| Backgrounds: Rotator cuff tendon-to-bone healing occurs by formation of a scar tissue interface after repair, which makes it prone to failure. As many as 94% of rotator cuff tendons fail to heal back to the bone after repair. Given the relatively high failure rate of surgical repair, there is a strong need for new repair strategies to improve rotator cuff tendon-bone healing. Bone marrow-derived mesenchymal stem cells(MSCs) are transplanted to the injured sites and have the potential to augment the regeneration of bone, cartilage, muscle, ligament, and tendon in vivo, which makes MSCs an ideal tool for engineered tissue repair and cell therapy in different disease types. Tob1(transducer of ERBB2, 1) is a member of the Tob/B-cell translocation gene(BTG) family, which is a negative regulator of BMP/Smad signaling and negatively regulates osteoblast proliferation and differentiation by suppressing the activity of receptor-regulated Smad proteins. In the present study, we determined if transplantation of MSCs with Tob1 deficiency improved tendon-bone healing after rotator cuff injury. Considering that little is known about upstream regulators of Tob1, we further investigated if mi R-218, a potent osteo-mi R expressed in MSCs, could regulate Tob1 expression and affect tendon-bone healing.Objectives: 1. To explore the biological characteristics of MSCs with Tob1 silencing and lay the foundation for the following research of tendon-bone healing. 2. To investigate the effect of MSCs with Tob1 silencing on tendon-bone healing in a rat supraspinatus repair model. 3. To investigate if mi RNA could regulate Tob1 expression. 4. To explore the effect of MSCs with mi R-218 on tendon-bone healing in a rat supraspinatus repair model.Methods: 1. MSCs were isolated from 12 weeks old healthy male Sprague-Dawley rats. A recombinant lentiviral vector, encoding sh RNA against Tob1 was constructed and used to infect MSCs. The proliferative capacity, differentiation potential and surface markers were tested to evaluate the effects of gene modification on MSCs.2. Sprague-Dawley rats were randomly divided into three groups, each of which received one of three different treatments at the interface between tendon and bone: fibrin glue carrier plus MSCs, fibrin glue carrier plus short hairpin RNA(sh RNA)-Tob1-transduced MSCs and control group. Animals were sacrificed at 4 and 8 weeks, and the tissues were analyzed using histologic, biomechanical testing and q RT-PCR. 3. Screening the candidated mi RNAs for regulating Tob1 expression in MSCs and identifying their target genes. Tob1 expression was detected by q RT-PCR and western blot when mi R-218 was upregulated or downregulated. 4. A recombinant lentiviral vector, encoding mi R-218 precursor was constructed and used to infect MSCs. Sprague-Dawley rats were randomly divided into three groups, each of which received one of three different treatments at the interface between tendon and bone: fibrin glue carrier plus MSCs, fibrin glue carrier plus mi R-218+MSCs and control group. Animals were sacrificed at 8 weeks, and the tissues were analyzed using histologic and biomechanical testing.Results: 1. sh RNA-Tob1 virus, but not the control virus, efficiently reduced the expression of Tob1 gene and Tob1 protein in MSCs. MTT assay showed that knockdown of Tob1 significantly increased the proliferative activity of MSCs on the second and third days. Induced differentiation test showed that MSCs-sh Tob1 have the ability to differentiate into osteoblast and adipocyte. MSCs-sh Tob1 were positive for CD29, CD44, and were negative for CD34, CD45. 2. At 4 weeks the sh RNA-Tob1 group exhibited significantly higher ultimate load to failure compared to the MSC or the control group. However, there were no differences at 4 weeks among the three groups in terms of stiffness. At 8 weeks, the ultimate load to failure was greater in the sh RNA-Tob1 group compared to the MSC or control group. The stiffness of the sh RNA-Tob1 group was significantly higher than that of the MSC or control group. At 4 weeks after surgery, the control group specimens showed a loose fibrovascular interface between tendon and bone consisting mainly of fibroblasts. In the MSC group and sh RNA-Tob1 group, small numbers of cartilage-like cells were visible at the tendon-bone junction. At 8 weeks after surgery, sparse and unorderly collagen fibers were observed in the control group. In the MSC group, perpendicular collagen fibers resembling Sharpey’s fibers and small numbers of chondrocytes were observed at the interface between tendon and bone. Furthermore, more chondrocytes and fibrocartilage were observed at the tendon-bone interface in the sh RNA-Tob1 group compared to the MSC group. We determined gene expression of both collagen type I and II at the injured tendon-bone junction by real-time PCR. At 4 weeks the expression level of collagen type I m RNA was significantly upregulated by treatment with MSCs. Moreover, collagen type I m RNA expression was further enhanced in tendons of the sh RNA-Tob1 group compared to tendons in the MSCs group. There were no significant differences at 4 weeks among the three groups in the expression of collagen type II. However, at 8 weeks, expression of both collagen type I and II m RNA in the MSC group was significantly stronger compared to the control group, and was further increased in the sh RNA-Tob1 group compared with the MSCs group. 3. Using two different target prediction programs, Target Scan and mi Randa, we derived a putative mi R-218 target site within the Tob1 3′UTR(170–177 bp). Luciferase reporter assays showed that after transfecting the Tob1 3′UTR reporter construct in the presence of mi R-218, relative luciferase activity was significantly reduced. Furthermore, Tob1 protein was significantly downregulated by mi R-218 mimic and upregulated by mi R-218 inhibitor. 4. We stably infected MSCs with lentivirus containing mi R-218. The highly upregulated expression of mi R-218 was confirmed by real-time PCR. The Tob1 protein level was significantly downregulated by overexpression of mi R-218. Overexpression of mi R-218 effectively improved tendon-bone healing, as evidenced by elevated levels of ultimate load to failure and stiffness, a greater number of chondrocytes and increased amount of fibrocartilage at 8 weeks.Conclusions: This study showed that Tob1 deficiency enhanced the effect of MSCs on tendon-bone healing in a rat rotator cuff repair model and demonstrated that expression of Tob1 may be regulated by mi R-218. These findings offer a theoretical basis for better application of MSCs to tendon-bone healing. |
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