Porous hydrogel loaded with BMSCs overexpressing IL-4 promotes tendon-bone healing by activating the TGF-β/Smad2/3 pathway

Abstract

Background: rotator cuff injury (RCI) is a common shoulder injury that is difficult to completely repair with surgery. Therefore, new strategies are needed to promote tendon-bone healing. The purpose of this study was to explore the possible mechanism of the protective effect of a porous hydrogel loaded with BMSCs overexpressing IL-4 on tendon-bone healing. Methods: the cell counting kit-8 assay was used to evaluate the cytotoxicity of the porous hydrogel and the protective effects of bone marrow mesenchymal stem cells (BMSCs) overexpressing IL-4. Green fluorescence was detected through immunofluorescence analysis after the porous hydrogel loaded with BMSCs was transfected with the IL-4 adenovirus. The expression of M1 and M2 polarization-related proteins was detected using an immunofluorescence assay. Additionally, apoptosis-associated proteins and TGF-β/Smad2/3 signaling pathway-related proteins were evaluated through immunofluorescence, immunoblotting and PCR analyses. ECMs were evaluated via RT-PCR and immunofluorescence analyses. In vivo CT and histological analyses were performed to evaluate the protective effects of the BMSC-overexpressing IL-4 porous hydrogel. Results: BMSCs were isolated and cultured and met the criteria for the identification of mesenchymal stem cells. Immunofluorescence detection was performed after the transfection of adeno-associated viruses overexpressing IL-4, and it was found that fluorescence intensity was strongest with an MOI value of 80. More importantly, after loading the porous hydrogel, the resulting hydrogel complex could further promote the polarization of M2 cells and inhibit the polarization of M1 cells in vitro. After treatment with the BMSC-overexpressing IL-4 porous hydrogel, the TGF-β pathway was significantly activated in the IL-4/BMSC group, while the protective effect was significantly weakened after SB431542 treatment. The results of in vivo CT and histological analyses showed that therapy with IL-4/BMSCs could partially repair rotator cuff injury. Conclusion: our research demonstrated that the BMSC-overexpressing IL-4 porous hydrogel could not only activate the TGF-β pathway to promote M2 macrophage polarization in vitro but could also facilitate cartilage tissue regeneration in vivo, providing a novel idea for the therapy of rotator cuff injury.