Role Of Mesenchymal Progenitor Cells In Human Articular Cartilage Affected By Primary Osteoarthritis

BackgroundOsteoarthritis (OA) is the most common disease affecting bones and joints, the second leading cause of disability in middle-aged and elderly (especially complicated with cardiovascular and cerebrovascular diseases). OA is catergorized into two types: primary OA and secondary OA. Primary OA is caused by an imbalance of articular cartilage damage and repair. Mesenchymal progenitor cells (MPCs) with self-renewal and differentiation potential can be activated under certain conditions to proliferate and differentiate into osteoblast cells, cartilage cells and fat cells, which are critical in maintaining tissue stability and repairing damages. It has been confirmed that bFGF and TGF-β1 play important roles in promoting the proliferation of mesenchymal stem cells (MSCs). Nuclear binding proteins SOX6 and SOX9, essential for the expressions of genes related to chondrogenic differentiation of MSCs, are key switch for cartilage differentiation. Recent studies have found the presence of MPCs in human normal and primary OA articular cartilage, and the quantity of MPCs in primary OA articular cartilage is much more than that in normal articular cartilage. However, we still know little about proliferation and differentiation potential of MPCs in primary OA and normal articular cartilage as well as their roles in the genesis and development of OA, about effects of bFGF and TGF-β1 on promoting the proliferation of MSCs, and about the role of SOX6 and SOX9 to induce MSCs into cartilage differentiation. So we attempt to isolate MPCs from primary OA and normal articular cartilage by immunomagnetic cell separation method and flow cytometry, study their proliferation features and differentiation potential into adipogenic, osteogenic and chondrogenic lineages as well as their roles in the genesis and development of primary OA, thus exploring the pathogenesis of primary OA. Simultaneously different concentrations of bFGF and TGF-β1 both alone or together were used to promote cell proliferation of MPCs from primary OA cartilage, and adenovirus vectors loading with SOX6 or SOX9 genes infected multipotential MPCs from primary OA cartilage to explore how these genes affect the differentiation. This study aims to better understand the pathogenesis of OA and find out new and effective methods to prevent and treat OA.Objective: To observe the growth and proliferation capabilities of MPCs in primary OA and normal articular cartilage, and their differentiation properties into chondrocytes, osteoblasts and fat cells so as to elucidate the important role of MPCs in the pathogenesis of primary OA by applying cytokines such as bFGF and TGF-β1 and related genes SOX6 and SOX9 to induce cell proliferation and differentiation, aiming to provide theoretical evidence in preventing and curing primary OA.Methods:1. Wet weight of unit cell content was compared between primary OA and normal articular cartilage after modified enzyme digestion. MPCs were sorted out from primary OA and normal articular cartilage through immunomagnetic separation based on the expressions of CD105 and CD166, then further identified by flow cytometry for molecular phenotypes. The characteristics of growth and proliferation of MPCs in normal and OA cartilage were determined by studying cell morphology and cell growth curve.2. The potential of MPCs derived from normal and primary OA cartilage for chondrogenic differentiation was analyzed by means of morphological observation under inverted microscope, TB staining as well as collagen typeⅡand Aggrecan staining, and determination of GAG contents and quantitative RT-PCR for collagenⅡmRNA; that for osteogenic differentiation by means of morphological observation and Von Kossa staining, ALP content determination and quantitative PCR for BGP mRNA; that for adipogenic differentiation by means of oil red O staining and determining relative contents of TG.3. Different concentrations of bFGF and TGF-β1 alone or together were used to treat primary OA cartilage and their effects on proliferation of MPCs were tested by MTT method. SOX6 and SOX9 genes were respectively ligated into adenovirus shuttle plasmids pAdTrack-CMV and