At 4 weeks after implantation, the repair was assessed by the Wakitani method

At 4 weeks after implantation, the repair was assessed by the Wakitani method. treated with C/GP gel and Matrigel-engineered Ad-mock-GFP-transfected chondrocytes, and group C (n=4) were treated with C/GP gel and Matrigel-engineered Ad-hBMP7-GFP-transfected chondrocytes. Rabbits were sacrificed at 4 weeks after transplantation, and the repair effect was PHA 408 measured by the Wakitani scoring method. On the basis of the RT-PCR and western blot results, hBMP7 was efficiently overexpressed in the Ad-hBMP7-GFP-transfected chondrocytes. The ELISA results showed that the yields of collagen II and hyaluronic acid in Ad-hBMP7-GFP-transfected chondrocytes were significantly higher than those in Ad-mock-GFP-transfected chondrocytes. Chondrocytes have a better morphology and arrangement in a Matrigel scaffold than in C/GP, as assessed by H&E staining and scanning microscopy. According to the Wakitani score, Matrigel combined with Ad-hBMP7-GFP-transfected chondrocytes successfully promoted the repair of cartilage defects in rabbit knees. Keywords: cartilage defect, bone morphogenetic protein 7, Matrigel scaffold == Intro == As an inflammatory and degenerative rheumatic disease, cartilage defects are characterized by joint pain, locking phenomena and even severe disability (1). As a result of cartilage not readily self-healing, cartilage repair continues to be challenging intended for clinical doctors and scientists. Tissue engineering technology has provided great opportunities intended for cartilage repair (2, 3). For example , autologous chondrocyte transplantation (ACT) has been successfully tested in several pet experiments and clinical trials by expanding autologous chondrocytesin vitroand injecting them back into the defective cartilage site (4, 5). However , some problems have been reported for this technique. Firstly, obtaining chondrocytes from the joint surface might introduce additional injury to the joint. Secondly, chondrocytes expandedin vitroreadily undergo PHA 408 a process of dedifferentiation (6). The chondrocytes might suffer a reduction in the production of hyaluronic acid and type II collagen, which are two important components for cartilage regeneration. The loss of phenotypic traitsin vitromight result in the loss of ability to regenerate stable and healthy cartilagein vivo(7). Various efforts have been made to prevent chondrocytes from dedifferentiationin vitroandin festn. Some gene therapies have achieved success in chondrocyte tissue culture and pet cartilage repair experiments. It has been reported that bone morphogenetic protein 2 (BMP2) treatment might help chondrocytes to maintain their phenotypesin vitro(8). Moreover, short-term exposure to fibroblast growth factor 2 (FGF-2)in vivois also effective in stimulating cartilage repair or enlargement (912). Furthermore, 3-dimensional (3D) tissue culture exhibits huge advantages in maintaining the phenotype of chondrocytes. Different scaffold materials have been tested for cartilage repair in the last few years. Chitosan/glycerophosphate (C/GP) gel (13), Matrigel (14), collagen (15) and fibrin gel (16) have exhibited excellent performances in cartilage cell culturein vitroand cartilage repair in pet experiments. Bone morphogenetic protein 7 (BMP7) is a member of the transforming growth factor (TGF)- MMP7 superfamily (17) that is crucial in the regulatory pathways intended for embryogenesis, tissue repair and regeneration (18). There is evidence that BMP7 is able to induce the differentiation of bone marrow-derived PHA 408 mesenchymal cells into chondrocytes (19) and might serve as an anabolic activator intended for chondrocytes in tissue culture (20). In addition , clinical studies have PHA 408 revealed that BMP7 is able to prevent the degeneration of cartilage during inflammatory arthritis (21, 22). These observations suggest that BMP7 has great potential for use in cartilage repair and regeneration. In the present study, gene therapies and 3D culture techniques were combined for use in the repair of cartilage defects. Two types of 3D culture gel-engineered Ad-hBMP7-GFP-transfected chondrocytes were prepared. By implanting the engineered gels into rabbit cartilage defects, their ability to improve cartilage repair in rabbit knees was evaluated and compared. == Materials and methods == == == == Animals == A total of 12 adult male New Zealand White rabbits (50 weeks old; weight, 2 . 02. 5 kg) and 12 female New Zealand White rabbits (6 weeks old, weight, 1 . 52. 0 kg) were provided by the Laboratory Pet Center of Nantong University (Nantong, China). All animals were housed individually. Food and water were givenad libitum. The animals’ general health and care conditions were monitored and recorded separately by the laboratory animal welfare officer. This study was approved by the laboratory pet ethics committee of Nantong University. == Primary cell culture and hBMP7 transfection == Rabbit articular knee cartilages were.