Modulation Of Homeostasis In The Intervertebral Disc: Implications For Intervertebral Disc Degeneration And Regeneration

Intervertebral disc degeneration(IDD)is a leading factor of degenerative spinal disorders,which include intervertebral disc herniation,spine canal stenosis,spine instability,slippage and scoliosis.Hence,IDD and its complications have been a primary problem affecting people’s health and quality of life.In normal human intervertebral disc,there are many significant homeostasis states,mainly including immune homeostasis,cellular homeostasis,extracellular matrix homeostasis and nutrition homeostasis,etc.Meanwhile,these homeostasis states are closely related to each other and are at a dynamic physiological balance in different life stages.The maintenance of these homeostasis states is vital to ensure normal physiological function of the disc.However,the process of IDD begins when the homeostasis is broken due to different internal and external causes.Also,this pathological process can be accelerated with homeostasis missing.To our knowledge,most present studies of IDD mechanism are focused on the modulation of homeostasis in the intervertebral disc as its fundamental core.As for the regeneration of the disc,it is an essential foundation to reconstruct the homeostasis of the disc through the application of biomaterials,stem cells and stimulating factors.In the current study,different homeostasis states of the disc were studied to elucidatethe mechanism of IDD from immunological,cytological and nutritional views.Additionally,the intervention and reconstruction of homeostasis states in the disc were also studied to clarify the mechanism for intervertebral disc regeneration through the application of biomaterials and adipose-derived stem cells.The whole study is divided into the following four parts(the fourth part includes three experiments).Part 1: Fas L on nucleus pulposus cells induces Fas-mediated apoptosis of vascular endothelial cells: implications for the maintenance of disc immune homeostasisBackground and objective: As the largest avascular organ,the intervertebral disc is in an immune homeostasis state.However,the immune homeostasis is broken by immunocytes and inflammatory factors invading,which is mediated by angiogenesis in IDD.Studies have shown that both Fas L and Fas were expressed in nucleus pulposus(NP)cells and vascular endothelial cells.However,it is not clear as to whether Fas L/Fas apoptosis palys a role in preventing angiogenesis in the disc.Methods: Human NP cells and vascular endothelial(HMEC-1)cells were cultured and the expression of Fas L on NP cells was up-regulated by lentiviral vector.ELISA was used to detect soluble Fas L(s Fas L)expression in NP cell cultures.Then,the co-culture system of NP cells and HMEC-1 cells established.After 2 days of co-cultures,HMEC-1 cells were harvested.Flow cytometry was performed for apoptosis analysis and western blot was used for Fas and Fas-related apoptosis pathway detection.Results: We found that the apoptosis of HMEC-1 cells could be induced by NP cells and the apoptosis ratio was higher in normal NP cells compared to degenerate NP cells.The apoptosis ratio of HMEC-1 cells was increased after effectively up-regulating the Fas L expression in degenerate NP cells,with the activation of FADD and caspase-3 pathway.Furthermore,the expression of Fas was increased in HMEC-1 cells following co-cultured with NP cells,which was induced by Fas L produced by NP cells.Conclusions: Our results showed that Fas L expressed on NP cells could induce apoptosis of vascular endothelial cells,indicating its essential role in inhibition of vascular invasion and blocking immune cells infiltration.This finding might shed light on the modulation ofimmune homeostasis of the intervertebral disc.Part 2: Downregulation of cytokeratin 8(CK8)by compressive loads in NP cells:implications for the maintenance of cellular homeostasis in the intervertebral discBackground and objective: Based on its unique structure,CK8 is regarded as an important factor to cell shape and signal pathway.The abnormal expression and phosphorylation of CK8 has been observed in many diseases.Previous studies have shown that CK8 is expressed in human nucleus pulposus(NP)cells and decreases in NP tissues during intervertebral disc degeneration(IDD).Nevertheless,the