BACKGROUND: Tendon injuries result in over 300,000 repairs performed each year in the U.S. However, proper healing is not guaranteed and many require revision. Cell-based tissue engineering is one approach that can address this limitation in tendon tissue restoration. Specifically, the utilization of mesenchymal stem cells (MSCs) is critical in the future development of tissue engineered tendons. BMP-12 is a member of the TGF-β/BMP superfamily and has been implicated to induce MSCs to undergo tenocyte-like differentiation and form ectopic tendon-like tissue when MSCs overexpressing BMP-12 are implanted in vivo. In this study, we tested the hypothesis that BMP-12 treated MSCs seeded in a collagen scaffold and implanted into a tendon defect will augment tendon healing by inducing the formation of tendon-like tissue within the defect.
METHODS: Adult male Sprague-Dawley rats (N=24, 6-9 months old, [390±20g]) were anesthetized and a longitudinal incision was made in the left hind-limb, exposing the calcaneal tendon. The calcaneal tendon was then isolated, and a 5mm long, half-width partial defect was created on the lateral border of the tendon. A collagen scaffold either (1) unseeded (control), (2) seeded with murine MSCs only, or (3) seeded with murine MSCs and directly exposed to rBMP-12 (10ng/mL for 12hrs) was sutured into the defect using 10-0 nylon suture. The skin incision was then closed using 4-0 vicryl suture. Fourteen days post-surgery, the implant and surrounding tissue was harvested and processed for histochemical staining with Toluidine blue and immunohistochemical analysis against an early [scleraxis (Scx)] tendon marker. Sagittal sections were imaged using a light microscope and Axiovision v4.6 software (Zeiss). Harvested implants were also processed for real-time PCR analysis, assessing mRNA expression of Scx and a late [tenomodulin (Tnmd)] tendon marker.
RESULTS: The morphology of implants containing BMP-12-treated MSCs was characteristically different than those of non-BMP-12-treated MSCs and unseeded implants (Fig 1). Specifically, there were increases in cell longitudinal alignment/organization along the tensile axis, matrix deposition, cell elongation, and Scx staining in response to BMP-12 treatment relative to the other conditions. Furthermore, Scx and Tnmd relative mRNA expression was significantly increased in BMP-12-treated implants compared to non-BMP-12-treated and unseeded implants (p<0.001) (Fig 2).
CONCLUSIONS: Based on the morphology and molecular expression results, BMP-12 treated MSCs seeded in a collagen scaffold induced tendon-like tissue formation when implanted in a tendon defect, thus augmenting tendon repair. The morphology recapitulated tendon tissue and the expression of tendon phenotypic genes suggests that the MSCs differentiated into tenocytes. Moreover, a 12-hour, direct exposure of BMP-12 was sufficient to induce tendon-like tissue formation in the implanted seeded-scaffold. The ability to utilize direct chemical induction can have strong implications for BMP-12 in tendon tissue engineering and developing novel drug-delivery systems to treat tendon injury.