The Study On Infrapatellar Fat Pad Derived-Stem Cells In Cartilage Repair

Objectives:Articular cartilage lacks self-regeneration and self-healing ability,and the repair of cartilage lesion is still a major clinical problem.The development of regenerative medicine has shown great promise for the treatment of cartilage lesions.Clinical trials of stem cell therapy for articular cartilage repair have been carried out in some countries and regions.At present,these studies are still in the stage of exploration and trial and error,so many researchers focus on strategies that can optimize and improve stem cell-therapy,including the cell sources selection,preconditioning and co-delivery.The purpose of this study was to evaluate the feasibility of IPFP-SCs as a source of stem cells for repairing cartilage,study the optimization of PRP and cell separation,and to explore the methods of repairing articular cartilage by IPFP-SCs so as to provide ideas and references for clinical application.Methods and results:This study consisted of three experimental parts.In the first part,rabbit IPFP-SCs were isolated by enzyme digestion,cultured in vitro,and identified by phenotypical characterization.Furthermore,their differentiation ability was detected by tridirectional differentiation method.Two-centrifugation method was used to prepare leukocyte-containing PRP(L-PRP)and pure PRP(P-PRP).In vitro experiments were performed to compare the effects of two different forms of PRP on IPFP-SCs,such as cell proliferation rate,cell phenotype and gene expression,and to detect the effects of L-PRP and P-PRP on the chondrogenic capacity of IPFP-SCs.In the second part,IPFP-SCs were isolated,extracted and cultured by non-enzymatic digestion method,and compared with IPFP-SCs obtained by traditional enzymatic digestion method in cell proliferation capacity,characterization and three-way differentiation ability.Additonally,the differences in chondrogenic differentiation between the two groups were compared.In the third part,24 New Zealand white rabbits were randomly divided into 4 groups: IPFP-SCs isolated from non-enzymatic method co-delivery with P-PRP group(Group A),IPFP-SCs isolated from enzymatic method co-delivery with P-PRP group(Group B),SVF isolated from enzymatic method group(group C)and saline group(group D).The full-thickness cartilage defect on the rabbit femoral condyle was used as the animal model.Groups A,B and C were treated with the intra-articularly injection using different formulas of cells,and the blank control group was injected with normal saline only.Rabbit jointspecimens were taken 6 weeks and 12 weeks after the operation for macroscopical and histological analysis.Statistical methods for histological analysis were used to evaluated the effects of the IPFP-SCs in different forms for cartilage regeneration.Results: 1.The IPFP-SCs isolated by enzymatic digestion method were characterized in the terms of cell morphology,immunophenotype and multilineage differetiation capacities,and the results met the criteria of mesenchymal stem cells(MSCs);2.IPFP-SCs were cultured in the presence of L-PRP and P-PRP prepared by two centrifugation methods,both of which had pro-proliferation effects,and the pro-proliferation rate was correlated with PRP concentration.After the intervention,no significant difference was found in the flow detection of cell phenotype,and the expressions of COL2,ACAN and SOX9 in the P-PRP group were higher than those in the L-PRP group and the control group.As a result of chondrogenic pellet induction culture,COL2,ACAN and SOX9 expressions were increased in chondrogenic pellets after the intervention of PRP,and more proteoglycan deposition and type II collagen production were observed histologically.3.The time required to obtain SVF by enzyme-free non-digestion method was significantly less than that of enzyme digestion,and the acquisition rate of primary cells was significantly lower than that of enzyme digestion.After culture and amplification,IPFP-SCs from two different sources showed no significant difference in cell proliferation,cell phenotype and tri-differentiation ability.4.In animal experiments,the repair effect of cartilage defect area in group A and B was the best,and there was no significant difference between the two groups.The repair effect of group C was weaker than that of the first two groups,but it was better than group D.Conclusion:1.IPFP-SCs have the basic characteristics of stem cells and have the potential for chondrogenic differentiation,Infrapatellar fat pad can serve as an excellent stem cell source for regeneration medicine and have brilliant prospect in the future.2.L-PRP and P-PRP with different leukocyte content could promote the proliferation of IPFP-SCs,but did not affect the cell phenotype of IPFP-SCs.Compared with L-PRP,P-PRP was more conducive to the promotion of IPFP-SCs into chondrogenic differentiation.3.The time required for isolation process of SVF by non-enzymatic method was much less that of enzymatic digestion method,the amount of total primary cellsyielded by enzymatic digestion method was much greater.IPFP-SCs obtained with the two method had the similar level when characterizing the cells in the terms of the proliferation ratio,cell phenotype and differentiation potential.4.The use of non-enzymatically isolated SVF in rabbit animal cartilage defect model was beneficial for cartilage repair,while the combination of amplified IPFP-SCs and P-RPP had the best repair effect.