Bone regenerative medicine aims at designing biomimetic biomaterials able to guide stem cells fate towards osteoblast lineage and prevent orthopaedic common pathogen adhesion. Owing to bone inorganic/organic composition, we herein report, using a versatile process based on simultaneous spray coating of interacting species, a calcium phosphate (CaP) / chitosan (CHI) / hyaluronic acid (HA) functionalized collagen membrane as a new strategy for bone regenerative medicine. Physicochemical characterizations of CaP-CHI-HA coating were performed by scanning electron microscopy, X-ray photoelectron and infrared spectroscopies and high-resolution transmission electron microscopy, revealing the formation of a thin coating mainly composed of non-stoichiometric crystalline hydroxyapatite dispersed into polymorphic organic film. Biocompatibility of CaP-CHI-HA coated membrane, evaluated after 7 days in contact with human mesenchymal stem cells (MSCs), showed spread, elongated and aligned cells. Metabolic activity and DNA quantification studies showed an increase in MSCs proliferation on coated membrane compared to uncoated membrane over the study time. Similarly, cytokines (IL-6, IL-8, osteoprotegerin) and growth factors (VEGF, bFGF) release in supernatant, as well as endothelial cells recruitment, were significantly increased in presence of CaP-CHI-HA coated membrane. Thus, CaP-CHI-HA coated membrane provides a suitable environment for MSCs to induce bone healing. Moreover, pro-inflammatory cytokines (IL-1β and TNF-α) secretion by human monocytes was significantly reduced on CaP-CHI-HA coating compared to LPS stimulation. CaP-CHI-HA coating also reduced significantly Staphylococcus aureus and Pseudomonas aeruginosa adhesion on the membrane, conferring a bacterial anti-adhesive surface. Based on our results, CaP-CHI-HA functionalized collagen membrane provides an interesting material for bone regeneration.

Mesenchymal stem cells (MSCs) are tissue-resident stroma cells capable of modulating immune cells through the secretion of paracrine factors. However, the comparison of MSCs potential, from different sources and submitted to hypoxia within a 3D scaffold, in secreting pro-healing factors has never been investigated. With a chemical composition similar to type I collagen, a major component of connective tissues retrieved in dental pulp, bone and umbilical cord, Hemocollagene® haemostatic foam presented porous and interconnected structure (> 90%) and a relative low elastic modulus of around 60 kPa. All these criteria meet basic requirements for tissue engineering based material. Herein, we assessed and compared the effect of hypoxia (3% O₂) on the regulation and release of pro-angiogenic factors (VEGF, b-FGF and IL-8) from bone marrow (BM), Wharton's jelly (WJ) and dental pulp (DP) derived MSCs cultured in Hemocollagene®. After 10 days of culture, qRT-PCR analysis showed an up-regulation of b-FGF and VEGF mRNA in BM- and WJ-derived MSCs, but not in DP-derived MSCs. Furthermore, hypoxia highly up-regulated IL-8 expression in WJ-derived MSCs and moderately in both BM and DP-derived MSCs. In contrast, ELISA analysis showed a higher amount of VEGF and IL-8 in supernatant provided from DP-derived MSCs culture compared to BM and WJ-derived MSCs. B-FGF was not detected whatever the experimental condition. In conclusion, MSCs derived from several tissues were able to release pro-angiogenic factors under hypoxic conditions. There was no clearly superior type of MSCs for therapeutic use, however DP-derived MSCs are likely to be more advantageous.