Nucleus Pulposus-related Extracellular Matrix For Differentiation Of Adipose-derived Stem Cells And Intervertebral Disc Regeneration

Intervertebral disc(IVD)degeneration and consequent low back pain is extremely common in clinical medicine.The degeneration of nucleus pulposus(NP)caused by NP cell dysfunction is thought to initiate IVD degeneration.Stem cell-based tissue engineering is a promising treatment of NP degeneration,and adipose-derived stem cell(ADSC)is widely used as seeding cell for its ease of access and ability of NP-like differentiation.However,the suboptimal microenvironment in degenerated NP inhibits the survival and differentiation of ADSCs and therefore limit the clinical use of ADSCs.Bio-scaffold can transplant ADSCs into NP while provide specific microenvironment for cells,so it is regard as a key factor in stem cell-based tissue engineering.Because the existing bio-scaffolds have various defects,it is necessary to create novel bio-scaffold which is more perfect and suitable in NP regeneration.NP-related extracellular matrix(ECM)type ? collagen(CII)and chondroitin sulfate(CS)are native components in healthy NP,and they can also regulate the survival and differentiation of stem cells.So,they are good choices to fabricate bio-scaffold used in NP.This study aimed at fabricating an injectable hydrogel-like ADSCs delivery system based on CII and CS,and investigating its biological effects on ADSCs in vitro.The molecular mechanism of NP-like differentiation of ADSCs induced by this delivery system has also been discovered by determining the interaction between integrin,transcription factor,and related signaling pathway.We have also verified the regenerative effects of our ADSCs delivery system on degenerated IVDs in a rat tail disc degeneration model.This study has been divided into three parts:(1)The fabrication of injectable hydrogel-like cell delivery system based on nucleus pulposus-related extracellular matrix;(2)The molecular mechanism of nucleus pulposus-like differentiation of adipose-derived stem cells induced by type ? collagen;(3)The regenerative effects of injectable hydrogel-like cell delivery system on intervertebral disc degeneration.Chapter ? The fabrication of injectable hydrogel-like cell delivery system based on nucleus pulposus-related extracellular matrixObjective:To demonstrate the biological activities of CII and CS on ADSCs,and fabricate an injectable hydrogel-like cell delivery system based on CII and CS.Methods:ADSCs were cultured in hydrogel fabricated by CII or CS with different concentrations in low pH environment,and ADSCs in pellets were used as a control.After cultured for 3,7,and 14 days,cell proliferation was determined by CCK-8 assay,cytotoxicity of CII and CS were measured by LDH assay,genes of inflammatory were detected by RT-qPCR,and NP-specific protein marker were analyzed by Western blot.Then proper ratio of CII and CS was chosen to fabricate an injectable hydrogel-like cell delivery system.Technologies such as scanning electron microscopy,rheological analysis,and ninhydrin assay were used to determine the characteristic of cell delivery system.In addition,RT-qPCR and Western blot were used to measure the NP-like differentiation efficiency of ADSCs loaded in the cell delivery system.Results:Both CII and CS showed no cytotoxicity on ADSCs.CII at a concentration of higher than 3 mg/mL could significantly improve the protein expressions of NP specific markers,while CS at a concentration between 5-10 mg/mL could significantly inhibit the gene expressions of inflammatory marker.We used 3 mg/mL CII and 8.1 mg/mL CS to fabricate an injectable CII/CS composite hydrogel-like ADSC delivery system under the cross-linking of 0.02%genipin.The delivery system we fabricated showed good gelation property,biostability,and mechanical property.In addition,the delivery system maintained the biological activities of CII and CS,and significantly increased the gene expressions of NP specific markers.Conclusion:The injectable hydrogel-like ADSC delivery system based on CII and CS has good properties,and can stimulate the differentiation of ADSCs into an NP-like phenotype.Chapter ? The molecular mechanism of nucleus pulposus-like differentiation of adipose-derived stem cells induced by type ?collagenObjective:To demonstrate the molecular mechanism of NP-like differentiation of ADSCs induced by CII and mediated by integrin.To clarify the interaction between integrin,forkhead box(Fox)-A2,and the sonic hedgehog(Shh)signaling pathway.Methods:The expression of FoxA2 in ADSCs were overexpressed and knocked down by using lentiviral vectors,and then the ADSCs were cultured on CII.Immunofluorescence,RT-qPCR,and Western blot were used to measure the expression of integrins,NP specific markers,Shh,and Glil.Alcian blue staining,DMMB assay,and hydroxyproline assay were used to measure the synthesis of glycosaminoglycan and collagen.Small molecular inhibitor and block antibody were used to investigate the interaction between integrin a2,FoxA2,and the Shh signaling pathway.Results:The activation of FoxA2 depended Shh signaling pathway was involved in NP-like differentiation of ADSCs induced by CII.FoxA2 overexpression significantly enhanced NP-specific gene expression and the synthesis of glycosaminoglycan and collagen,whereas FoxA2knockdown decreased NP-like differentiation and inhibited the activation of Shh signaling pathway.The enhanced NP-like differentiation related to FoxA2 overexpression was partially rescued by Shh signaling pathway inhibitor GDC-0449.In addition,FoxA2 inhibited the expression of Itga2 and further promoted NP-like differentiation induced by CII.Conclusion:FoxA2 plays an essential role in the NP-like differentiation of ADSCs by activating the Shh signaling pathway,and the inhibition of integrin a2 increases the expression of FoxA2 and the activation of the Shh signaling pathway.Chapter ? The regenerative effects of injectable hydrogel-like cell delivery system on intervertebral disc degenerationObjective:To determine the regenerative effects of the injectable CII/CS composite hydrogel-like ADSCs delivery system on degenerated IVDs in a rat tail disc degeneration model.Methods:Rat tail disc degeneration model was performed by needle puncture.After two weeks,the rats were divided into five groups:the control group(without needle puncture and other treatments);the degeneration group(with needle puncture and DMEM injection);the CCS group(with needle puncture and 0.02%genipin cross-linked CII/CS composite hydrogel injection);the ADSC group(with needle puncture and ADSCs injection);and the CCSA group(with needle puncture and injectable CII/CS composite hydrogel-like ADSCs delivery system injection).0,4,8,and 16 weeks after injection,the rats were evaluated by MRI,radiological,histological,immunohistochemical,and biochemical analysis to determine the NP water content,disc height,IVD structure,and ECM content.In addition,histological grading score was used to quantify the degeneration of IVD.Results:16 weeks after injection,the disc height,NP water content,ECM content of NP in the CCSA group were improved compared with the degeneration group,and the structure of IVD became regular.The regenerative effects of the CCSA group was better than the ADSCs and CCS groups,and inferior to the control groupConclusion:The injectable CII/CS composite hydrogel-like ADSCs delivery system has the ability to regenerate the degenerated IVD to some extent.