In Vitro Experimental Study On Interaction Between Human Degenerative Nucleus Pulposus Cells And Bone Marrow-derived Mesenchymal Stem Cells

Part I Isolation and culturing of human degenerative intervertebral nucleus pulposus cellsObjective: To investigate the isolating and culturing method of nucleus pulposus cells from degenerative human intervertebral discs in vitro. Methods: Nucleus pulposus from 15 human degenerative intervertebral disc samples were collected to extract NP cells by 0.025% collagenaseⅡand 0.004% deoxyribonucleaseⅡdigestion. After culturing and generation, the morphological change of nucleus pulposus cells was observed by continuous observation under inverted microscope. The proteoglycan and collagenⅡexpression were detected by toluidine blue and immunocytochemistry staining respectively, to note the chondrocyte-like phenotype of nucleus pulposus cells. Results: All nucleus pulposus samples were successfully digested by 0.025% collagenaseⅡand 0.004% deoxyribonucleaseⅡand the extracted nucleus pulposus cells were cultured and expanded. The round or polygonal primary NP cell had an average adherence time of 7 days and took approximately 4 weeks to reach 95% confluence. The proteoglycan was stained as blue and the collagenⅡas brown respectively by toluidine blue and immunocytochemistry staining. Conclusion: The 0.025% collagenaseⅡand 0.004% deoxyribonucleaseⅡdigestion can successfully isolated the nucleus pulposus cells from degenerative intervertebral discs. Nucleus pulposus cells demonstrated the chondrocyte-like phenotype in terms of proteoglycan and collagenⅡexpressions. PartⅡExperimental study on isolation, culture and differentiation of human bone marrow-derived mesenchymal stem cellsObjective: To investigate the isolating and culturing method of human bone marrow-derived mesenchymal stem cells in vitro, and to detect their pluripotentiality. Methods: Density gradient centrifugation and different time adherent methods were performed to isolated and purified human bone marrow-derived mesenchymal stem cells from adult bone marrow. During the culturing, the morphological characteristics of bone marrow-derived mesenchymal stem cells were observed by continuous observation under inverted microscope. The surface antigens of bone marrow-derived mesenchymal stem cells were analysed using the FACS. Parts of bone marrow-derived mesenchymal stem cells were induced in osteogenic, adipogenic, chondrogenic medium. Alkaline phosphatase activity, Von Kossa staining, oil red staining, safranin’O-fast Green staining, alcian blue staining,Ⅱcollagen immunohistochemistry were performed to evaluate the osteogenic, adipogenic and chondrogenic differentiation. Results: The bone marrow-derived mesenchymal stem cells were mostly fusiform in shape. Cultures of bone marrow-derived mesenchymal stem cells express CD29, CD44 and CD90 positively, but CD34, CD45 and HLA-DR negatively. After 14 days of induction, the cells were positively stained by alkaline phosphatase activity, Von Kossa staining, oil red staining, safranin’O-fast Green staining, alcian blue staining,Ⅱcollagen immunohistochemistry. Conclusions: Density gradient centrifugation and different time adherent methods can isolate and culture a homogenous bone marrow-derived mesenchymal stem cells population. Multilineage differentiation in the bone marrow-derived mesenchymal stem cells can be confirmed. PartⅢExperimental study on interaction between human degenerative nucleus pulposus cells and bone marrow-derived mesenchymal stem cellsObjective: To investigate the interaction between human degenerative nucleus pulposus cells (NPCs) and bone marrow-derived mesenchymal stem cells (BMMSCs) cocultured in a noncontact system. Methods: The 3th passage BMMSCs cocultured with the first passage NPCs in a transwell plate. Three experimental groups were designed as follows: BMMSCs group, NPCs group and cocultured group. In each group,the changes of TGF-β1, IGF and PDGF in cell supernatants were detected by the enzyme-linked immunosorbent-essay. The proliferation of both BMMSCs and NPCs were observed using cell counting kit-8 and the expression of type I