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

ABSTRACT 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.