Observation on A-PRF promoting regeneration of osteochondral defects in rabbit knee joints / 上海交通大学学报（医学版）

ABSTRACT

Objective·To explore the role of advanced platelet-rich fibrin(A-PRF)in osteochondral regeneration.Methods·Bone-marrow mesenchymal stem cells(BMSCs)and knee joint chondrocytes were obtained from New Zealand rabbits.A-PRF was obtained by low-speed centrifugation of the heart blood of rabbits.The histological structure of A-PRF was observed by an optical microscope.The release of growth factors in A-PRF was detected by ELISA,including platelet-derived growth factor,transforming growth factor-β,insulin-like growth factor,vascular endothelial growth factor,epidermal growth factor and fibroblast growth factor.A-PRF's cytotoxicity and capability for promoting the proliferation of rabbit BMSCs were detected by live/dead double staining and MTT methods.The effect of A-PRF on the gene expression of type Ⅱ collagen,aggrecan,alkaline phosphatase(ALP)and osteocalcin(OCN)in rabbit BMSCs was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Transwell chambers were used to determine the effect of A-PRF on the migration ability of rabbit BMSCs and the chondrocytes.Rabbit knee osteochondral defect models were established,and 18 rabbits were randomly divided into 3 groups.The A-PRF group(n=6)was implanted with A-PRF in the defect,the A-PRF+BMSCs group(n=6)was implanted with rabbit BMSCs on A-PRF,and the control group(n=6)did not undergo implantation.The rabbits were sacrificed 12 weeks after surgery and the knee joint specimens were stained with hematoxylin-eosin(H-E),toluidine blue and safranin O/fast green.Based on the surface morphology and histology of the knee joints,the International Cartilage Repair Society(ICRS)scoring system was used for macroscopic and histological scoring.Results·A-PRF had a loose network structure and can slowly release growth factors.No cytotoxicity to rabbit BMSCs was observed after adding A-PRF,and the the capability for promoting the proliferation of rabbit BMSCs was significantly increased at 24,48 and 72 h after adding A-PRF(all P<0.05).Chondrogenesis-related gene Ⅱ collagen and aggrecan,as well as osteogenesis-related genes ALP and OCN were significantly up-regulated(all P<0.05).After adding A-PRF,the migration abilities of rabbit BMSCs and chondrocytes were significantly enhanced(both P<0.05),and the migration ability of rabbit BMSCs was significantly higher than that of chondrocytes(P=0.025).The joint surface morphology in the rabbit knee joint defect models was observed.It can be seen that the defects in the A-PRF group and the A-PRF+BMSCs group were basically restored,while the the defects in the control group were only covered by soft tissue.In the ICRS macroscopic score,there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group,but the scores of the two groups were all significantly higher than those of the control group(all P<0.05).According to the histological results,both the A-PRF group and the A-PRF+BMSCs group formed osteochondral repair,but the cartilage in the A-PRF group was more mature,while the control group formed fibrous repair.In the ICRS histological score,there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group,but the scores of both the groups were significantly higher than those of the control group(both P<0.05).Conclusion·Autologous A-PRF has good biocompatibility and the capability for promoting the proliferation of BMSCs.It can promote the repair of cartilage and subchondral bone both in vitro and in vivo.