Cell-free bilayer functionalized scaffold for osteochondral tissue engineering.

Osteochondral tissue engineering using layered scaffolds is a promising approach for treating osteochondral defects as an alternative to microfracture procedure, autologous chondrocyte implantation, and cartilage-bone grafting. The team previously investigated the chondrogenesis of mesenchymal stem cells (MSCs) on a polycaprolactone (PCL)/acetylated hyaluronic acid scaffold. The present study first focused on fabricating a novel osteoconductive scaffold utilizing bismuth-nanohydroxyapatite/reduced graphene oxide (Bi-nHAp/rGO) nanocomposite and electrospun PCL. The osteoconductive ability of the scaffold was investigated by evaluating the alkaline phosphatase (ALP) activity and the osteogenic genes expression in the adipose-derived MSCs. The expression of Runx2, collagen I, ALP, and osteocalcin as well as the result of ALP activity indicated the osteoconductive potential of the Bi-nHA-rGO/PCL scaffold. In the next step, a bilayer scaffold containing Bi-nHAp/rGO/PCL as an osteogenic layer and acetylated hyaluronic acid/PCL as a chondrogenic layer was prepared by the electrospinning technique and transplanted into osteochondral defects of rats. The chondrogenic and osteogenic markers corresponding to the surrounding tissues of the transplanted scaffold were surveyed 60 days later by real-time polymerase chain reaction (PCR) and immunohistochemistry methods. The results showed increased chondrogenic (Sox9 and collagen II) and osteogenic (osteocalcin and ALP) gene expression and augmented secretion of collagens II and X after transplantation. The results strongly support the efficacy of this constructed cell-free bilayer scaffold to induce osteochondral defect regeneration.