Construction of Tissue-Engineered Cartilage Combining Chondrocytes Induced by rAAV2-TGF-β1 Transfected MSCs with RGD-Modified Nano-PCL-b-PLLA Scaffolds In Vitro

Tissue engineering technology offers a novel approach for cartilage regeneration to treat osteoarthritis. However, cartilage engineering has not mixed the following advantageous factors together: chondrocytes differentiated from recombinant adeno-associated virus 2-transforming growth factor-beta 1 (rAAV2-TGF-beta 1) transfected mesenchymal stem cells (MSCs) and Arg-Gly-Asp (RGD)-modified nano-poly (epsilon-caprolactone)-block-poly(L-lactic acid) (PCL-b-PLLA), both of which may benefit future clinical application. This study aims to evaluate properties of this cell-scaffold compound in vitro. Canine MSCs obtained from bone marrow was isolated and cultured in vitro, then it was transfected by rAAV2-TGF-beta 1 with the multiplicity of infection (MOI) of 5 x 10(5) v.g./cell and 1 x 10(5) v.g./cell respectively. Chondrification markers, including TGF-beta 1, collagen II and Aggreacan were detected by ELISA, Western blot, RT-PCR and immunocytochemistry respectively. Compared with RGD-modified PCL-b-PLLA scaffold, ultra-structure of RGD-modified nano-PCL-b-PLLA scaffold was determined under scanning electron microscopy (SEM). Third generation of chondrocytes derived from rAAV2-TGF-beta 1 transfected MSCs was seeded on both scaffolds. Cellular morphology, protein quantity and DNA content were compared between the two scaffolds. CanineMSCs appeared spindle-shape and revealed active proliferation. Following rAAV2-TGF-beta 1 transfection, contents of TGF-beta 1, collagen II and Aggreacan were increased. The group transfected by 5 x 10(5) v.g./cell showed higher expression of chondrification markers than the group of 1 x 10(5) v. g./cell at each time point (P < 0.05). Under SEM, poriness of RGD-modified nano-PCL-b-PLLA scaffold was 93%, which was higher than that of non-nanostructured scaffold (86%). Following co-culture with third generation of chondrocytes, cells in RGD-modified nano-PCL-b-PLLA scaffold bound more tightly with each other and had increase in extracellular matrix (ECM) secretion than those in non-nanostructured scaffold. Similarly, DNA and protein contents of chondrocytes increased more on nanostructured scaffold than those on the counterpart (P < 0.05). Visual observation found the size and hardness of cell-nanostructured scaffold compound were similar to its previous status, while its surface was smoother and glossier. A large quantity of chondrocytes entered into the nanostructured scaffold and secreted massive ECM. Engineered cartilage combining rAAV2-TGF-beta 1 transfected MSCs-derived chondrocytes with RGD-modified nano-PCL-b-PLLA scaffold can be successfully constructed in vitro with satisfying properties, revealing a preferable choice in cartilage tissue engineering.