Polysaccharide Complex Hydrogel Promotes Regeneration Of Degenerated Discs Through Mechanotransduction Pathways And Macrophage Immunomodulation

[Objective] A methacryloylated dextran(Dex MA)light-responsive injectable composite hydrogel loaded with rockweed polysaccharide(Fucoidan)was prepared to investigate its mechanical properties and evaluate its biocompatibility;analyze its restorative and anti-inflammatory effects on intervertebral disc degeneration,and observe its ability to retard disc degeneration.[Methods] In this project,photosensitive injectable polysaccharide composite hydrogel scaffolds were prepared using Fucoidan and Dex MA as raw materials,and their morphological,hydrophilic,and mechanical properties were investigated.In vitro,the biocompatibility of the hydrogels was evaluated by cytoskeleton staining,cell proliferation,and viability.To assess the effects of co-culture of the composite hydrogels with IL-1β-stimulated myeloid cells(NPCs)on cell proliferation and anti-inflammation,cell viability and expression of relevant inflammatory factors were measured by experiments;meanwhile,the CAV1-YAP signaling pathway was examined for CAV1,YAP,p-YAP,The expression of CAV1,YAP,p-YAP,COL II and Agg proteins in the CAV1-YAP signaling pathway was also examined to investigate the effect of the mechanotransduction pathway on cellular ECM.In vivo,a rat intervertebral disc degeneration model was established through a two-layer fibrous ring method and randomly divided into Sham,Control,Fucoidan,Dex MA,and Dex MA-Fucoidan groups.the effect of the composite hydrogel on the repair of degenerated IVD was analyzed by imaging,histology,and immunohistochemistry,and the corresponding modulation was explored.[Results] We successfully prepared a photoresponsive injectable polysaccharide composite hydrogel scaffold,characterized by morphological and mechanical properties.Meanwhile,cellular experiments showed that the Dex MA-Fuc4 composite hydrogel significantly promoted cell proliferation,and cellular activity of NPCs;IL-1β-stimulated NPCs co-cultured in the composite hydrogel not only promoted cell proliferation but also showed significant anti-inflammatory effects;meanwhile,the activation of CAV1-YAP mechanotransduction axis promoted the metabolism of ECM.After injection into the rat IDD model,the composite hydrogel inhibited the local inflammatory response and gradually prevented ECM degradation by inducing the polarization of M2 macrophages.In addition,the Dex MA-Fuc4 composite hydrogel group showed the lowest decrease in intervertebral space height on X-ray and the highest T2-weighted signal on MRI.These results suggest that Dex MA-Fucoidan composite hydrogel is an effective method to promote tissue regeneration in degenerative discs.[Conclusion] The photoresponsive injectable Dex MA-Fucoidan polysaccharide composite hydrogel exhibits superior biocompatibility for local administration of the IVD by the injectable route,achieving in situ cross-linking and curing within tens of seconds through photo responsiveness,not only for effective filling of the nucleus pulposus region but also for closure of the local gap in the fibrous ring.More importantly,it has a delaying effect on the inflammatory response in the degenerated area,allowing the mechanical stability of the IVD to be maintained and thus achieving relief from the progression of IDD.In the microenvironment of the excessive local inflammatory response of the disc,this composite hydrogel scaffold offers a new way of thinking about tissue regeneration and,a promising therapeutic tool for delaying disc degeneration.