Synergistic impact of platelet rich plasma-heparin sulfate with hydroxyapatite/zirconia on the osteoblast differentiation potential of adipose-derived mesenchymal stem cells.

3D porous hydroxyapatite (HA) has been reinforced by zirconia (ZrO2) coating and impregnation with a combination of platelet rich plasma (PRP) as a source of growth factors (GFs) and Heparin sulfate (HS) to sustain the release of GFs. Adipose mesenchymal stem cells (ADMSCs) were characterized by flow cytometry for CD (cluster of differentiation) 44, CD105, CD106, CD34 and CD144, along with checking the multipotency by differentiation into the adipocytes and osteoblasts. Then, they were cultured on the scaffold treated with and without osteogenic media on days 7, 14 and 21. Electron micrograph and PKH staining show that the ADMSCs have a fusiform phenotype in the absence of osteogenic induction. Cell viability assay shows a higher number of the viable cells on the PRP-containing scaffolds than PRP-free scaffolds on day 7. Colorimetric evaluation, quantitative RT-PCR and immunocytochemistry demonstrate that PRP and HS significantly elevate the alkaline phosphatase enzyme activity and also accelerate the production of both early and mid-osteogenic markers, including collagen I and osteopontin expression with and without osteogenic conditions. The PRP-HS also accelerates the expression of the late osteogenic marker, osteocalcin, in both mRNA and protein level expression with a peak on day 21. In conclusion, supplementation of HA/ZrO2 with PRP/HS has a synergistic impact on the ADMSCs, even in the absence of chemical induction. It seems that HA/ZrO2/PRP/HS scaffold provides a higher osteoconductive microenvironment for stem cell differentiation to osteoblasts.