collagen,Ⅱcollagen, aggrecan and Sox-9 gene of these two types of cells were detected by RT-PCR at 4 days, 8 days, 12 days, respectively. The morphological characteristics of BMMSCs and NPCs were observed using inverted microscope. Results: The amount of TGF-β1, IGF and PDGF in cocultured group were significantly higher than those in the two control groups from the 4 th day to 12 th day(P<0.05). Cell numbers of BMMSCs and NPCs in cocultured group were significantly higher than those in two control groups at each time point. Higher levels of aggrecan and Sox-9 expression were detected in NPCs from the cocultured group than those in control group from the 4th day to 12th day(P<0.05). Expression of collagenⅡwas increased significantly in NPCs from the cocultured group than those in control group at the 12th day(P<0.05). Additionally, expression of collagenⅡ, aggrecan and Sox-9 was significantly enhanced in the cocultured group than those in the control group from the 8th day to 12th day. After 12 days of coculturing, morphologic changes of the two types of cells were not noted. Conclusions: During coculturing process, the microenvironment that NPCs exsiting can diffierentiate BMMSCs into NPC-like cells; BMMSCs have ability to regulate the extracellular matrix synthesis of degenerative disc cells through secreting active cytokine TGF-β1、IGF、PDGF.Part IV The effects of TGF-β1 on biological potential of human nucleus pulposus cells from different stages of intervertebral disc degeneration in vitro.Objective: To mimic the trophic effect of bone marrow-derived mesenchymal stem cells using exogenous TGF-β, and to investigate the trophic effect on biological potential of human nucleus pulposus cells (NPCs) from different stages of intervertebral disc degeneration in vitro. Methods: GradeⅡ、Ⅲ、IV intervertebral disc tissues were collected to isolate and culture NPCs in vitro. The experimental groups were designed as follows: gradeⅡdegeneration group, gradeⅢdegeneration group and grade IV degeneration group, and the respectively control groups were established. Administration of 10ng/ml TGF-β1 was performed in each experimental group. After 4 days of TGF-β1 treatment, the proliferation of NPCs in each group was evaluated. The expression of type I collagen,Ⅱcollagen, aggrecan and Sox-9 gene of NPCs were detected by Real-time PCR at 4 days. The protein levels of aggrecan and p53 were evaluated using Western-blot analysis.β-galactosidase staining was performed to evaluate cell senescence. Results: After 4 days of TGF-β1 treatment, the numbers of NPCs in each group increased significantly. Cell numbers of NPCs in gradeⅡdegeneration group were significantly higher than those in gradeⅢand IV degeneration group, respectively(P<0.05). Prior to TGF-β1 treatment, the expression of type I collagen,Ⅱcollagen, aggrecan and Sox-9 gene of NPCs was not different significantly in each experimental group. The expression of each gene increased significantly after 4 days of TGF-β1 treatment. Higher level ofⅡcollagen, aggrecan and Sox-9 expression was observed in NPCs from gradeⅡdegeneration group than those in gradeⅢand IV degeneration group, respectively(P<0.05). The protein level of aggrecan also increased significantly in gradeⅡdegeneration group than those in gradeⅢand IV degeneration group, respectively. The percentage of cells positive forβ-galactosidase staining was higher in gradeⅢand IV degeneration group than that in gradeⅡdegeneration group. The protein level of p53 enhanced significantly in gradeⅢand IV degeneration group than that in gradeⅡdegeneration group. There was no significant difference between the gradeⅢand IV degeneration group. Conclusions: A certain amount of TGF-β1 can significantly stimulate degenerative nucleus puposus cells and enhance the biological potential of nucleus puposus cells, but the severity of disc degeneration can affect the effectiveness of TGF-β1 treatment. These results indicate that the severity of disc degeneration play a role in effectiveness of bone marrow-derived mesenchymal stem cells treatment on intervertebral disc degeneration by affecting trophic effect of bone marrow-derived mesenchymal stem cells.