| Background:Macrophages are cells that play an important role in tissue repair,and different polarization types of macrophages play different roles in the process of tissue repair;previous studies have shown that M1 macrophages play an important role in the regulation of tendon-bone healing,so it is of great research significance to explore the role of M1 macrophages on tendon-bone healing and the regulatory mechanism.Objective:A transgenic mouse ACLR model with enhanced polarization of M1 macrophages was constructed to investigate its effect and mechanism on tendon-bone healing by secreting interferon-stimulated gene 15 in vitro and in vivo.Methods:Common C57 mice and Tsc1KO(M1 macrophage-polarized)transgenic mice were used to construct ACLR models to observe the polarization of macrophages at the tendon-bone interface,as well as the effect of enhanced polarization of M1 macrophages on tendon-bone healing;the changes in the amount of new bone at the tendon-bone interface were observed by Micro-CT;the connection between the graft and the bone tunnel was evaluated biomechanically to preliminarily determine the tendon-bone interface healing in M1 macrophage-polarized enhanced mice.Then by transcriptome sequencing analysis,Isg15 protein expression was decreased in macrophages of Tsc1KO mice,and then combined with in vitro and in vivo experiments,the expression levels of Isg15 protein in macrophages of different polarization types were further verified;an ACLR model of wild-type C5 7 mice was constructed to verify the effect of Isg15 protein on tendon-bone healing and explore the possible mechanism of promoting tendon-bone healing.Results:Immunofluorescence staining results showed that M1 macrophages were polarized and M2 macrophages were polarized at the tendon-bone interface in common C57 mice,while M1 macrophages were polarized and M2 macrophages were polarized at the tendon-bone interface in Tsc1KO mice.Histological staining results showed that the tendon-bone interface healing score of Tsc1KO mice was significantly reduced at 4 and 8 weeks;Micro-CT results showed that at 8 weeks,the bone tunnel new bone mass of Tsc1KO mice was lower than that of littermate control mice;biomechanics showed that the maximum graft failure tensile force of Tsc1KO mice at 8 weeks was significantly lower than that of the control group;transcriptome sequencing results suggested that there was a significant difference in the expression of Isg15 between Tsc1KO mouse cells and WT mouse cells,and the expression of Isg15 in M1 macrophages was verified to be significantly decreased by in vivo and in vitro experiments;in order to further verify the effect of Isg15 protein,we used sustainedrelease hydrogel to sustained-release Isg15 protein in mouse bone tunnels,histological staining showed that the histological score of mice with sustained-release Isg15 at the tendon-bone interface was significantly increased,and biomechanics suggested that mice with sustained-release Isg15 at 8 weeks had higher maximum failure tensile force and stiffness;immunohistochemical staining showed that sustained-release Isg15 in bone tunnels promoted the expression of Runx2 and OCT at the tendon-bone interface Isg15 sustained-release promoted the formation of new bone mass in the tunnels of mice at two times:4 and 8 weeks;suggesting that Isg15 can promote the formation of bone in the bone tunnel thereby promoting tendon-bone healing.Conclusions:Combined data analysis showed that enhanced polarization of M1-type macrophages resulted in decreased expression of Isg15 protein,resulting in decreased new bone mass at the tunnel,which in turn led to weakened connection at the tendonbone interface,decreased maximum failure load and stiffness of reconstructed tendons,and ultimately led to poor tendon-bone healing;we promoted tendon-bone healing by supplementing rIsg15 protein in the bone tunnel to promote the expression of osteogenic protein at the tendon-bone interface,which suggested that Isg15 protein supplementation may be a potential therapeutic target for the treatment of tendon-bone healing and has certain clinical significance. |
