| Intervertebral disc degeneration is a crucial reason for low back pain, which can lead to a disability that reduces patient quality of life and increases health-care costs. Several studies have demonstrated that alterations in extracellular matrix (ECM) biosynthesis and a decreased number and function of cells in the nucleus pulposus are initial triggers of disc degeneration. Cell transplantation is a treatment of intervertebral disc degeneration causes, andadipose-derived stem cell(ADSC) has been carried out in previous studies. With the proposed method of preconditioning/predifferentiation, mesenchymal stem cells which were differentiated into nucleus pulposus celltype are ideal source of cells for cell therapy. In addition, collagentype II, which is an important component of the extracellular matrix innucleus pulposus, mainly has good biocompatibility and degradation characteristics, and could be a natural carrier for cell therapy. In this study, the effects of TGF-β3 and IGF-1 on ADSC differentiation into nucleus pulposus, and the effects of collagen type II on ADSC biological activities, such as proliferation and differentiation, have been studied in vitro, and the effects of collagen typeⅡ carried with ADSCs by differentiation into nucleus pulposus cell type in porcine disc degeneration model have been studied in vivo. This study has been divided into four parts:(1) TGF-β3 and IGF-1 synergy ameliorates adipose derivedstem cell differentiation towards the nucleus pulposus cell type;(2) Proportion of collagen type II in the extracellular matrix promotes the differentiation of adipose-derived stem cells into nucleus pulposus cells;(3) Transcription factor FoxA2 is crucial regulator of collagen type II induced adipose-derived stem cell differentiation towards nucleus pulposus cell type;(4) Collagen type II carried ADSCs of differentiation into nucleus pulposus cell type in porcine intervertebral disc regeneration.Chapter I TGF-β3 and IGF-1 synergy ameliorates adipose derived stem cell differentiation towards the nucleus pulposus cell typeObjective:This study aimed to investigate the synergy between transforming growth factor beta 3 (TGF-β3) and insulin-like growth factor 1 (IGF-1) on ADSCs and the underlying mechanism using a serum-free culture system.Methods and results:ADSC proliferation and viability was measured using a CCK-8 assay and annexin V-FITC/propidium iodide, respectively. ADSC in micromasses were investigated for differentiation towards nucleus pulposus cells. SOX9, collagen-I, collagen-II, aggrecan, and decorin expression was detected by RT-PCR and immunoblotting. Matrix deposition was assessed by sulfated glycosaminoglycan analysis. Novel chondrogenic and nucleus pulposus genes were detected to distinguish differentiated cell types. MAPK/ERK and TGF/Smad signaling pathways were also examined. As a result, the synergy between TGF-β3 and IGF-1 enhanced ADSC viability, extracellular matrix biosynthesis and differentiation towards nucleus pulposus cells, partly through activation of the MAPK/ERK signaling pathway.Conclusion:Therefore, the synergy between TGF-β3 and IGF-1 ameliorates ADSC viability, differentiation, and promotes intervertebral disc regeneration.Chapter II Proportion of collagen type II in the extracellular matrix promotes the differentiation of adipose-derived stem cells into nucleus pulposus cellsObjective:During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells.Methods and results:Adipose-derived stem cells (ADSCs) were cultured as a micromass or in in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility and cell proliferation increased after collagen type Ⅱ treatment. Expressions of SOX9, aggrecan and collagen type Ⅱ were increased in a collagen type Ⅱ dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type Ⅱ concentration. Only ADSCs highly expressed NP cell marker KRT19 in collagen type Ⅱ culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type Ⅱ-stimulation.Conclusion:The concentration and type of collagen affect ADSCs differentiation into NP cells. Collagen type Ⅱ significantly ameliorates ADSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type Ⅱ may attenuate the differentiation and biosynthesis of transplanted stem cells.Chapter Ⅲ Transcription factor FoxA2 is crucial regulator of collagen type Ⅱ induced adipose-derived stem cell differentiation towards nucleus pulposus cell typeObjective:Type Ⅱ collagen is the vital collagen component of normal nucleus populsus, and affects mesenchymal stem cell differentiation. FoxA transcription factors are essential for the development of dorsal axial structures and required for formation of the intervertebral discs. We investigated the potential mechanism of FoxA family members involving in type II collagen induced adipose-derived stem cell (ADSCs) differentiation into nucleus pulposus cell type.Methods andresults:Rat adipose-derived mesenchymal stem cells were obtained and cultured on plate coated with variable concentration of type II collagen. Cell viability and proliferation were detected by CCK-8 assay. Differentiation related gene and protein expression were examined by RT-qPCR and western blotting, including SOX9, aggrecan, type I collagen, type II collagen, and KRT19. FoxA family members, including FoxAl, FoxA2, and FoxA3, were analyzed by RT-qPCR. And immunofluorescence was used to analyze Foxa2 expression. Overexpression and knockdown of Foxa2 by lentivirus with FoxA2 plasmid and shRNA were presented to explore underlying mechanism of collagen type II induced cell differentiation. Cell viability and proliferation had no significant difference among variable concentration collagen. Differentiation associated gene and protein expression of SOX9, aggrecan, type II collagen, and KRT19 were found to increase significantly in ADSCs on type II collagen coated plate with a dose dependence manner and a time dependence manner in 14 day period. Among FoxA family members, FoxA2 increased significantly in type II collagen induced cells. Overexpression of FoxA2 ameliorated the differentiation into nucleus pulposus cell type, on the contrary, knockdown of FoxA2 attenuate the differentiation process. And FoxA2 involved in MAPK/ERK signaling associated differentiation.Conclusion:Type II collagen mostly affects ADSC differentiation into nucleus pulposus cell type with the time and dose dependence manner. FoxA2 is crucial regulator in type II collagen induced differentiation.Chapter IV Collagen type II carried ADSCs of differentiation into nucleus pulposus cell type in porcine intervertebral disc regenerationObjective:To study the effects of collagen type II carried ADSCs of differentiation into nucleus pulposus cell type in porcine intervertebral disc regeneration.Methods andresults:Bama miniature pig ADSCs have been isolated, cultured, and identified. Porcine disc degeneration model was performed by acupuncture, and the process was according to grouping:negative control group (NC), no acupuncture, no treatment; degeneration control group (DC), injection of DMEM with high glucose after acupuncture; stem cell group (SC), injection of ADSCs of predifferentiation into nucleus pulposus cell type after acupuncture; collagen type II group (CII), injection of collagen type II after acupuncture; stem cell and collagen type II group (SC-CII), injection of collagen type II carried ADSCs of predifferentiation into nucleus pulposus cell type after acupuncture. After treatment, discs were assessed by MRI at each time point. After 24 weeks, intervertebral discs were been evaluated by histology and immunohistochemistry, and the nucleus pulposus were determined by biochemical, RT-qPCR and Western blotting. The results showed that the discs were degenerative in DC group. In SC, CII, and SC-CII group, the discs had regenerated, compare to DC group. And in SC-CII group, the discs also performed similar with NC group.Conclusion:Collagen type Ⅱ carried ADSCs of differentiation into nucleus pulposus cell type can be effective in treating degenerative disc of Bama miniature pigs. |
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