Pro osteon/collagen hydroxylapatite hybrid scaffold is able to induce human mesechymal stem cells (hMSCs) to osteogenic differentiation

Aim. Innovative scaffolds are of paramount importance for bone grafting and re-growth. Distinct enhancements of scaffold properties may optimize the product performance for different applications in the fields of maxillofacial and odontoiatric sciences/clinics. The most common scaffold improvements were obtained for their biocompatibility, mechanical properties in osteo-conductive and osteo-inductive properties and healing rate. In this study an innovative hydroxylapatite hybrid scaffold composed of granular hydroxylapatite (Pro Osteon ® 200, Interpore Cross Irvine, CA, USA) and collagen Hemostat (Bard Warwick, Rhode Island, USA) (Coll/HA) was investigated using human bone marrow-derived mesenchymal stem cells (hMSCs) of adult donors (1-3). Materials and Methods. In order to evaluate (i) biocompatibility, (ii) osteoconductivity and (iii) osteoinductivity hMSC cultures were grown on the innovative scaffold. The cellular morphology, cytoskeleton organization, and cell viability were studied by immunohistochemistry (IHC), and AlamarBlue metabolic assay respectively. Osteocalcin and osteopontin expression proteins were detected by IHC. The temporal osteocalcin expression protein in hMSCs grown on the biomaterial and in osteogenic condition (OC), and the control (TCPS), were quantified by Human Osteocalcin Instant E.L.I.S.A assays. Expression of osteogenic genes were evaluated by quantitative PCR (Q-PCR) array technologies; the Human Osteogenesis RT² Profiler PCR Array (Qiagen) was used to analyze the expression of 84 genes related to osteogenic differentiation at day 21. Results. Cell morphology of hMSC–eGFP cells was indistinguishable from that of parental hMSC. Indeed, hMSC-eGFP grown on the scaffold showed a normal morphology. Metabolic activity was increased during the 21 days of experiments (P<0.05). The cytoskeleton architecture seemed to be well organized, whereas its integrity remains uninfluenced by the scaffold during the time course. The biomaterial induced the matrix mineralization in hMSCs at day 14. Osteogenic proteins, such as osteocalcin and osteopontin were detected at day 21.The biomaterial induced the up-regulation of osteocalcin protein expression levels, quantified in E.L.I.S.A assay at day 21, compared to control (TCPS) and at day 14. Gene expression analyzed in hMSCs allowed us to detect the upregulation of mRNAs of 16 genes, belonging to the osteogenic differentiation pathway. Specific genes were for (i) the ossification process: BMP2/3, COL2A1, MMP9, NOG, SPP1, TNFSF11, TGFB3, (ii) osteoblast differentiation were: GLI1, SMAD3, SP7, (iii) whereas for the extracellular matrix (ECM) and cell adhesion molecules were: MMP10, ICAM1, ITGAM, CD36. In addition, the growth factor CSF3 was also up-regulated compared to the control, ad day 21. The transcription factor SP7 was the highest gene modulated by the biomaterial with a 3 Log 2 fold increase. It has been reported that during the development of the skeletal bone and tooth, SP7 is a key mesenchymal factor necessary for cell fate decisions in the differentiation of specialized cells. Down-regulated genes were that encoding ECM and cell-to cell adhesion molecules such as BGN, CDH11, COL1A1, COL5A1, COMP, CSF2, CTSK, IGF1/2, IGF1R, ALPL. Early transcription factors, such as RUNX2, SMAD1, TWIST1 were down-regulated, at day 21. In addition, FGFR2 and BMPR2 genes were also down-regulated compared to the control. Discussion. Our data demonstrate that the innovative scaffold provides a good microenvironment for the hMSCs adhesion and proliferation. The scaffold demonstrated biocompatibility in term of morphology, viability and cytoskeleton architecture of hMSC grown on the biomaterial. Gene expression profile analyses by array technology demonstrated that, in hMSCs, the scaffold induces up-regulation in specific genes that are involved in ossification process, such as BMP2/3, SPP1 and SP7, at d 21 post-cell seeding. The scaffold induces a up-regulation of the osteocalcin protein with improvement in matrix mineralization, indicating a good osteoinductivity performance. In conclusion, our experimental cell biology and epigenetic analyses suggest that the Coll/HA hybrid scaffold is an excellent biomaterial for the bone repair and bone tissue engineering.