Improvement Of Intervertebral Disc Degeneration By HAMA Hydrogel Loaded With Salvianolic Acid B And Bone Marrow Mesenchymal Stem Cells

[Objective]To investigate the mechanism by which salvianolic acid B（SalB）inhibits H₂O₂-induced apoptosis of bone marrow mesenchymal stem cells（BMSCs）and the activation of the JAK2-STAT3 signalling pathway in them.;synthesis of hyaluronic acid hydrogels（HAMA）and verification of their physicochemical properties;exploring the biocompatibility of HAMA hydrogels with loaded BMSCs;to investigate the feasibility of SalB combined with BMSCs piggybacking on HAMA hydrogel scaffolds to delay intervertebral disc degeneration（IDD）.[Methods]Cell-based tissue engineering approaches have emerged as a realistic alternative for regenerative disc tissue repair,and bone marrow-derived BMSCs offer an excellent seed cell option for cell therapy of IDD.The potential of bone marrow mesenchymal stem cells（BMSCs）to differentiate into nucleus pulposus cells（NPCs）to replenish the nucleus pulposus cells lost during disc degeneration and thereby slow the progression of IDD has also become a viable option.We first verified whether SalB could reduce H₂O₂-induced apoptosis in BMSCs in vitro using CCK8 assay and flow cytometry,verified the activation of JAK2-STAT3 signalling pathway by SalB by Western-Blotting,and examined the effect of SalB on chondrogenic differentiation of BMSCs by using Alcian Blue staining.The pore size and porosity as well as water retention and degradability of HAMA hydrogels were examined using scanning electron microscopy and lysozyme digestion,and then the biocompatibility of HAMA hydrogel scaffolds was examined by live-dead staining and skeletal staining of BMSCs within the hydrogels.It was also verified that SalB inside the HAMA hydrogel still had a protective effect on BMSCs and could promote the proliferation of BMSCs using the CCK8 method.A rat model was then established using the pinprick method and paraffin sections were taken at 4W and 8W to assess the extent of disc degeneration and the treatment effect of each treatment group by histological staining and immunohistochemistry.[Results]In vitro experiments have demonstrated that SalB can reduce H₂O₂-induced apoptosis in BMSCs by activating the JAK2-STAT3 signalling pathway and increasing its phosphorylation level,while promoting chondrogenic differentiation of BMSCs.HAMA hydrogels have a large pore size and porosity as well as good water retention properties,providing the space required for the growth of BMSCs with good biocompatibility.In contrast,the addition of SalB to HAMA hydrogels still improved the survival of BMSCs within the hydrogels.The results of the rat tail spine sections showed that the HAMA+SalB+BMSCs group had lower histological scores and higher levels of type II collagen and aggrecan compared to the other treatment groups,both at4W and 8 weeks,significantly delaying the progression of disc degeneration in the rat tail spine.[Conclusion]In conclusion,SalB combined with BMSCs encapsulated together in a HAMA hydrogel carrier and implanted into the degenerated disc showed good therapeutic results,and this modality is a very promising treatment strategy for IDD with positive effects on the morphological and functional recovery of the disc.