Role Of Yes-associated Protein(YAP)in Topography-and Mechanical Property-induced Differentiation Of Annulus Fibrosus-derived Stem Cells

Chapter Ⅰ Regional expression of YAP in annulus fibrosus and its relationship with elastic moduli and fiber diameter of collagen fibrilsObjective:To explore the regional variations of YAP expression in annulus fibrosus(AF)and its relationship with elastic moduli and fiber diameter of collagen fibrils.Methods:The AF of a three-week-old New Zealand white rabbit and three-month-old SD rat was harvested respectively.The explanted samples were divided into inner AF(iAF),middle(mAF)and outer(oAF)scanning areas.Atomic force microscopy(AFM)technique were used to measure the fibril diameter of iAF,mAF and oAF.Nanoindentation technique was used to measure the elastic modulus of AF tissue at micro/nano-level.The expression of YAP and phospho-YAP were tested by immunohistochemistry,immunofluorescence and Western blot to explore the regional expression of AF.PCR were performed to evaluate the expression of AF phenotypic marker genes.Results:The diameter of collagen fibrils were observed in AFM increased significantly in the outer layer compared to that of the inner layer.Immunohistochemistry and immunofluorescence staining for YAP in the oAF showed high degree of nuclear localization.In contrast,cells throughout the iAF showed low levels of nuclear YAP.While staining for cytosolic,phosphorylated YAP was more pronounced.The result of Western blot was in accordance with immunohistochemistry that the phosphorylated YAP was higher in iAF than the oAF.The same findings were also observed in mouse AF of 3 month.PCR analysis showed that Col-Ⅰ,Col5a1,Adamts17,Sfrp2,Coll2al were highly expressed in oAF while the Col-II and Aggrecan were expressed in iAF significantly.Conclusion:YAP was mostly nuclear in cells in the oAF which consist of relatively large diameter of collagen fibrils and high elastic moduli,while cells of the iAF showed high levels of phosphylated,cytosolic YAP which consist of relatively small diameter of collagen fibrils and low elastic moduli.These data indicated that YAP activation is necessary for topography and mechanical mediated differentiation of AFSC.Chapter Ⅱ Role of yes-associated protein(YAP)in topography-induced differentiation of annulus fibrosus-derived stem cellsObjective:To achieve diversified differentiation of annulus fibrosus-derived stem cells(AFSCs)by varying the fiber size of cell culture scaffolds and to decipher the possible role of YAP-associated mechanosensing activity on the regulation of cell behaviors through the topographical features of cell culture substrates.Methods:A series of electrospun fibrous scaffolds,whose fiber size approximated that of native AF tissue,were fabricated from biodegradable poly(ether carbonate urethane)urea(PECUU)materials.AFSCs were cultured on the scaffolds with different diameters for 7 days.Following that,Western blot,PCR and immunofluorescence were performed to evaluate the expression of AF marker genes and the nuclear translocation of YAP.Results:After incubation,AFSCs were almost uniformly oriented along the fiber direction of scaffolds.Cells on the scaffolds with small fibers appeared round,while they were spindle-shaped on scaffolds of large fibers.The expression of oAF markers(Col-Ⅰ,Col5a1,Adamts17,Sfrp2,Col12a1)in AFSCs increased with the fiber size of scaffold,while the expression of iAF phenotypic markers(Col-Ⅱ and aggrecan)showed an opposite trend.Yap was translocated to the nucleus in AFSCs on scaffolds with large fibers,while it remained mostly phosphorylated and cytosolic in cells when cultured on scaffolds with small fibers.Inhibition of YAP led to decreased expression of tendon/ligament-related genes,such as Mkx,Bgn and Dcn.Furthermore,increasing fiber size induced cell spreading and promoted the formation of mature focal adhesions of AFSCs.Overall,tuning YAP transcriptional activity by fiber size led to the modification of focal adhesions,and determined cell shape,spreading area and expression of extracellular matrix.Conclusion:Findings from this study indicate that AFSCs,depending on the fiber size of scaffolds,may be differentiated into various types of AF-like cells which resembled the phenotypes of cells in native AF tissue.These provide new insights into the mechanism