pAdEasy-1, then the recombinant plasmids were used to infect MPCs derived from primary OA articular cartilage. TB and the expressions of collagen type II protein and mRNA in differentiated MPCs were compared between the infected group and the uninfected group. Results:1. The wet weight of unit cells isolated from primary OA articular cartilage (1.55±0.16 g/106 cells) was only 41.3% of normal articular cartilage (3.57±0.18 g/106 cells). The rate of CD105+ cells in primary OA and normal articular cartilage chondrocytes was 95.7%±1.16% and 95.2%±1.18%, that of CD166+ was 6.8%±0.26% and 6.6%±0.19%, and that of CD105+/CD166+ was 4.5%±0.09% and 4.6%±0.07%. There was no significant difference between them (P>0.05). The survival rate pf sorted cells was 93%-95%. In MPCs derived from primary OA and normal articular cartilage, CD105+ cells were 96.5±1.12 and 95.5±1.16 respectively, CD166+ cells were 96.8±1.26 and 97.5±1.31, CD105+/CD166+ cells were 93.5±1.09 and 94.6±1.13, while CD34+ cells and CD45+ cells were respectively 0.5±0.012 and 0.4±0.010, 0.8±0.014 and 0.6±0.011. The cell growth and proliferation of MPCs derived from primary OA cartilage was higher than those of normal articular cartilage.2. After induction for chondrogenic differentiation, CD105+/CD166+ MPCs derived from primary OA and normal articular cartilage formed cartilage tissue in micromass pellet cultures, whose TB, collagen typeⅡ, aggrecan staining were all positive, but GAG contents and collagen typeⅡmRNA expression were significantly lower in MPCs from primary OA articular cartilage (P<0.05); After induction for osteogenic differentiation, MPCs were positive by Von Kossa staining, while ALP contents and BGP mRNA expression were decreased in MPCs from primary OA articular cartilage (P<0.05); After induction for adipogenic differentiation, MPCs were positive by red oil O staining, while TG contents increased in MPCs from primary OA articular cartilage (P<0.05).3. Either bFGF (10-50 ng/ml) or TGF-β(0.1-1.0 ng/ml) alone can significantly promote the proliferation of MPCs derived from primary OA cartilage (P<0.05), and with their increased concentration, the proliferation rate was of no significant changes (P>0.05). The combination of 10 ng/ml bFGF and 1.0 ng/ml TGF-βsignificantly increased the proliferation of MPCs from primary OA (P<0.05). Either SOX6 gene or SOX9 gene could stably infect MPCs from primary OA cartilage. TB and Collagen typeⅡwere strongly positive in the SOX6- or SOX9-infected MPCs, while they were weekly positive in the uninfected MPCs. Collagen typeⅡmRNA expression in SOX6-infected MPCs derived from primary OA cartilage was up to 3.8 times higher than that in uninfected cells (P<0.01), and that in SOX9-infected MPCs was 5.15-fold of the uninfected cells (P<0.01). Conclusion:1. Chondrocytes in primary OA articular cartilage were significantly less than those in normal articular cartilage. Multipotential MPCs existed in primary OA and normal articular cartilage. The same quantity of MPCs could be acquired from the same amount of primary OA and normal articular cartilage. The growth and proliferation of MPCs derived from primary OA cartilage were more active than those from normal articular cartilage. So we estimated that MPCs isolated from unit volume of primary OA articular cartilage were remarkably less those from the same volume of normal articular cartilage, indicating the important role of MPCs in pathogenesis of primary OA.2. MPCs have the potential of chondrogenic, osteogenic, and adipogenic differentiation. MPCs derived from primary OA cartilage showed decreased capability of chondrogenic and osteogenic differentiation, but enhanced adipogenic differentiation, suggesting that MPCs in primary OA cartilage is abnormal in differentiation, leading to decreased ability of cartilage cells in repairing articular cartilage damage.3. Both bFGF and TGF-β1 play important roles in the proliferation of MPCs in primary OA cartilage. The stable transfection of SOX6 and SOX9 genes into MPCs derived from primary OA cartilage could significantly promote chondrogenic differentiation of MPCs. There must be feasible methods of gene technology to promote cell proliferation and differentiation of MPCs for repairing articular cartilage injury.