokes the improved in vitro biological outcomes described in the literature. Results revealed first an equivalent solubility of Si0.5HA and HA pellets, and second that cells do not react favourably to the pure SiHA surface.

Synthesis and Characterization of Cx-Siy-HA for Bone Tissue Engineering Application

The main goal of this work is to prepare carbon and silicon co-substituted calcium hydroxyapatite (Cx-Siy-HA) for bone tissue engineering application. This study includes the synthesis of pure powders with a controlled amount of carbonate (x) and silicate (y) ions within the apatite structure, their characterization with the establishment of database for different compositions, and the manufacture of dense bioceramics. Carbon-silicon co-substituted hydroxyapatite (C0.5-Si0.5-HA) powders are synthesized by aqueous precipitation. According to structural, spectroscopic and elemental characterizations, silicate and carbonate are included in the apatite lattice and their stoichiometries are controlled. The heat treatments under CO2 atmosphere allow the sintering of pellets without decomposition of the apatite structure.