| BackgroundThe discovery of the nucleus pulposus stem cells(NPSCs)of the intervertebral disc opens the study of the endogenous regenerative repair of the degenerated nucleus pulposus.However,there is still no systematic study on the biological characteristics and the regenerative potential of NPSCs.At the same time,the phenomenon of damage degeneration has been drawn an increasing attention.Therefore,the regeneration of tissue engineering based on NPSCs is expected to be a new method for the treatment of degenerative intervertebral disc.Objectives1.Identification and biological characteristics of human degenerative NPSCs.2.The biological effect of intervertebral disc degeneration on the characteristic of NPSCs.3.BMP-7 active fragment peptide hydrogel RADA-RKPS protects TNF-alpha induced apoptosis of the degenerated nucleus pulposus stem cells.Methods1.Collecting the nucleus pulposus specimens of lumbar degenerative disease and the homologous bone marrow blood specimens.The differential adhesion method was used to obtain the NPSCs.Then the stem cells were identified by morphological observation,cell proliferation,cell cycle,flow cytometry,stem cell markers,three lines induced differentiation and related detection.And compared with the homologous BMSCs,to further clarify the specific biological characteristic of NPSCs.2.In order to study the degeneration process effect of intervertebral disc on biological characteristics of NPSCs.NPSCs with different Pfirrmann degeneration classification were compared by cell morphology,proliferation,colony formation,cell senescence.3.TNF-alpha was used to induced NPSCs apoptosis in vitro,explore the active fragment of BMP-7 self-assembling peptide hydrogel compared to RADA-RKPS whether RADA 16-I cell activity,NPSCs proliferation and degeneration of human nucleus pulposus cell apoptosis and the protective effect of differentiation.Results1.NPSCs and BMSCs in cell morphology,proliferation,cell cycle,dry gene expression has a similar characteristic.Although most of the two cells were similar in cell immunophenotype,the expression of CD 166 in NPSCs was significantly lower than that in BMSCs.In addition,there was no difference in osteogenic and adipogenic ability between the two groups,but NPSCs had a strong potential for chondrogenesis.2.With the aggravation of Pfirrmann degeneration grades.NPSCs showed a decreasing trend in cell morphology,proliferation,colony formation and cell senescence.And the most obvious decrieasing changes were observed in PfirrmannIV,V grades.3.In the RADA 16-I group,the apoptosis inducing group and the control group were confirmed by TUNEL staining and BAX/BCL-2 ratio in vitro.As compared with the RADA 16-I group,RADA-RKPS group showed an increasing effect in cell activity,promoting cell proliferation,reducing cell apoptosis and promoting differentiation of NPSCs,demonstrateing a protective effect on the apoptosis of NPSCs in the degenerated environment.Conclusions1.In this study,we confirmed that NPSCs has similar characteristics of stem cells with BMSCs,but the expression of NPSCs was lower and the chondrogenic differentiation potential was stronger.These results suggest that NPSCs is an ideal mesenchymal source,rather than an embryonic source.2.with the increasing of Pfirrmann degeneration grades,the biological characteristics and function of NPSCs were significantly decreased,which may be the pathological basis and initiating factors of early degeneration of intervertebral disc.3.BMP-7 bioactive peptide self-assembling peptide hydrogel RADA-RKPS can holds the biological effects of protecting NPSCs in inflammatory environment,suggesting that it can be used as an ideal material for degenerative intervertebral disc tissue engineering. |
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