regulatory mechanism of CK8 in IDD has not been clarified.Methods: Firstly,the expression of CK8 and its phosphorylation in the NP tissue was analyzed by Western blot and immunofluorescence.Then,the NP cells were cultured in compressive load cultures for different time and pressure.Using Western blotting and immune blotting,the expression of CK8 and its soluble expression level in the NP cells was detected.At the same time,the activation of PKC was analyzed.Finally,CK8 phosphorylation and its relationship to PKC activity were analyzed by applying PKC activator and inhibitor.Results: The expression of CK8 decreased and its phosphorylation increased in NP cells with IDD.Compressive load was found to be an important factor inducing the phosphorylation and disassembly of CK8 with a time dependent and degree dependent manner in vitro.Additionally,protein kinase C(PKC)could be activated by compressive load,indicating that PKC was a significant molecular factor contributing to the alternation CK8 expression.Conclusions: To our knowledge,the current study is the first to focus on the molecular mechanisms of CK8 alternation with regard to IDD.The activation of PKC,which is mediated by compressive load,is found to be an essential link between biological force and CK8 alterations,which shed a light on the maintenance of cell structural homeostasis in the intervertebral disc.Part 3: Effect of perfluorotributylamine-enriched alginate on NP cell: implications for the regulation of oxygen homeostasis in intervertebral disc regenerationBackground and objective: Studies have explored many scaffolds for intervertebral disc regeneration.However,the efficiency of applied scaffolds was restricted by nutrient loss.Studies have shown that deficiency of oxygen could lead to IDD.Hence,the regulation of oxygen homeostasis might be helpful in disc regeneration.Accordingly,the aim of this study was to use perfluorotributylamine(PFTBA)as a monitor of oxygen level and explore its role of in oxygen homeostasis for disc regeneration.Methods: Firstly,PFTBA-enriched alginate was formulated.The swelling,degradation and oxygen release behavior of the alginates were measured.Scanning electron microscopy was used for morphological detection.Then,human nucleus pulposus(NP)cells were 2D/3D cultured in the PFTBA-enriched alginates.The survival and proliferation were examined by live/dead staining,CCK-8 assay and cell counting assay.Extracellular matrix synthesis property and NP cell markers alternation were measured by rt-PCR and immunofluorescent staining.Finally,the PFTBA-enriched alginate was used in a mouse disc degeneration model for in vivo validation.Micro-CT and histological assay were used in the animal evaluation.Results: The physical properties of alginate were not influenced by PFTBA.The level of oxygen was up-regulated in alginate with higher PFTBA concentration.The survival and proliferation of NP cells were protected and increased respectively with PFTBA addition.Meanwhile,it was found that 2.5% PFTBA was able to regulate extracellular matrix to a NP-like tissue graft with modest impact on markers of NP cell.In the animal IDD model,alginate with 2.5% PFTBA was found to restore the height and extracellular matrix of the degenerated disc.Conclusions: For the first time,PFTBA was applied as an oxygen regulator in alginate scaffold for disc regeneration in vitro and in vivo.Our results suggest that PFTBA could be a promising option in the regulation of oxygen homeostasis for intervertebral disc regeneration.Part 4: The role of adipose-derived stem cells(ADSCs)in the reconstruction of homeostasis: implications for intervertebral disc regenerationCurrently,options of stem cell treatment for IDD aiming for reconstruction of homeostasis in the intervertebral disc are receiving more and more attentions.Among the stem cell types,the adipose-derived stem cells(ADSCs)gain intensive interest based on its accessibility and abundance.In this part,the role of ADSCs was studied with regard to its function in disc cellular homeostasis and extracellular matrix homeostasis reconstruction in m RNA and micro RNA levels.Meanwhile,the regulatory role of ADSCs in homeostasis protection under un-physiological pressure environment was also studied.Hence,this part was divided into three experiments as follows:Experiment 1: Impact