Shanghai Jiaotong Daxue xuebao. Yixue ban (Jan 2024)
Observation on A-PRF promoting regeneration of osteochondral defects in rabbit knee joints
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.
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