| Background and Objective:The key to the treatment of osteoarthritis(OA)is the effective intervention for early cartilage degeneration.Cartilage tissue engineering is a research hotspot,in which chondrogenic progenitor cells(CPCs)may be the key to cartilage repair.Fibronectin(FN),as one of the extracellular matrix,can be combined with the engineering vector Pluronic f-127 to be able to be applied as injectable FN gel with the potential of clinical application.Research on tissue engineering cartilage construction in our preliminary studies showed that,Traditional Chinese hurb radix clematidis(Weilingxian,WXL)can play to the role of the kind of growth factors,promote cartilage cell(CC)proliferation and collagen type ? polysaccharide and protein secretion and TGF-beta 1 expression,therefore,it can help to solve the existing problems.Therefore,we wonder whether WLX can promote the proliferation of CPCs in vitro and promote cartilage regeneration.The objective of our study was to fabricate a novel injectable fibronectin(FN)/Pluronic F-127 hydrogel with potential clinical application and explore its effect on chondrogenesis of CPCs and whether Weilingxian could promote CPCs proliferation,and the underlying mechanism at the early stage of OA.Methods:(1)Cells isolation and culture.(2)CPCs were isolated from the knee joint cartilage of mice and cultured at various concentrations(0,10,20,30 or 40 ?g/ml)of fibronectin.And best concentration of FN was Selected through the CCK-8 assay.(3)CPCs and CCs were cultured at various concentrations(0,50,100,150 or 200 ?g/ml)of Weilingxian(WLX).(4)To block the integrin ?5?1 signaling,the anti-integrin ?5?1 antibody was added.The effects on proliferation,migration and differentiation of CPC induced by integrin ?5?1 were evaluated through the EdU incorporation assay,CCK-8 assay,western blot,transwell assay,wound healing assay and cell pellet assay and RT-PCR.(5)Mixed Pluronic F-127 and the soluble fibronectin(1:1,v/v)was used as a novel injectable hydrogel.The mice were divided into groups of those not subjected to anterior cruciate ligament transection(sham group and sham with FN/Pluronic group)and those that were subjected to anterior cruciate ligament transection(OA group,OA with FN/Pluronic group and OA with Pluronic group).The different treatments on cartilage were measured via histological scores,collagen content and dynamic distribution of CD105-and CD 166-positive cells.Results:(1)The first generation of cells insolated from full-thickness cartilage was spindle or fibroblast-like,monolayer arranged and maintaining a stable form under inverted phase contrast microscopy,after 7 days of culture in vitro.Most cartilage progenitor cells express specific cell markers:Notch-1,integrin ?5?1,cartilage-specific transcription factor SOX-9 and transcription factor RUNX-2 for bone and cartilage development.Specific stem cell surface antigens were also expressed:CD105(94.30%positive)and CD166(95.11%positive).Hematopoietic stem cell-derived surface antigen and leukocyte common surface antigen were negative.(2)The proliferation of CCs treated with 100,150 or 200 ?g/ml Weilingxian after 48 hours was significantly increased(p<0.05),which reached the highest at 100 ?g/ml,and then going into the platform.Fibronectin has no significant effect on the proliferation of CPCs.(3)The proliferation of CPCs treated with 20,30 or 40 ?g/ml fibronectin was significantly increased(p<0.05),which reached the highest at 20 ?g/ml,and then going into the platform.So 20 ?g/ml was chosen as the optimal cell proliferation concentration for subsequent experiments.Fibronectin has no significant effect on the proliferation of CCs.(4)Proliferation:Compared with the control group,FN group significantly promoted the proliferation of CPCs(514 ± 14.2 vs.381 ± 12.5,p<0.05).Compared with FN group,Mitosis of the addition of FN and integrin ?5?1 antibody group was significantly inhibited(362 ± 13.1 vs.514 ± 14.2,p<0.05).Migration:Compared with the control group,the average number of cells per field(± variance)in FN group increased to 153(±4.2)and 256(± 6.2)at 12h and 24h respectively(p<0.05);Compared with FN and integrin ?5?1 antibody group,cell migration was significantly inhibited(p<0.05).Differentiation:no matter whether integrin ?5?1 pathway was blocked,type ? collagen expression had no difference among the groups.Compared with FN group,type ? collagen and aggrecan decreased significantly in FN and integrin ?5?1 antibody group.