| Osteoarthritis is the most common degenerative joint diseases in the elderly, whichusually cause joint pain and deformity, and eventually lead to chronic disability of patients.With the aging of the population of the World, osteoarthritis is rapidly becoming a majormedical and ecomomic burden. Its pathological characteristics include the erosion ofarticular cartilage, osteophyte formation, subchondral bone sclerosis, as well as a series ofbiochemical and morphological changes of the synovium and joint cavity. Currently, thereis no effective treatment to control and slow the progression of osteoarthritis, and explicitits etiology and pathogenesis can provide a scientific basis for the clinical treatment ofosteoarthritis.Objective:The aim of this study was to investigate how static mechanical force alone, as well asCa2+channel block verapamil influence the expression of MCP-1, Pro MMP-1, MMP-3,and MMP-13in chondrocytes of osteoarthritis patient cutured within agarose hydrogelsand made into cell/agarose constructs, and to understand the role of menchanical force andCa2+in the pathogenesis of osteoarthritis, thus to provide a theoretical basis for effectiveclinical prevention strategies and treatment of osteoarthritis.Methods:The cartilage of tibial plateau were selected from12patients with knee osteoarthritisafter knee replacement. The chondrocytes were isolated and cultured from cartilage oftibial plateau，using DMEM medium. After amplification, chondrocytes of every patientwere embedded in agarose hydrogels of37℃to obtain a final concentration of2×106cells/ml respectively. After gelling, cell/agarose constructs were made into cylindricalplugs with5-mm diameter and3-mm height which were put into6holes plates, andconstructs of6patients were divided into experimental group randomly, while another6patients for control groups. Verapamil was added to the experimental group with theconcentration of40μM. Repeated experiments confirm that chondrocytes/agaroseconstructs could keep its integrity under45kPa static pressure within2hours period whenthe agarose concentration was0.5%. The stasis compression of15kPa,30kPa and45kPawere applied on control groups respectively for0.5h,1h and2h by using Compression PlusSystem, and the compression of0kPa was only applied for2h. While for experimental groups, the stasis compression from0kPa to45kPa were all applied for2h. Aftercompression, the cell/agarose constructs were continued cultured at37℃in the presence of5%CO2for24h. At the different time points studied, the medium of constructs werecollected and saved in-20℃. the concentration of MCP-1, Pro MMP-1, MMP-3, andMMP-13were detected with ELISA. And the date were analysed by SPSS17.0usingfactorial analysis, including the expression of MCP-1, Pro MMP-1, MMP-3, and MMP-13under the two factors of mechanical stress and culture time alone or together, as well as thetwo factors of mechanical stress and verapamil.Results:1. Firstly, we analysised the influence of different mechanical force (15kPa～45kPa)and culture time under pressure, on the expression of MCP-1, MMPs.(1) There weresignificant differences of Pro MMP-1expression in three groups of different pressure,P<0.01, and Pro MMP-1levels gradually decreased with the increase of mechanicalpressure.(2) There were also significant differences of Pro MMP-1under the differentpressure times, P=0.04, and they reduced with the extension of culture time.(3)There wasno interactive influence between mechanical force and culture time on Pro MMP-1production, P=0.71.(4) As for MCP-1, MMP-3, MMP-13secretion, there was nostatistical difference on varying pressure, P=0.88,0.94,0.44respectively.(5) And therewere no statistical difference of MCP-1, MMP-3, MMP-13secretion on different culturetime, P=0.31,0.43,0.12respectively.2. Secondly, we analysised the influence of different mechanical force (15kPa～45kPa) and Verapamil, on the expression of MCP-1, MMPs.(1)There were significantdifferences of Pro MMP-1expression in four groups of different pressure, P<0.01, andthere was an declined trend of Pro MMP-1levels gradually with the adding of mechanicalpressure.(2) There was statistical difference between experimental and control group,P<0.01, Verapamil can reduce the secretion of Pro MMP-1.(3) There was no interactionbetween mechanical force and Verapamil, P=0.64.(4) There was no significant differenceof MCP-1levels under different mechanical loading (the group of0kPa was discardedbecause of error).(5) Verapamil can significantly down-regulate MCP-1expression,P=0.05.(6) There was no interaction on MCP-1secretion between mechanical pressureand Verapamil, P=0.09.(7) As for MMP-3, MMP-13, there was no statistical difference onvarying pressure, P=0.32,0.61.(8) Verapamil had no statistical influence on MMP-3,MMP-13secretion either, P=0.51,0.77. Conclusions:1. Chondrocytes/agarose constructs with0.5%concentration of agarose and2×106mlconcentration of cell were appropriate for cell mechanics study in vitro.2. Chondrocytes/agarose constructs could afford the suitable static pressure under45kPa within2hours period via using Compression Plus System.3. Under the suitable mechanical pressure (≤45kPa), the levels of Pro MMP-1secreted by chondrocytes cultured in agarose hydrogels significantly decreased with theamplified pressure.4. Within a certain time (≤2h), the levels of Pro MMP-1was down-regulated with theextension of culture time.5. Verapamil (40μmol/L) can reduce the procuction of Pro MMP-1, MCP-1ofOsteoarthritic chondrocytes in vitro.6. There was no significant influence of mechanical force (≤45kPa) on Pro MMP-1levels secreted by Osteoarthritic chondrocytes.7. And mechanical loading, culture time and Verapamil, Whoever all had no influenceneither on MMP-3secretion nor MMP-13. |
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