| Background and objectiveRotator cuff injury is the most common cause leading to shoulder disability, despite the advancement of repair techniques, rotator cuff retear rates after repair remain high which are about 20% to 94%. According to the results of histopathological researches, a histologically normal insertion site does not regenerate after rotator cuff repair. The main problem is that the fibrocartilage zone at the insertion site can’t regenerate, only reactive scar appears there. Some studies have reported that the repaired interface had weaker mechanical strength compared with the normal one so that it may be vulnerable to retears after repair. Recently, more and more researches around the world have reported the biologic augmentation for cartilage regeneration at the interface of tendon to bone after rotator cuff repair trying to make the histologically normal insertion site regenerate, which has become the hot topic of research.Kartogenin(KGN) is a recently characterized drug with small molecule that promotes the selective differentiation of endogenous mesenchymal stem cells(MSCs) into chondrocytes, thus stimulating cartilage regeneration without exogenous seed cells and scaffolds. In addition, limb skeletal growth and tendon development both can be enhanced by KGN. Therefore, KGN has positive effects on cartilage and tendon regeneration, which makes it possible that KGN was used to promote tendon to bone healing in our study. Otherwise, it has been reported that MSCs from adjacent synovium, tendons and other tissues will promote tendon to bone healing after rotator cuff injury. Therefore, we had the hypothesis that KGN could induce the differentiation of endogenous MSCs into chondrocytes, thus stimulating cartilage regeneration at the interface of tendon to bone healing, which would reproduce the normal morphology of the interface, and the improved quality of tendon to bone healing would reduce the retear rate of repaired rotator cuff. To verify the hypothesis, we performed this study. Research contents and methodsSynovium specimens and bone marrow tissues from patients were collected during surgery at the Chang Zheng Hospital Affiliated to the Second Military Medical University from July 2014 to June 2015. After the primary culture and identification, synovial-derived mesenchymal stem cells(SMSCs) and bone marrow-derived mesenchymal stem cells(BMSCs) were obtained. The effects of different concentrations of KGN on the proliferation ability of SMSCs and BMSCs were detected by CCK-8 cell counting. q RT-PCR was used to detect the effects of different concentrations of KGN on the expression of cartilage phenotype genes of SMSCs and BMSCs, and the ability of differentiating into cartilage of SMSCs and BMSCs with KGN intervention was compared.Fibrin glue(FG) was prepared as KGN carrier, and the feasibility of the drug system was determined by fluorescence spectroscopy via monitoring the loss of KGN in the preparation and the slow release of KGN from the system. Rabbit models of rotator cuff injury were established with the right shoulder joint operated. The drug system of KGN was used to promote tendon to bone healing, then the effects of different concentrations of KGN on cartilage regeneration in the interface of tendon to bone healing at 6 weeks after rotator cuff repair were evaluated by histological staining and the tendon maturing scoring system, the optimal concentration of KGN would be found. In this phase, 15 New Zealand White rabbits were randomly divided into 5 groups(no implant, only FG, 100μmol/L KGN with FG, 500μmol/L KGN with FG, 1mmol/L KGN with FG), there were three rabbits in each group. In the second phase, KGN with the optimal concentration was used to improve tendon to bone healing, in which the sample size increased to verify the efficacy of KGN, 36 rabbits were randomly divided into 3 groups(no implant, only FG, KGN at the optimal concentration with FG), there were 12 rabbits in each group, and the results were observed at 3, 6 and 12 weeks after rotator cuff repair by histological analysis. 