Multimodal Mechanical Stimulation Regulate The Differentiation Of Rabbit Annulus Fibrosus-derived Stem Cells

Part I: Effects of cyclic tensile strain on the metabolism on annulus fibrosus-derived stem cells with magnitude-dependenceObjective: The degeneration of annulus fibrosus(AF)is an important cause of intervertebral disc degeneration(DDD).The extracellular matrix(ECM)of AF is rich in collagen,proteoglycan and water,and endures different types of loading,such as compression,torsion,and especially strain.Generally speaking,the imbalance of metabolism in the ECM can lead to the degeneration of AF under excessive biomechanical stimulation.The aim of our research is to probe the possible mechanism of AF degeneration at the cellular level and to determine the effects of cyclic tensile strain(CTS)with different magnitudes on anabolicand catabolic gene expression in annulus fibrosus-derived stem cells(AFSCs).Based on that,it will be possible to find a suitable biomechanical stimulation to fabricate a tissue-engineered AF with the ideal biomechanical performance.Methods: Using enzymatic hydrolysis to isolate and culture annulus fibrosus-derived stem cells from the annulus fibrosus of New Zealand white rabbit,then seeded the cells on the PDMS,adopt cyclic tensile strain(0%,2%,5%,12%,0.5 Hz,4 h/d)to stimulated the cells,then observed the morphology of cells after F-actin and DAPI staining and analyzed the synthetic anabolic genes(Col-I,Col-II,Aggrecan)and catabolic gene(MMP-3,MMP-13,TIMP-1)expression in different groups by RT-q PCR and ELISA.Results: The expression of anabolic genes(aggrecan,type I collagen,and type II collagen)and related proteins in AFSCs of moderate and low mechanical stimulation groups(2%,5%)increased after CTS,especially in the 5% stimulation group.However,there was no obvious difference in catabolic gene expression.The expression of catabolic genes(MMP-3,MMP-13,TIMP-1)and related proteins in AFSCs of the excessive mechanical stimulation group(12%)increased,but anabolic genes(aggrecan,type I collagen,and type II collagen)and related proteins decreased.There were no obvious changes in cell morphology in the 0%,2%,and 5% stimulation groups,however,the morphology of cells in the 12% stimulation group was smaller than other groups;CTFM showed that the CTFs decreased gradually with respect to the magnitude of strain.Conclusion: Our results showed that moderate mechanical stimulation can increase the expression of anabolic genes and related proteins;excessive mechanical stimulation can increase the expression of catabolic genes and related proteins,but decrease the expression of anabolicgenes and related proteins.Part II: The elastomers with different elastic modulus which fabricated with genipin crossing-linked Decellularized Annulus Fibrous Matrix(DAFM)and chitosan regulating the differentiation of rabbit annulus fibrosus-derived stem cellsObjective: Annulus fibrosus(AF)injures is an important cause of intervertebral disc degeneration(DDD).Tissue engineering method has become an ideal approach for AF regeneration recently,which remains great challengs because of heterogeneity of AF tissue in cellular,mechanical,and biochemical aspects.Scaffold is an important part in tissue engineering,however,the current scaffolds for AF tissue engineering were easy to degenerate because of the uniform structure.We aimed to reveal the combined effect of decellularized porcine annulus fibrous matrix(DAFM)and substrate rigidity on biological characterization of rabbit annulus fibrous derived stem cells(AFSCs).Methods: We used genipin crossing-linked DAFM with chitosan to fabricate biomimetic scaffolds for AF tissue engineering which have different elasticity.Tensile tests,SEM and AFM were used to study the mechanical properties and surface characteristics of scaffolds.After being cultured on the scaffolds,the proliferation of AF-derived stem cells(AFSCs)was examined by CCK8 tests.The morphology of AFSCs was observed by SEM and cytoskeleton staining.The genes expression(Col-I,Col-II,Aggrecan)was measured using RT-q PCR.Cell traction forces(CTFs)of AFSCs were determined using CTF microscopy(CTFM).Results: After AFSCs cultured on these scaffolds for 7 days,the gene expression of collagen-II and aggrecan decreased from low elasticity