Effects Of Strontium Ranelate, HEGF, NPY And WISP3 In Human Articular Chondrocytes In Vitro

BackgroundOsteoarthritis (OA) is a slowly progressive degenerative disease characterized by gradual loss of articular cartilage, but its mechanism is still unknown. Biochemical and genetic factors contribute to cartilage lesions in OA by disrupting chondrocyte-matrix associations and altering metabolic responses in the chondrocyte. Spondyloepiphyseal dysplasial tarda with progressive arthropathy (SEDT-PA) is an autosomal-recessive hereditary disorder of cartilage homeostasis and characterized by progressive joint stiffness, pain, limited mobility, soft tissue swelling and joint deformities. The pathogenesis of SEDT-PA is unkown though it has been demonstrated that WISP3 is the causing gene.The degradation of articular cartilage is the main pathological changes in OA and SEDT-PA. Matrix metalloproteinases (MMPs) are long considered to play a central role in the degradation of cartilage aggrecan and collagens, especially for MMP-1, MMP-3 and MMP-13. Recent biochemical studies demonstrated that one of the aggrecanases, the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families play a significant role in the progression of OA. However, ADAMTS-5 (aggrecanase 2) has generally been considered to be the most important aggrecanase in OA, while Wnt-1-induced signaling protein 3(WISP3), an essential protein for mainting cartilage intergrity by regulating the expression of collagen II and aggrecan, was recognized to be the causative factor related to SEDT-PA.Some studies have demonstrated that Strontium Ranelate, hEGF, NPY and WISP3 played siginificant roles in the formation and metabolic homeostasis of bone, whether they have functions in the progression of OA is not clear. Aim of the study is to understand the effects of these factors on articular chondrocytes and three conclusions could be drawn from the present investigation:(1) articular chondrocytes cultured in embedded board containing rat tail collagen type I is an excellent differentiation technique for chondrocytes study in vitro and we have found that the proliferation of SEDT-PA chondrocytes is the fastest while OA chondrocytes proliferate slowestly; (2) increased expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 is likely to be linked to the cartilage degeneration in many disordered conditions; (3) SR, NPY, WISP3 and hEGF can prevent the cartilage from degradation in OA and SEDT-PA by inhibiting the expression of ADAMTS-5, MMP-3 and MMP-8.PART ONEImmunohistochemial Expression and Significance of ADAMTS-5,WISP3,MMP-1,MMP-3 and MMP-13 in human articular cartilageObjective:To ivestigate the relationship between cartilage degeneration and the expression of aggrecanase 2 (ADAMTS-5), Wnt-1 inducible secreted protein 3 (WISP3), matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3) and matrix metalloproteinase (MMP-13) in osteoarthritis.Methods:The histological changes of cartilages by hematoxyllin-eosin staining and Immunohistochemical expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 were studied in 14 osteoarthritis cases and 4 normal controls. Results:Articular tissue became predominantly fibroplastic complicated by a decrease in the number of chondrocytes and with the appearance of clustered and hypertrophic ones which subjected to apoptosis, as well as a reduction of extracellular matrix. The immunohistochemical expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 were higher in OA articular cartilage compared to that of the normal subjects.Conclusions:The increased expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 was likely linked to cartilage degeneration.PART TWOCulture of human articular chondrocytes in embedded board containing rat tail collagen type I in vitroObjective:To investigate the proliferation of chondrocytes cultured in embedded board containing rat tail collagen type I.Methods:Chondrocytes were isolated by enzymic digestion from the articular cartilage from patients with knee replacement surgery, and cultured in monolayer system until the second generation, then cells were cultured in embedded board containing rat tail collagen type I. The cells were used for evaluation cell growth by phase-contrast microscope, toluidine blue method, and cell proliferation by MTT method.Results:The chondrocytes cultured in embedded board containing rat tail collagen type I grew much better than cultured in monolayer system. The SEDT-PA chondrocytes proliferated fastestly and showed the strongest heterochromia to toluidine blue staining,while the OA chondrocytes proliferated slowestly and showed the weakest heterochromia when compared to normal chondrocytes which proliferated with a medium speed and showed middle heterochromia.Conclusions:OA chondrocytes cultured in embedded board containing rat tail collagen type I presented the nature phenotypes characterized by human OA articular chondrocytes and can be regarded as the better techniques for experimental approach of OA.PART THREEThe Effects of Strontium Ranelate, WISP3, hEGF and NPY on Human Articular Chondrocytes in VitroObjective:To investigate the effects of Strontium Ranelate(SR), WISP3, hEGF and NPY in human articular chondrocytes in vitro.Methods:Normal, OA, and SEDT-PA articular chondrocytes were cultured in vitro. Appropriate concentrations of SR, WISP3, hEGF and NPY were added to these cells culture medium. The effects of these factors on cultured cells were evaluated by MTT, Western-blot and ELISA methods.Results:A promoted proliferation of SR and WISP3 on OA chondrocytes but not for SEDT-PA cells was repeatedly observed with MTT. The results of ELISA and Western blotting were supported by the following findings:①SR, WISP3, hEGF and NPY could reduce the expression of ADAMTS-5,and WISP3 increased the expression of ADAMTS-5；②SR, hEGF could increase the expression of singalling molecules JNK in OA, and hEGFas well as NPY showed to increase the expression of singalling molecules ERK in OA;③SR reduced the expression of MMP-3, NPY, hEGF, SR, WISP3 increased the expression of MMP-8 in OA, but NPY, hEGF, SR reduced the expression of MMP-8 in SEDT-PA, NPY, hEGF, SR, WISP3 reduced the expression of MMP-13 in OA and SEDT-PA.Conclutions:SR, NPY, WISP3 and hEGF could prevent the OA and SEDT-PA cartilage from degradation by inhibiting the expression of ADAMTS-5, MMP-3 and MMP-8.