Changes Of Distribution Of Type Ⅵ Collagen In Osteoarthritis Cartilage Based On Three- Dimensional Images In Situ And The Influence Of Upregulation Of HtrA1 On It In OA Cartilage

Objectives:To explore the changes of spatial distribution of type VI collagen in osteoarthritis cartilage compared to the healthy cartilage. And to explore the role of HtrA1 in OA pathogenesis by studying the distribution of HtrA1 in OA cartilage and its influence on the changes of distribution of type VI collagen.Materials and Methods:Frozen sections of full thick cartilage from osteoarthritis and healthy knees were performed type VI collagen immunofluorescence staining, and then were scanned by Delta-Vision Elite system. The scanning data were processed and analyzed in Imaris software, on which the volume of chondrocyte(Vc) and the thickness and volume of the type VI collagen (Vp) of two groups were measured. Meanwhile, paraffin sections of articular cartilage, harvested from model group and control group, were stained with Safranin O, HtrAl immunohistochemical staining and double immunofluorescence staining of type VI collagen and HtrAl to compare the gross morphology and the positive cell numbers of HtrAl immunohistochemistry of cartilage of two groups and to analyze the relationship as well as the distribution of HtrAl and type VI collagen.Results:In healthy cartilage, every chondrocyte wore a thin coat of type VI collagen, whose thickness had no significant difference (P>0.05) and volume increased(P<0.01) when the depth increased. In osteoarthritis cartilage, the volume of type VI collagen coat decreased in transition layer and radiation layer(P<0.01). Moreover type VI collagen coat worn out irregularly while there were scattered distribution of type VI collagen in the extracellular matrix. Along with the degeneration of cartilage, the proteoglycan content of the cartilage showed by Safranin O decreased significantly in OA group compared with the healthy control group, which was consistent with other researches. The remained proteoglycan concentrated in the PCM and TM. Less normal articular cartilage cells showed positive HtrA1 staining, and significantly increased (P<0.01) in the transitional layer and radiation layer than the surface layer; Almost all chondrocytes in OA cartilage were stained with HtrA1, and the positive rate of each layer showed no significant difference (P>0.05), and the positive stained area was limited to nucleus and cytoplasm and the extracellular matrix was negatively stained. In healthy cartilage, type Ⅵ collagen and HtrA1 co-existed in the transitional layer and radiation layer. In OA cartilage, the fluorescent of type Ⅵ collagen weakened while the corresponding intracellular HtrA1 fluorescence enhanced, indicating that HtrA1 has an important role in changing the distribution of type Ⅵ collagen.Conclusions:The irregular distribution of type Ⅵ collagen in the osteoarthritis cartilage may be one of the factors which induce of chondrocyte degeneration. Chondrocytes of OA cartilage upregulate the secretion of HtrA1 and distributed in the cytoplasm and nucleus, which is likely to play an important role in the change of the spatial distribution of type VI collagen. By inducing the differentiation of chondrocyte and matrix degradation, HtrAl may accelerate the articular cartilage degeneration and OA pathogensis.