of direct cell co-cultures on human ADSCs and NP cellsBackground and objective: Based on the accessibility and abundance,ADSCs have been proposed as an ideal option in disc regeneration for homeostasis reconstruction.However,the interaction between ADSCs and NP cells still remains unclear,particular with direct co-cultures.Accordingly,this study was aimed for elucidate the influence of direct co-cultures on both ADSCs and NP cells for the first time.Methods: Human ADSCs and NP cells were obtained and cultured.CFDA was used to label ADSCs.Then,ADSCs and NP cells were cultured in a direct co-culture system.After7 days,the cells were harvested and separated by flow cytometry.Rt-PCR was performed to analyze cell phenotypic changes of the two kinds of cells.Results: ADSCs showed directed differentiation to the NP cell phenotype with a significant increased expression of multiple genes in extracellular matrix(ECM),NP markersand pertinent growth factors.Meanwhile,the gene expression of COL2A1,ACAN,and COL6A2 of degenerated NP cells was also increased after co-cultures.Conclusions: The current study suggests that direct co-cultures of ADSCs and NP cells could stimulate ADSCs differentiation toward NP cells,as well as induce degenerated NPcells to recover function.Therefore,ADSCs showed a promissing potential in the reconstruction of homeostasis for the intervertebral disc regeneration.Experiment 2: microRNAs analysis of ADSCs after its differentiation to NP cell phenotypeBackground and objective: In Experiment 1,ADSCs were found to differentiate to NP cells following co-culture with NP cells.However,it remains unknown in the change of micro RNAs of the ADSCs during differentiation.Accordingly,the present study is aimed to clarify the micro RNAs alternation after its differentiation to NP cell phenotype.Methods: ADSCs were obtained from Experiment 1 and their RNA was settled to the mi RCURYTM LNAArray(v.18.0)for micro RNAs analysis.Results: It was found that 30 microRNAs were differentially expressed in the ADSCs following differentiation,including 11 up-regulated micro RNAs and 19 down-regulated micro RNAs.Conclusions: For the first time,the current study explored the preliminary screening of micro RNAs alternation in ADSCs after the differentiation to NP cell phenotype.It provides basic data for further studies in the investigation of ADSCs of disc homeostasis from the aspect of micro RNAs field.Experiment 3: ADSCs protect NP cells from apoptosis and degradation in compressive load environment: implications of homeostasis reconstruction for intervertebral disc regenerationBackground and objective: In experiment 1,we found the trophic influence of ADSCs on degenerated NP cells.As abnormal load can lead to NP cells apoptosis and degeneration,whether ADSCs restore the detrimental influence of disc homeostasis in compressive load environment remains open.Accordingly,the present study is aimed to evaluate ADSCs impact on NP cell in abnormal load settings.Methods: With the co-culture of ADSCs,NP cells were cultured in compressive load at3.0MPa for 48 h.NP cells cultured in the same condition were performed as control group.Flow cytometry,live/dead staining and scanning electron microscopy were applied toassess cell death.The expression of apoptotic pathways was examined.Rt-PCR and immunostaining were used to verify the expression of NP extracellular matrix,matrix degradation mediators and inflammatory factors.To further examine the phenotype of NP cell,the expression of NP markers was also detected.Results: With the suppression of caspase-9 and caspase-3,the apoptosis of NP cells induced by compressive load was inhibited by ADSCs co-cultures.Additionally,ADSCs showed a protective impact on NP cells from the adverse effects of compressive load:ADSCs were found to be able to up-regulate the expression of extracellular matrix genes,TIMPs and cytokeratin 8 expression.On the other hand,ADSCs were also found to inhibite the increase of MMP-3,MMP-13,ADAMTS-1 and-5,as well as inflammatory factors including IL-1β,IL-6,TGF-β1 and TNF-α.Conclusions: ADSCs protect compressive load induced NP cell apoptosis and degradation.The protective impact of ADSCs found in this study could make an essential understanding to our knowledge in their role of homeostasis reconstruction for intervertebral disc regeneration.