High-purity magnesium screws modulate macrophage polarization during the tendon–bone healing process in the anterior cruciate ligament reconstruction rabbit model

Abstract

Magnesium (Mg) screws perform clinical potential in anterior cruciate ligament (ACL) reconstruction, and promote fibrocartilaginous entheses regeneration at the femoral entrance. We aim to prove that high-purity Magnesium (HP Mg) screws modulate macrophage polarization in fibrocartilage interface regeneration both in vitro and in vivo. HP Mg extracts performed good cytocompatibility and significantly promoted M2 macrophage polarization in the flow cytometry and ELISA assays. M2 macrophages stimulated fibrochondrocyte differentiation of co-cultured hBMSCs, and HP Mg extracts had synergistic effect on the process. Then we applied HP Mg screws, with Ti screws as control, in the ACL reconstruction rabbit model. In the histological and immunofluorescence analysis, HP Mg screws inhibited M1 polarization at 2 weeks and highly promoted M2 polarization at 2 and 4 weeks at the tendon-bone interface. Furthermore, regeneration of fibrocartilaginous entheses, rather than the fibrovascular scar interface, was detected in the HP Mg group at 12 weeks. For further mechanism study via RNA-seq detection and WB assays, we found that AKT1 was highly activated in M2 polarization, and HP Mg could stimulate AKT1 expression, rather than AKT2, in the early phase of tendon-bone healing. Our study elucidated macrophage polarization during tendon-bone healing process and emphasized HP Mg on M2 polarization and fibrocartilage interface regeneration via the selective activation of AKT1 and PI3K/AKT pathway.

A strategy for mechanically integrating robust hydrogel-tissue hybrid to promote the anti-calcification and endothelialization of bioprosthetic heart valve

“…In terms of HUVECs, a western blot (WB) assay was performed to characterize the growth state by analyzing the expression of HUVECs markers, including von Willebrand factor (vWF), interleukin-1β (IL-1β), endothelial nitric oxide synthase (eNOS) and platelet endothelial cell adhesion molecule-1 (CD31) [ 20 , 35 ]. In addition, the polarization and the secretion of inflammatory factors of RAW 264.7 were quantified using flow cytometry (LSRFortessa SORP (BD)) and enzyme-linked immunosorbent assay (ELISA) kits, respectively ( Supplementary Method S3 ) [ 36 , 37 ].…”