Growth differentiation factor 5(GDF5)-induced mesenchymal stromal cells enhances tendon healing.

Abstract

Mesenchymal stromal cell(MSCs) has immense potential for use in musculoskeletal tissue regeneration, however, there is still a paucity of evidence on the effect of tenogenic MSCs(TMSC) in tendon healing in vivo. This study aimed to determine the effects of GDF5-induced rbMSCs in infraspinatus tendon healing in a New Zealand White rabbit model. In this study, bone marrow-derived rbMSCs were isolated, and 100 ng/ml GDF5 was used to induce tenogenic differentiation in rbMSC. The effects of GDF5 on rbMSC in vitro were assessed by total collagen assay, gene expression analysis and immunofluorescence staining of tenogenic markers; native tenocytes isolated from rabbit tendon were used as a positive control. In in vivo, a window defect was created on the infraspinatus tendons bilaterally. After three weeks, the rabbits(n=18) were randomly divided into 6 groups and repaired with various interventions: (i)surgical suture; (ii)fibrin glue; (iii)suture and fibrin glue; (iv)suture, fibrin glue and tenocytes(rbTenocyte); (v)suture, fibrin glue and MSCs as well as, (vi)suture, fibrin glue and TMSC. All animals were euthanized at 6 weeks postoperative. The in vitro GDF5-induced rbMSCs (or TMSC) showed increased total collagen expression; augmented scleraxis(SCX) and type-I Collagen(COL-I) mRNA gene expression levels. Immunofluorescence showed similar expression inGDF5-induced rbMSC to that of rbTenocyte. In vivo histological analysis showed progressive tendon healing in TMSC treated group; cells with elongated nuclei aligned parallel to the collagen fibers and the collagen fibers were in more organized orientation, along with macroscopic evidence of tendon callus formation. Significant differences were observed in cell treated groups compared to the non-cell treated groups. Histological scoring showed a significantly enhanced tendon healing in TMSC and MSC treated groups compared to rbTenocyte group. The SCX mRNA expression levels, at 6 weeks following repair, were significantly up-regulated in the TMSC group. Immunofluorescence showed COL-I bundles aligned in parallel orientation; this was further confirmed in AFM imaging. SCX, TNC and TNMD were detected in the TMSC group. In conclusion, GDF5 induces tenogenic differentiation in rbMSCs, and TMSC enhances tendon healing in vivo compared to conventional suture repair.