(5)HE staining:cartilage surface of sham group and sham + FN/Pluronic group was smooth.Synovial cell proliferation,inflammatory cell infiltration,and cartilage degenerative changes were observed in OA and OA+ FN/Pluronic groups.In OA+ FN/Pluronic group,there was a slight trembling of the cartilage surface,but cartilage or cartilage progenitor cells increased.The surface cartilage layer was discontinuous in the OA+ Pluronic group.Safranine green staining:OA mice have loss of proteoglycan(saffron staining fades),cartilage surface is rough and discontinuous,and there is a certain lack of chondrocyte layer.OA + FN/Pluronic group showed slight cartilage trembling but no cartilage loss or discontinuity.The vertical fracture in the OA +Pluronic group extended below the cartilage surface(<25%).Type ? collagen immunohistochemical staining:the type II collagen staining of sham group and sham + FN/Pluronic group were strongly positive,and had no significant changes.OA + FN/Pluronic group was strong stained,suggesting protective effect of FN/Pluronic,and staining of O A group obvious decreased(88.8 ± 5.9/45.6 ± 6.2 vs.25.6± 4.7,p<0.05).The number of positive cells in OA +Pluronic group was significantly lower than that in OA + FN/Pluronic F-127 treated group(45.6± 6.2 vs.88.8 ± 5.9,p<0.05).OARSI score:OA + FN/Pluronic F-127 treatment group significantly inhibited joint degeneration(2 ± 0.62,p<0.05),while OA non-treatment group had the highest score.The OA + Pluronic group had a decreased score(6 ± 0.73 vs.10 ± 0.58,p<0.05)but remained higher than the OA + FN/Pluronic group(6 ± 0.73 vs.2 ± 0.62,p<0.05).Biomechanically,the strength of the cartilage in the OA with FN/Pluronic group was more superior to those in the other two OA groups but still lower than those in the sham groups.Dynamic Distribution of CPCs:Immunofluorescence:2-3 weeks of CD 105 positive cells in the cartilaginous surface of the OA-untreated and OA+ FN/Pluronic F-127 treated groups were uniformly arranged two weeks after the operation.After 6 weeks,the number of CD105 positive cells accumulating on the cartilage surface of OA + FN/Pluronic F-127 group reached 4-5 layers.However,in the OA-untreated group,the number of CD 105 positive cells at the site of injury was significantly decreased.After 2 weeks,the proportion of surface positive cells of OA + FN/Pluronic F-127 treatment group increased from 41.8%(standard deviation 7.2)to 87.5%(standard deviation 8.7).However,the OA-untreated group showed no significant change from 2 weeks to 6 weeks(42.6%to 32.7%,p = 0.16).Flow cytometry experiments:It was found that in Separation of superficial cells from the cartilage surface using sequential pronase/collagenase digestion,CD105 and CD166 were overexpressed at 6 weeks postoperatively in OA + FN/Pluronic F-127 treated groups(mean positive cell fraction 80.9%And 81.6%,p<0.05).However,there was no significant change in OA group(p = 0.20).Western blot results:Compared with 2 weeks after operation,the expression of integrin ?5?1 in OA fibronectin/Pluronic F-127 group was significantly increased 6 weeks after operation,indicating that intra-articular injection of certain concentration of fibronectin/Pluronic F-127 hydrogel alleviate joint damage by up-regulating integrin ?5?1.However,there was no significant difference in the OA-treated group at 2 weeks and 6 weeks after operation.The exogenous fibronectin appeared as a dense region without cells but had strong staining for FN by immunohistology assay on the cartilage surface in the early stage OA model.Conclusion:These findings suggest that FN enhances CPC proliferation,migration and chondrogenic differentiation via the integrin ?5?1 signaling pathway.Based on these results,we prepared a novel injectable fibronectin/Pluronic F-127 hydrogel to repair cartilage lesions that could offer an important theoretical basis for the treatment of cartilage degeneration. |
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