A. L. Titan1, E. J. Fahy1, R. E. Jones1, A. Salhotra1, D. S. Foster1, S. Menon1, C. Chan1, N. Quarto1, M. T. Longaker1 1Stanford University,Department Of Surgery,Stanford, CA, USA
Introduction: The tendon-to-bone junction, which facilitates movement of a joint, is a specialized interface that is composed of a structurally continuous gradient from uncalcified tendon to calcified bone. Unfortunately, many common partial tendon-to-bone interface injuries heal in a mechanically inferior fashion and its morphology is never fully recapitulated. The origin of the cells involved in tendon-to-bone healing has yet to be fully elucidated.
Methods: 30 wild type (C57BL/6) and 9 Rainbow (ActinCreER ;R26VT2/GK3) mice were used in this study. All study procedures were approved by Stanford’s APLAC. We performed unilateral experiment procedures in which we created a partial laceration of Achilles tendon at the tendon-to-bone interface. A sham skin incision procedure was performed on the contralateral limb. Mice were allowed free cage activity and all animals were euthanized at either 3 days or 1 week post-operatively. Gross evaluation and histology was performed to confirm the tendon-to-bone interface was intact. A total of 18 Achilles were used for confocal microscopy, histological analyses, 30 were used for Fluorescence-activated cell sorting (FACS) of Skeletal Stem Cells (SSCs) using the following antibody profile: CD45−, Ter119−, CD202b−, Thy1−, 6C3−, CD51+, CD105−, CD200+. Osteogenesis assays were performed from SSCs harvested at post injury day 7.
Results: Gross inspection confirmed that the tendon-to-bone interface remained intact at post-injury day 3 and 7 (Fig 1A). By post injury day 7, all tissue gaps caused from the partial laceration appeared fully closed macroscopically. Confocal microscopy demonstrated an increasing number of clones and size of clones between post injury day 3 and 7 (Fig 1B). FACS analysis revealed an increase in proportions of SSCs at the tendon-to-bone interface that were SSCs from post injury day 3 to day 7 (Fig 1C). SSCs obtained from post injury day 7 successfully underwent osteogenesis, which was confirmed with alizarin red staining (Fig 1D).
Conclusion: Skeletally originated cells including SSCs appear to play an important role in tendon-to-bone interface healing. Additional investigation is required to continue to elucidate the mechanisms of skeletally originated cells’ involvement in tendon-to-bone healing post-operatively.
