Biomimetic Bilayer Scaffold as an Incubator to Induce Sequential Chondrogenesis and Osteogenesis of Adipose Derived Stem Cells for Construction of Osteochondral Tissue.

Toward osteochondral tissue construction, the present study introduced a bilayer scaffold to induce sequential chondrogenesis and osteogenesis of stem cells in vitro. Two scaffolds that are both based on poly(l-glutamic acid) (PLGA) and chitosan (CS) were combined to form the bilayer scaffold. The cartilage region was the covalently cross-linked PLGA/CS hydrogel with a tubular pore structure, possessing a swollen network to prevent cellular adhesion, while inducing spontaneous cellular aggregate formation. The bone region was the electrostatically cross-linked PLGA-grafted nano hydroxyapatite (nHA--PLGA)/CS scaffold, which supported cellular adhesion and spreading. Human adipose derived stem cells (hASCs) were seeded into the cartilage region and observed to aggregate, formimg multicellular spheroids, which subsequently fused to rod-like aggregates with a larger size. At the same time, hASCs in aggregates crossed the interface and entered the bone region, presenting adhesion and spreading. With the induction of bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) during the first 14 days and BMP-2 alone during the last 14 days, hASCs aggregates in the cartilage region underwent chondrogenesis, expressing an abundant cartilage matrix including glycosaminoglycans (GAGs) and type II collagen (COL II) at 28 days. The chondrogenic induced hASCs migrated in the bone region turned to osteogenesis at 28 days, which was associated with their large spreading area and the switch of the induce factor. Thus, the present bilayer scaffold induced the different distribution of hASCs, resulting in subsequent chondrogenesis and osteogenesis, realizing osteochondral tissue construction in vitro.