Microsphere-Based Hierarchically Juxtapositioned Biphasic Scaffolds Prepared from Poly(Lactic-co-Glycolic Acid) and Nanohydroxyapatite for Osteochondral Tissue Engineering.

This study aims to prepare biphasic osteochondral scaffolds based on seamless joining of sintered polymer and polymer/ceramic microspheres for co-culture of chondrocytes and bone marrow stem cells (BMSCs). Poly(lactide--glycolide) (PLGA) microspheres and 10% nanohydroxyapatite (nHAP)-incorporated PLGA (PGA/nHAP) microspheres were prepared through the oil-in-water precipitation method. Virgin (V) and composite (C) scaffolds were prepared from 250⁻500 µm PLGA and PLGA/nHAP microspheres, respectively, while osteochondral (OC) scaffolds were fabricated through the combination of V and C scaffolds. Physico-chemical properties of scaffolds were characterized through microscopic-spectroscopic evaluations. The effect of nHAP in scaffolds was investigated through thermogravimetric analysis and mechanical testing, while surface hydrophobicity was tested through contact angle measurements. Rabbit chondrocytes and BMSCs were used for cell culture, and cell morphology and proliferation were determined from SEM and DNA assays. Alizarin red and Alcian blue stains were used to identify the in vitro bone and cartilage tissue-specific regeneration, while cetylpyridinium chloride was used to quantitatively estimate calcium in mineralized bone. For co-culture in OC scaffolds, BMSCs were first seeded in the bone part of the scaffold and cultured in osteogenic medium, followed by seeding chondrocytes in the cartilage part, and cultured in chondrocyte medium. High cell viability was confirmed from the Live/Dead assays. Actin cytoskeleton organization obtained by DAPI-phalloidin staining revealed proper organization of chondrocytes and BMSCs in OC scaffolds. Immunofluorescent staining of bone (type I collagen and osteocalcin (OCN)) and cartilage marker proteins (type II collagen (COL II)) confirmed cellular behavior of osteoblasts and chondrocytes in vitro. Using an ectopic osteochondral defect model by subcutaneous implantation of co-cultured OC scaffolds in nude mice confirmed cell proliferation and tissue development from gross view and SEM observation. IF staining of OCN and COL II in the bone and cartilage parts of OC scaffolds and tissue-specific histological analysis exhibited a time-dependent tissue re-modeling and confirmed the potential application of the biphasic scaffold in osteochondral tissue engineering.