Ad-HGF Gene Transduced-MSCs Combined Double-lay polymer scaffold to repair osteochondral defects simultaneously

摘要

METHODS Bone marrow-derived stem cells of a five-month-old New Zealand White rabbit were isolated and culture-expanded inDMEM supplemented with 15% FBS at 37 ℃, 5%CO2 atmosphere incubator. The 5th passage MSCs were treatedaccording the groups: (1) DMEM with 15%FBS (2) DMEM with 15%FBS containing TGF-β 1 at 10ng/ml, bFGF at25ng/ml and dexamethasone at 10-7M, as induced MSC. (3) Adenovirus carrying hypotocyte growth factor cDNA(adHGF)transduced MSC, as gene modified MSC. (4) adHGF transduced MSC which was induced with cytokines. Rabbitchondrocytes served as the control. The morphological change in five kinds of cells was observed with phase chontrastmocroscope. Cell proliferation capacity was tested by MTT; quantitative secreation of GAG was tested by alcin blue;collagen I, II were tested by immunohistochemical and RT-PCR; FCM for the transduction efficency after adGFP modifiedMSCs; and ELISA for HGF expression of adHGF transfected MSCs. The morphology of double-layer PLGA scaffold andMSCs was obsrevied with SEM. MSCs exposed to different treatments were loaded into PLGA double-layer scaffold(diameter 3.5mm, thickness 3 to 4 mm)to form composite osteochondral grafts, which were incubated for 24 hours at 37 ℃,5%CO2 incubator, and then implanted respectively into subcutaneous pockets on the backs of 5 five-weeks-old femaleathymic mice. The composites were harvested and examined with histology at 3 weeks after implantation. Fifty three tofour-month-old New Zealand White rabbits were divided into six groups: MSCs/PLGA(Group A); MSCs induced withcytokines/PLGA(Group B); MSCs transfected with adHGF/PLGA(Group C); MSCs transfected with adHGF afterinduced with cytokines/PLGA(Group D); PLGA scaffold alone or no treatment as control(Group E); as well astransplantation of autologous osteochondral grafts(Group F). The osteochondral defect of diameter 3.5mm and depth of 3 to4mm was created in the patellar grove of distal femur with a hand drill, and the different composites were implantedaccordingly. Specimens were harvested at 4, 8, 16 and 24 weeks postoperatively, and examined with histology formorphologic features, and stained immunohistochemically for type I, II collagen. Each sample at 24 weeks was graded withuse of a histological evaluation. RESULTS MSC was havested after passaged three to four weeks in vitro. Type I collagen was identified in all MSCs, whether induced,transfected or not. Type II collagen was identified in aggregated culture MSC, cytokins induced and adHGF transfectedMSCs. The proliferation of these cells was same. The quantity of secreting GAG in induced MSC, gene transfected MSCis higher than MSC while lower than cartilage cells. The expression of HGF of transductive MSC was stable at least 4weeks. MSCs attached to PLGA scaffold and scattered into pores. Histological examination showed that the compositequalified osteochondrogenic capacity in nude mice model.In rabbit models, repair cartilage was observed in the upper-layer, and bone in the lower-layer of most specimens at 8weeks postoperation. At 24 weeks after implantation, the subcondral bone was completely repaired with overlying maturehyaline-like cartilage in group induced MSC/PLGA(group B, 4/6), adHGF-MSC/PLGA(group C, 2/6) and induced,adHGF/PLGA(group D, 2/4). The gross view of group B , C and D was similar to that of group auto-cartilage graft(group