of YAP mechanosensing activity and imply that it may play a key role in the regulation of cell differentiation in response to topographical variations such as fiber size.Chapter Ⅲ Substrate stiffness-and topography-dependent differentiation of annulus fibrosus-derived stem cells is regulated by Yes-associated proteinObjective:To examine the combined effect of both mechanical and microstructural features on the gene expression of AFSCs.Methods:AFSCs were cultured on four types of poly(ether carbonate urethane)urea(PECUU)scaffolds with controlled elasticity and fiber diameter:soft,small diameter(SS);stiff,small diameter(FS);soft,large diameter(SL)and stiff,large diameter(FL),then incubated for 7 days.Results:AFSCs were almost uniformly oriented along the fiber direction of scaffolds.AFSCs on the scaffolds of small diameter were round,while they were spindle-shaped on scaffolds of large diameter regardless of substrate elasticity.Mature focal adhesions were clustered around the periphery of cells on large diameter scaffolds,especially on stiff scaffolds.On the small diameter scaffolds,unmature focal adhesions were distributed in a much diffused manner.PCR results revealed that when the diameter of scaffold was kept constant,the expression of oAF markers(Col-Ⅰ,Col5a1,Adamts17,Sfrp2,Col12a1)in AFSCs increased with scaffold elasticity,while the expression of iAF phenotypic markers(Col-Ⅱ and Aggrecan)genes showed an opposite trend.Moreover,when scaffold elasticity was controlled,the gene expression of oAF markers in AFSCs increased with fiber diameter.In contrast,the expression of iAF markers decreased.Such substrate elasticity and microstructure dependent changes of AFSCs were similar to the gradient characteristics of native AF tissue.In addition,increasing elasticity and fiber diameter of scaffolds promoted YAP activation and its nuclear translocation.Conclusion:The results illustrate that the mechanical property is a potent regulator of AFSCs differentiation.Moreover,we reveal that microstructure of scaffold affects spreading area,focal adhesion and differentiation of AFSCs.Therefore,both mechanical property and microstructure of scaffold regulate AFSCs differentiation,possibly through a YAP-dependent mechanotransduction mechanism.Chapter Ⅳ Biphasic nanofibers can construct effective tissue-engineered intervertebral discs for therapeutic implantationObjective:To explore the in vivo performances of topographical and mechanical biomimetic tissue engineered(TE)intervertebral disc(IVD)in a rat tail IVD model.Methods:The TE IVD is comprised of an alginate hydrogel-based nucleus pulposus(NP)and hierarchically organized,concentric ring-aligned electrospun poly(ether carbonate urethane)urea(PECUU)-based AF.The outer phase of the scaffold was soft with small diameter(SS)and the inner phase of the scaffold was stiff with large diameter(FL).We inplanted the TE-IVD into the caudal spine of a rat to evaluate its function.Nuclear magnetic resonance imaging(MRI)and H&E analysis were performed to evaluate the stuctural(shape maintenance and integration with surrounding tissues)and functional(mechanical supporting and flexibility)performances of TE IVD.Results:MRI images showed that the brightness and shape of the biphasic PECUU TE-IVD were close to that of the adjacent normal IVDs at 1 month after operation.The height of the disc in the biphasic TE-IVD group was similar to the native IVD at 1 month while the height of the disc in the monophase TE-IVD group decreased significantly compared to the native IVD.H&E analysis showed that the interface between the vertebrae and the biphasic TE-IVD was integrated completely as the tissue interface of the native IVD.It was showed that 62%±5%of AF part was filled with newly formed cell matrix.Conclusions:We have built a three-dimensional tissue engineered total IVD construct containing NP and biphasic hierarchically organized mutilamellar AF with aligned PECUU scaffolds in each layer.The structure of the TE-IVD PECUU scaffolds fully resemble the corresponding native AF tissues.The long-term implantation of the TE-IVD in rat integrates well with the adjacent vertebrae and generates highly hydrated soft tissues.This work provides a novel approach to the goal of building a functional TE-IVD for the treatment of the terminal stage of degenerative disc disease.