scaffold to high elasticity scaffold,whereas the gene expression of collagen-I just was opposite.At the protein level,the content of collagen-II and GAG gradually decreased from low elasticity scaffold to high elasticity scaffold,whereas the content of collagen-I increased.The cell traction forces(CTFs)of AFSCs gradually decreased from low elasticity scaffold to high elasticity scaffold.Cytoskeleton staining and SEM showed that the morphology of cells were round or near round on the scaffold of low elasticity,while spindle on the scaffold of high elasticity.The gradual changes of AFSCs depending on the substrate elasticity were similar to the cellular,mechanical and biochemical characteristics of cells from outer region to inner region of native AF tissue.Conclusion: DAFM/CS composite scaffolds with different elasticity were fabricated using Genipin-crosslinking.These scaffolds mimicked the outer,middle and inner zones of native AF tissue and markedly affected the differentiation of AFSCs.Importantly,the substrate elasticity-dependent changes(cell morphology,gene expression,and CTF)of AFSCs were similar to the cellular,mechanical and biochemical characteristics of cells from outer region to inner region of native AF tissue.Part III: Multimodal mechanical stimulation regulate the differentiation of rabbit annulus fibrosus-derived stem cellsObjective: The annulus fibrosus tissue engineering has the following problems: cells cannot well adhesive to the scaffolds,and secreted less extracellular matrix(ECM)which lead to build the poor biomechanics of the tissue engineered annulus fibrosus,which cannot achieve the biomechanical performance of natural annulus fibrosus.Therefore,we intend to improve the biological activity of scaffolds to simulate the annulus fibrosus in the structure and function,then stimulated the secretation of ECM by mechanical stimulation.The research firstly fabricated the scaffold with different modulus,then combined with mechanical stimulation,namely multimodal mechanical stimulation regulate the differentiation of AFSCs.The work will provide the foundation for the annulus fibrosus tissue engineering.Methods: Using genipin crossing-linked DAFM with chitosan to fabricate biomimetic scaffolds which have different elasticity in the PDMS,SEM and AFM were used to study the mechanical properties and surface characteristics of scaffolds.AFSCs were being cultured on the scaffolds,applied the CTS on the AFSCs for 3 days,then used SEM and cytoskeleton staining to observe the morphology of AFSCs.The genes expression(Col-I,Col-II,and Aggrecan)was measured by RT-q PCR.Results: Have fabricated the scaffolds with different elasticity on the PDMS.After AFSCs dynamical cultured on these scaffolds for 3 days.In the 2%,5% stimulation gioups,the morphology of cells were round or near round on the scaffold of low elasticity,while spindle on the scaffold of high elasticity,in the 12% stimulation gioup,there were no obvious difference on all the scaffolds,but the cells aligned on the scaafolds.In the 2% stimulation gioup,the gene expression of collagen-I decreased on the low and middle elasticity scaffold,whereas there were no obvious difference in the gene expression of collagen-II and Aggrecan;the gene expression of collagen-I increased on the high elasticity scaffold,whereas the gene expression of collagen-II and Aggrecan decreased;In the 5% stimulation gioup,the gene expression of collagen-I decreased on the low elasticity scaffold,whereas the gene expression of collagen-II and Aggrecan increased;there were no obvious difference in the gene expression of collagen-I,collagen-II and Aggrecan on the middle elasticity scaffold;the gene expression of collagen-I increased on the high elasticity scaffold,whereas the gene expression of collagen-II and Aggrecan decreased on the high scaffolds;in the 12% stimulation group,the gene expression of collagen-I,collagen-II and Aggrecan increased on all the elasticity scaffold.Conclusion: The mechanical properties(modulus)of scaffolds and external mechanical stimulation can affect AFSCs differentiation,namely multimodal mechanical stimulation can regulate the differentiation of AFSCs.The study will provide a theoretical basis and reference value for annulus fibrosus tissue engineering.