Effect Of Secretory Leukocyte Protease Inhibitor On Tendon-bone Healing In Rat

BackgroundTendon-bone healing has always been a hot spot in the field of sports medicine research,because of the histological differences between tendon and bone,while the complex and slow process of tendon-bone healing often leads to the difficulty in restoring to the normal tendon-bone insertion after anterior cruciatc ligament(ACL)reconstruction,and the low biomechanical strength of healing always leads to reconstruction surgery failure,so it is essential to find an effective method to promote tendon-bone healing.ObjectiveTo explore the effect of secretory leukocyte protease inhibitor(SLPI)on tendon-bone healing after ACL reconstruction in rats in vitro and in vivo experiments.MethodsIn vitro experiments:First,osteogenic differentiation and SLPI overexpression were performed on rat bone marrow mesenchymal stem cells(BMSCs),and the expression levels of osteogenic differentiation-related genes Runx2,ALP,OCN,and OPN were detected by real-time PCR(qPCR)to preliminarily determine the effect of SLPI on the osteogenic differentiation ability of BMSCs;in addition,the effect of SLPI on the migration and invasion of rat BMSCs was additionally evaluated by cell scratch assay.In vivo experiment:A rat model of unilateral knee ACL reconstruction was used.Thirty-six SD rats were randomly divided into SLPI group and control group,in which fibrin gel mixed with SLPI recombinant protein was injected around the tendon graft,while only the equatorial fibrin gel was injected around the tendon graft in the control group.At the 2nd,4th and 8th week after reconstruction,the animals were sacrificed,then the effects of SLPI on tendon-bone healing after ACL reconstruction in rats were evaluated by histological observation,micro-CT analyses and biomechanical testing.ResultsThe qPCR results suggested that SLPI could up-regulate the expression levels of Runx2,ALP,OCN and OPN at different stages of osteogenic differentiation of BMSCs,preliminarily indicating that SLPI could improve the osteogenic differentiation ability of rat BMSCs.The relatively high scratch healing rate indicated that SLPI could improve the migration and invasion ability of rat BMSCs.The results of histological observation and scoring of tendon-bone interface showed that at each time point after reconstruction,there was more and thicker continuous new bone formation at the tendon-bone interface in the SLPI group,and orderly arranged collagen fibrous tissue and chondroid cells appeared earlier at the tendon-bone interface than control group.The histological score of tendon-bone interface in the SLPI group was higher than the control group,with significant differences at 4 weeks and 8 weeks.The results of immunohistochemical staining at the tendon-bone interface showed that the expression level of Runx2,an osteogenic differentiation-related marker,was higher in the interface of the SLPI group,and the expression of OCN began to be significantly up-regulated in the 4 and 8 weeks,respectively.Micro-CT analysis revealed more new bone formation around the tendon graft in the SLPI group,and the cross-sectional area of the tibial tunnel was smaller in the SLPI group than the control group at the same time points.Finally,the results of biomechanical tests evaluating the strength of tendon-bone healing showed that the transplanted tendons in the SLPI group had higher maximum tensile force and stiffness at 8 weeks after ACL reconstruction.ConclusionsBased on the results of in vitro and in vivo studies,SLPI can improve the migration and invasion ability and osteogenic differentiation ability of rat BMSCs.SLPI also can promote the process of osteogenic differentiation at the tendon-bone interface,so as to form more new bone around the tendon graft,and ultimately improve the biomechanical strength of tendon-bone healing.This study provides a new viewpoint for promoting tendon-bone healing and can provide a new therapeutic direction for promoting early stage of tendon-bone healing in clinical practice.