Abstract
Adipose-derived mesenchymal stem cells (ASCs) are easy to isolate from adipose tissues in abundance and have been widely employed in tissue engineering. However, ASCs appear to be inferior to bone marrow mesenchymal stem cells in terms of chondrogenic potential. We hypothesize that proper supply of transforming growth factor, low oxygen concentration and a dynamic environment could facilitate ASCs to differentiate into chondrocytes and to promote the engineered cartilage formation. Therefore, in this study we first developed a baculoviral vector that expressed TGF-β3 (Bac-CT3W) for the genetic modification of ASCs, which were then cultured in the two-phase rotating shaft bioreactor (RSB), hoping that the biological, environmental and physical stimuli altogether can facilitate the ASCs differentiation into chondrocytes and growth into engineered cartilages in vitro. We demonstrated that ASCs transduced with Bac-CT3W expressed high levels of TGF-β3 in a dose-dependent manner. After seeding the cells into 3D, porous scaffolds and culture in the RSB for 2 weeks, the cell/scaffold constructs grew into cartilage-like tissues with hyaline appearance and the cells resembled the authentic chondrocytes. The constructs were abundant in cartilage-specific extracellular matrix (ECM) including collagen II and glycosaminoglycan as demonstrated by histological staining and biochemical assays. Intriguingly, when the construct culture time exceeded 2 weeks, the abundance of ECM decreased, probably due to the degradation of the ECM molecules. These data demonstrated that combining the TGF-β3-expressing baculovirus and RSB for the genetic modification and dynamic culture of ASC enhances the chondrogenic differentiation of ASCs and cartilage formation, but the culture time appears to be critical to the cartilage quality. The chondrogenic differentiation of ASCs is time-consuming and requires sustained growth factor expression. We further developed hybrid baculoviruses that can persistently express TGF-β3 and bone morphological protein-6 (BMP-6) to genetically modify ASCs, hoping that TGF-β3 can suppress the hypertrophy of chondrocytes while BMP-6 can further enhance the chondrogenesis of ASCs. Furthermore, we developed new scaffolds that comprised PLGA, gelatin, chondroitin sulfate and hyaluronic acid (PLGA-GCH), hoping that these components futher synergistically promote ASCs chondrogenesis. The genetically engineered ASCs were seeded to the porous PLGA-GCH scaffolds and cultivated under low oxygen concentration condition that mimic the native cartilage environment. We demonstrated that transduction of ASCs with the hybrid baculovirus enabled sustained expression of TGF-β3 and BMP-6 at high levels in the 3D environment, stimulated the chondrogenesis of ASCs and formation of cartilage-like tissues. Transplantation of the engineered constructs into the osteochondral defects at the weight-bearing area of medial femoral condyle of rabbit knees accelerated and ameliorated the cartilage repair in 12 weeks, as evidenced by MRI. This study, for the first time, combines PLGA-GCH scaffolds and engineered ASCs persistently expressing grwoth factors for the formation of engineered cartilages and in vivo repair of osteochondral defects.