54 rabbits were randomly divided into 3 groups(no implant, only FG, KGN at the optimal concentration with FG), there were 18 rabbits in each group, and the results were observed at 3, 6 and 12 weeks after rotator cuff repair by biomechanical analysis.To explore the mechanism that KGN improved tendon to bone healing, based on related researches, 8 candidate target genes associated with cartilage and tendon regeneration were selected for q RT-PCR. Then the expression of BMP-7 gene(found by q RT-PCR screening) in SMSCs was down-regulated by si RNA technology, the effects of KGN on the expression of the genes in the downstream of signaling pathway were detected by q RT-PCR and Western blot. The relationship of BMP-7 and Smad5 in the pathway was verified by immunofluorescence staining. On the other hand, the expression of BMP-7 gene in SMSCs was up-regulated by Lentivirus technology, the effects of KGN on the expression of the genes in the downstream of signaling pathway were detected by q RT-PCR and Western blot. Finally, in order to verify BMP-7/Smad5 pathway in vivo, the expression of BMP-7 and Smad5 at the interface of tendon to bone healing under KGN intervention at 6 and 12 weeks after rotator cuff repair was evaluated by immunohistochemical staining analysis.Results and conclusionIn this research, we found the following results:1. Both human SMSCs and BMSCs had the multi-differentiation potential. KGN had notoxicity for SMSCs and BMSCs. KGN at 10nmol/L~10μmol/L promoted SMSCs proliferation at every timepoint of detection, especially KGN at 10μmol/L. KGN at 10μmol/L promoted BMSCs proliferation during the 2nd and 4th days after the intervention of KGN.2. The expression of aggrecan and type II collagen genes in SMSCs significantlyincreased under the intervention of KGN at 1μmol/L and 10μmol/L, and the expression of TIMP-1 gene in SMSCs also increased under the intervention of KGN at 100μmol/L. While only the expression of aggrecan and type II collagen genes in BMSCs increased under the intervention of KGN at 100μmol/L, but without statistical difference. It was proved that KGN could induce the differentiation of MSCs into cartilage, and the response of SMSCs to KGN was stronger than that of BMSCs, indicating the ability of SMSCs to differentiate into cartilage was better than BMSCs.3. KGN drug delivery system with fibrin glue as the carrier was successfully prepared.Fluorescence spectroscopy suggested that the loss of KGN was negligible during the preparation of KGN drug delivery system, and KGN could be released slowly from the system over time to impact on the surrounding microenvironment.4. The results of histological staining and the tendon maturing scores suggested thatdifferent concentrations of KGN could promote the healing of tendon to bone, especially KGN at 1mmol/L. At 3, 6 and 12 weeks after rotator cuff repair, KGN group(1mmol/L KGN) was superior to no implant group and fibrin glue group in terms of the regeneration of chondrocytes in the interface, the tendon maturing scores, ultimate load to failure and stiffness, all with statistical significance.5. Target genes BMP-7 and Smad5 were selected by q RT-PCR screening, which wereassociated with KGN promoting the selective differentiation of endogenous MSCs into chondrocytes to improve tendon to bone healing. Gene intervention technology,immunofluorescence staining, q RT-PCR and Western blot proved that KGN induced the differentiation of endogenous MSCs into cartilage by activating the BMP-7/Smad5 pathway.6. In vivo, the expression of BMP-7 and Smad5 at the interface of tendon to bonehealing under KGN intervention at 6 and 12 weeks after rotator cuff repair increased significantly, indicating that KGN improved tendon to bone healing by activating the BMP-7/Smad5 pathway.Based on the above results, we found that KGN could induce the selective differentiation of endogenous MSCs into chondrocytes to promote cartilage regeneration at the interface by activating the BMP-7/Smad5 pathway, the normal morphology of tendon-bone junction seemed to be reproduced. In addition, mechanical properties of the interface of tendon to bone healing also were improved. Finally, KGN successfully improved the quality of tendon to bone healing after rotator cuff repair. Therefore, this study found a new kind of biological intervention method for promoting tendon to bone healing after rotator cuff repair. The results of this study will help to further acknowledge the unique application value of KGN in the research field of tendon to bone healing, providing important evidence for more in-depth application researches and related mechanism researches in the future. |
PDF Full Text Request