Three-dimensional ultrastructural analysis of development at the supraspinatus insertion by using focused ion beam/scanning electron microscope tomography in rats.

To obtain a successful outcome after rotator cuff repair, the repaired tendon must be biologically anchored to the bone. However, the histological structure at the repaired tendon-bone interface differs from that of the site of normal tendon insertion. Therefore, analyzing postnatal development in detail will contribute to understanding the repaired tendon-bone interface after rotator cuff repair. In this study, we analyzed postnatal development at the tendon-bone insertion in terms of temporal changes in SOX9/SCX expression and three-dimensional (3D) ultrastructure with FIB/SEM tomography, a new scanning electron microscopic method. Sixteen postnatal Sprague-Dawley rats were used for the study. One-, two-, three-, and four-week-old rats were sacrificed and both right and left shoulders were removed; eight normal supraspinatus tendon insertions were isolated for each time point. At each time point, four specimens were evaluated with fluorescent immunostaining for SOX9/SCX expression, and the remaining four specimens were evaluated with FIB/SEM tomography. Even in postnatal development, SOX9(+) /SCX(+) expression was observed at the tendon insertion; expression gradually decreased with postnatal development at the normal tendon insertion. In 3D ultrastructure, the morphology of the cells and the number/orientation of the cell processes drastically changed by postnatal week 4. The pattern of SOX9/SCX expression and 3D ultrastructural changes obtained in this study contribute to an understanding of the complicated development of normal tendon-bone insertion. Therefore, this study helps elucidate the pathophysiology of tendon-bone insertion, especially in cases of rotator cuff tear and repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:969-976, 2016.