A composite bilayer scaffold functionalized for osteochondral tissue regeneration in rat animal model.

Osteochondral defects are defined most typically by damages to both cartilage and subchondral bone tissue. It is challenging to develop bilayered scaffolds that regenerate both of these lineages simultaneously. In the present study, an electrospun bilayer nanofibrous scaffold was designed to repair osteochondral lesions. A nanocomposite of hydroxyapatite, strontium, and reduced graphene oxide were combined with polycaprolactone polymer to fabricate the osteogenic differentiation layer. Additionally, the chondrogenic differentiation layer was also formed using polyethersulfone polymer and benzyl hyaluronan. The physical, mechanical, and chemical properties of the scaffolds were determined, and adipose-derived mesenchymal stem cells were cultured on each layer to evaluate their biocompatibility and differentiation potential. Cell viability, mineralization, alkaline phosphatase enzyme (ALP) expression, and extracellular calcium deposition were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, alizarin red staining, ALP activity, and calcium deposition. Real-time polymerase chain reaction (PCR) was used to assess the expression levels of osteogenic (Collagen I, Runx II, ALP, Osteocalcin) and chondrogenic (Sox9, Collagen II (Col II), Aggrecan) genes. Finally, the osteochondral scaffold was created by electrospinning these two layers for 2 days. The scaffold was grafted into the osteochondral defect of a Wistar rat's knee. 60 days after surgery, real-time PCR, immunohistochemistry (IHC), and hematoxylin and eosin staining were performed. The expression of chondrogenic and osteogenic genes was increased compared to the control group, as confirmed by real-time PCR. Furthermore, IHC revealed a rise in Col II and Collagen X expression. Finally, in vivo and in vitro studies have shown that the electrospun bilayer scaffold is biocompatible, which facilitates osteochondral healing.