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Serum Fibronectin Stimulates Chondrogenic Differentiation Of Human Mesenchymal Progenitor Cells Of Subchondral Bone In Late Stage Osteoarthritis

Posted on:2015-11-06Degree:DoctorType:Dissertation
Country:ChinaCandidate:C JiangFull Text:PDF
GTID:1224330431972883Subject:Clinical Medicine
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Objective:To analyze cell surface antigen pattern and multi-differentiation capacity of cells derived from human subchondral bone in patients of late-stage knee osteoarthritis; and also investigate the roles of serum fibronectin and bone morphogenetic protein-2on chondrogenic differentiation of those cells in vitro.Methods:Human progenitor cells were isolated and derived from subchondral cortico-spongious bone chips from13patients undertaking total knee arthroplasty due to late-stage osteoarthritis. Mesenchymal stem cell related cell surface antigens were analyzed by flowtometry. Multi-differentiation capacity in vitro were documented by histological staining of osteogenic, adipogenic and chondrogenic markers. Real-Time PCR was performed to confirm the related gene expression of chondrogenic differentiation. Besides,25ul/ml serum fibronectin (Fn group),100ng/ml BMP-2(BMP group) were added to the chondrogenic differentiation media separately and together (Co group) to analyze their possible roles on chondrogenic differentiation of those cells. After28days’chondrogenic differentiation inducing, immunohistochemistry staining of Collagen Ⅰ, Collagen Ⅱ, Aggrecan was performed, then total RNA was isolated from15pellets in each group and transcribed to cDNA, followed by Real-Time PCR to analyze the gene expression of Collagen Ⅰ, Collagen Ⅱ, Aggrecan, SOX-9in each group.Results:Cells started to grow out from bone chips in4-7days after placed in high-glucose DMEM in all selected patients, they firmly attached to the surface of culture plates, with spindle-like shape. Flowtometry in passage1cells showed that cell surface antigens in those cells were negative for CD14(0.24%), CD19(0.96%), CD34(0.47%), CD45(0.95%), HLD-DR (0.21%), homogenously positive for CD44(98.69%), CD73(97.94%), CD90(99.88%), CD166(91.46%), and CD105(6.13%), CD146(10.41%) were also detected in parts of cells in all selected patients. In vitro inducing of osteogenic, adipogenic and chondrogenic differentiation were successful in all cells from5patients, with osteogenic differentiation detected by analyzing alkaline phosphatase activity, Alizarin red staining, adipogenic differentiation by Oil Red0staining, chondrogenic differentiation by Alcian blue staining and immunohistochemical staining for CollagenI, Collagen Ⅱ and Aggrecan. In Real-Time PCR testing, the gene expression of Collagen Ⅱ, Aggrecan and SOX-9were all significantly elevated in serum fibronectin group, BMP-2group, compared to control group, with the gene expression of Collagen Ⅰ decreasd, which were more remarkable in Fn group and Co group compared to BMP-2group. But the gene expression had almost no significant differences between Fn group and Co group.Conclusion:multi-potential progenitor cells do exist in subchondral bone in the knee of patients with late-stage osteoarthritis. And serum fibronectin promotes the chondrogenic differentiation of those cells in vitro, which may not be further enchanted by co-action of BMP-2.
Keywords/Search Tags:late-stage osteoarthiritis, subchondral bone, cortico-spongiousprogenitor cells, chondrogenesis, serum fibronectin
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