The Study Of The Effect Of FGF2 On Promoting The Repair Of Articular Cartilage Defect By Modulating Subchondral Bone

Lesion of articular cartilage(AC)is difficult to repair due to the lack of blood supply,and the damage always leads to osteoarthritis(OA)and even disability.Articular cartilage regeneration is still a huge challenge in clinical medicine,and is extensively studied currently.Various growth factors are involved in the process of chondrogenesis and articular cartilage development,and they have been used in the research of articular cartilage repair.Previously,we constructed a collagen-based membrane,which can release the loaded Fibroblast Growth Factor 2(FGF2 or basic Fibroblast Growth Factor,bFGF)in a spatiotemporally controlled manner.Importantly,by implanting this membrane into articular cartilage defect of rabbit,we found that FGF2 promoted the repair through up-regulation the expression of multiple endogenous growth factors,such as Bone Morphogenetic Protein 2 and 4(BMP2 and 4).Recently,we found that the up-regulated BMP2 and 4 are mainly derived from subchondral bone via detecting the gene expression profiles of cartilage layer and subchondral bone by qRT-PCR,respectively.We also found that BMP3,an inhibitor of receptors of BMP2 and 4,could aggravate the damage of subchondral bone in vivo,ultimately blocked the progress of articular cartilage repair.These findings indicated that subchondral bone plays important roles in articular cartilage repair.And modulation of subchondral bone by FGF2,particularly by targeted deliver of it,via up-regulation the expression of BMP2 and 4 could enhance the repair.In the present study,we investigated the roles of subchondral bone and the mechanism of FGF2 during articular cartilage repair furtherly.Then we designed a kind of double-layered collagen membranes to targetedly deliver FGF2 into subchondral bone.Next,after implanting this membrane into articular cartilage defect,we determined the concentration of BMP2 and BMP4 in synovial fluid at early stage by ELISA.Subsequently,the effects of this targeted delivery system for FGF2 on articular cartilage repair were studied and the repair outcomes were evaluated.Finally,the mechanism of the targetedly delivered FGF2 to subchondral bone in articular cartilage repair was discussed.The main results and conclusions of this work are as follows:1.By injecting FGF2 solution at a low concertation into subchondral bone of a full-thickness articular cartilage defect locally and directly,we found although the defects were not completely restored 8 weeks after treatment,the repaired tissue was improved compared with the control group.Whereas,injection of dorsomorphin locally,an inhibitor of BMP signaling pathway,after FGF2 administration to subchondral bone,resulted in the worse repair outcome compared with FGF2 treated group.Therefore,these findings confirmed our hypothesis that subchondral bone played important roles during articular cartilage repair,and BMP signaling pathway was critical in the FGF2 promoted repair process.2.We prepared an inhomogeneous double-layered collagen membrane with one dense layer and one loose layer.Then FGF2 was dropped onto the loose layer of the membrane.It was found that an obvious initial burst release with-57%of the total bovine serum albumin(BSA),which was used as a model,in the first 2 hours;then the release was slowed down with approximately-75%of the total BSA released after 8 hours.Particularly,the loaded-BSA on the membrane was released in 8 hours from only the loose layer side,while the amount of it from the dense layer side was under detectable line.This result indicated that FGF2 could be directionally released from the membrane.3.At 1 day,3 day and 7 day after implanting of the double-layered membrane into articular cartilage defect,the synovial fluid was collected,and the amount of BMP2 and BMP4 were detected via Elisa.We found the amount of BMP2 and BMP4 in the synovial fluid of the FGF2 group were significantly higher than that of control group..4.Eight weeks after implantation of the inhomogeneous double-layered collagen membrane,the FGF2-loaded one resulted in the better repair outcome than that of control group.The restored tissues in the FGF2 group appeared much similar to neighboring normal cartilage.The scores of the overall evaluation for the FGF2 group were significantly higher than that of control group.Histologically,the FGF2 group showed the better performance of regenerative cartilage and subchondral bone.These results indicated that the reparative tissue in the targetedly delivered FGF2 group was significantly better than that of the control group at 8 weeks after implantation.In conclusion,we found FGF2 improved the repair of AC defect when directly injected it to subchondral bone might via upregulating BMPs,whereas inhibition of BMP signaling pathway impairs the regeneration.Finally,based on these findings,a kind of double-layered inhomogeneous collagen-based membrane for targeted delivery of FGF2 was prepared.This membrane was then transplanted with the FGF2 adsorbed layer facing the subchondral bone to repair rabbit articular cartilage defect,which induced high level of BMP2 in synovial fluid at early stage,consequently resulted in successful repair of AC defect.Thus,this targeted delivery system of FGF2 to subchondral bone provides a promising strategy for AC repair due to the relatively clear mechanism,the less amount of the growth factor and short duration of it.