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.

Crystallization processes at the surface of polylactic acid-bioactive glass composites during immersion in simulated body fluid.

We report on the crystallization processes occurring at the surface of PDLLA-Bioglass® composites immersed in simulated body fluid. Composites manufactured by injection molding and containing different amounts (0, 20, 30, and 50 wt %) of 45S5 Bioglass® particles were tested for durations up to 56 days and compared with Bioglass® particles alone. Crystallization processes were followed by visual inspection, X-ray diffraction (with Rietveld analysis) and scanning electron microscopy. Both calcite and hydroxyapatite were formed at the surface of all materials, but their relative ratio was dependent on the Bioglass® content and immersion time. Hydroxyapatite was always the major phase after sufficient immersion time, insuring bioactivity of such composites especially for Bioglass® content higher than 30 wt %. A scenario of crystallization is proposed. Rapid degradation of the composites with 50 wt % was also observed during immersion. Therefore, composites with 30 wt % of Bioglass® particles seem to exhibit the best balance between bioactivity and stability at least during the first weeks of immersion in contact with body fluids.

Elaboration of silica colloid/polymer hybrid support for oligonucleotide synthesis.

One way to increase the sensitivity of DNA diagnostic assays developed on microarrays is to improve the solid phase that allows the extraction of the target from a biological sample, before detection. Two parameters are influencing the performances of this capture step: (i) the specific surface area being offered for the capture and (ii) the number and the accessibility of oligonucleotide probes immobilized on the surface. In this context, we have developed an attractive approach which fulfills these two points. Our strategy was to elaborate a new material of high specific surface area, suitable to serve as support for both solid-phase oligonucleotide synthesis and in vitro diagnostic assay. This material has consisted of aggregates of colloidal amino-silica nanoparticles covalently linked by poly(ethylene oxide) (PEO) arms. The aggregation of amino-silica particles in the presence of reactive bis-isocyanate PEO was achieved in a controlled manner. The aggregate size and structure were examined by microscopy. The specific surface area of this material was measured by nitrogen adsorption technique. The composition of aggregate was studied by thermogravimetry and X-ray photoelectron spectroscopy. Then, this material has been successfully used as support for oligonucleotide synthesis of high yield and purity. The resulting system will be further evaluated in a diagnostic assay on a microarray.