The Effect Of Matrix Stiffness And Cellular Mechanical Properties On The Degenerative Intervertebral Discs And The Mechanism

Part 1 Matrix stiffness promotes cartilage endplate chondrocyte calcification in disc degeneration via miR-20a targeting ANKH expressionAbstract: The mechanical environment is crucial for intervertebral disc degeneration(IDD). However, themechanisms underlying the regulation of cartilage endplate (CEP)calcification by altered matrix stiffnessremain unclear. Methods: After that, we used miRNA-mRNA co-expressions to demonstrate that miR-20a was increased and ANKH was decreased when matrix became stiffer. The we demonstrated miR-20a directly targeted ANKH expression. Down-expression of miR-20a or overexpression of ANKH can inhibite high stiffness-induced calcification. Results: In this study, we found that matrix stiffness of CEP was positively correlated with the degree of IDD, and stiff matrix, which mimicked the severe degeneration of CEP, promoted inorganic phosphate-induced calcification in CEP chondrocytes. Co-expression analysis of the miRNA and mRNA profiles showed that increasing stiffness resulted in up-regulation of miR-20a and down-regulation of decreased ankylosis protein homolog (ANKH) during inorganic phosphate-induced calcification in CEP chondrocytes. Then we demonstrated that miR-20a directly targets 3'-untranslated regions of ANKH. The inhibition of miR-20a attenuated the calcium deposition and calcification-related gene expression, whereas the overexpression of miR-20a enhanced calcification in CEP chondrocytes on stiff matrix. The rescue of ANKH expression restored the decreased pyrophosphate efflux and inhibited calcification. In clinical samples, the levels of ANKH expression were inversely associated with the degeneration degree of CEP.Conclusion: Thus, our findings demonstrate that the miR-20a/ ANKH axis mediates the stiff matrix- promoted CEP calcification, suggesting that miR-20a and ANKH are potential targets in restraining the progression of IDD.Part 2 Cellular mechanical properties reflect the differentiation potential of nucleus pulposus-derived progenitor cellsAbstract: Cellular mechanical properties can reflect differences in cellular subpopulations, disease state, and tissue source. Previous work has shown that intervertebral disc (IVD) degeneration can lead to alterations in cell behavior and differentiation potency.Human nucleus pulposus-derived progenitor cells (NPPCs) are an attractive cell source for IVD regeneration. However, information regarding the relationship between cellular mechanical properties and differentiation potential of NPPCs is limited. Methods: In this study, we used AFM to detect each clonal population ofhuman nucleus pulposus-derived progenitor cells before and after differentiation. Results: Here we identified populations of progenitor cells, in the human degenerated nucleus pulposus, which are clonally multipotent and differentiated into mesenchymal lineages. Next, elastic and viscoelastic properties of undifferentiated NPPCs were measured via atomic force microscopy and correlated with differentiation potential. We found that elastic modulus, relaxed modulus, and instantaneous modulus positively were correlated with osteogenic potential. And apparent viscosity was correlated with chondrogenic potential. Conclusion: These results indicated the cellular mechanical properties were predictive of differentiation capability of NPPC clonal subpopulations, and could be used for enrichment based on lineage potential, which could dramatically improve the quality of IVD regeneration. Our results offer insights for a new cell sorting way for regenerative therapy in IVD tissue engineering applications.Part 3 Mesenchymal stem cells regulate mechanical properties of human degenerated nucleus pulposus cells through SDF-1/CXCR4/AKT axisAbstract: Mesenchymal stem cells (MSCs) transplantation to the degenerated intervertebral disc (IVD) have shown promise in decelerating or arresting the degenerative process of IVD. Cellular mechanical properties play crucial roles in regulating cell-matrix interactions, potentially reflecting specific changes that occur with cellular phenotype and behavior. However, the effect of co-culture of MSCs and nucleus pulposus cells (NPCs) on the mechanical properties of NPCs remains unknown. Methods: In this study, we used co-culture system to co-culture MSCs with NPCs. And we used AFM to detect the mechanical properties of NPCs. Then, we used inhibitors and CXCR4 siRNA to knockdown SDF-1 or CXCR4 expression to demonstrate the effect of SDF-1/CXCR4 in mechanical properties of NPCs regulated by MSCs. Results: In our study, we demonstrated that co-culture of degenerated NPCs with MSCs resulted in significantly decreased mechanical modulus (elastic modulus, relaxed modulus, and instantaneous modulus) and increased biological activity (proliferation and matrix gene expression) of degenerated NPCs, but not normal NPCs. SDF-1 was highly expressed in MSCs when co-cultured with degenerated NPCs. Inhibition of SDF-1 using AMD3100 or knocking-down CXCR4 in degenerated NPCs abolished MSCs-induced decrease in mechanical modulus and increased biological activity of degenerated NPCs, suggesting a crucial role of SDF-1/CXCR4. AKT and FAK inhibition attenuated MSCs- or SDF-1-induced decrease in mechanical modulus of degenerated NPCs. Conclusion: it was demonstrated in vitro that MSCs regulate the mechanical properties of degenerated NPCs through SDF-1/CXCR4/AKT signaling. These findings highlight a possible mechanical mechanism in which MSCs-directed cross-talk with degenerated NPCs in MSCs-based therapy of disc degeneration.