Ultrastructural characterization of cells in the tibial stump of ruptured human anterior cruciate ligament, their change
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ORIGINAL PAPER
Ultrastructural characterization of cells in the tibial stump of ruptured human anterior cruciate ligament, their changes and significance with duration of injury Mayur Nayak1 · Hira Lal Nag2 · Tapas Chandra Nag3 · Rahul Yadav1 · Vishwajeet Singh4 · Siddhartha Maredupaka2 Received: 14 June 2019 / Accepted: 17 September 2019 © The Japanese Society for Clinical Molecular Morphology 2019
Abstract Fibroblasts and myofibroblasts have been known to be present in both ruptured and intact human anterior cruciate ligament (ACL), and although their relevant histology and immunochemistry have been studied in the past, ultrastructural features of these cells are largely lacking. Therefore, we aim to characterise the ultrastructural details of these cells with the help of transmission electron microscopy (TEM) and to study the changes and their significance with duration of injury. Samples from 60 ruptured human ACL undergoing surgery were obtained and categorised according to duration of injury and observed under TEM with main focus on the following ultrastructural features: cellular morphology, presence of rough endoplasmic reticulum, Golgi apparatus, lamina, myofilaments, and presence of myofibroblasts. These features were further correlated with the duration of injury and association, if any, determined using appropriate statistical analysis. A total of 54 male and 6 female patients with mean duration of the injury of 23.01 ± 26.09 weeks (2–108 weeks) were included in the study and categorised into five groups based on duration of injury as follows: I ( 50 weeks). There was a significant association between the above-mentioned ultrastructural features and the duration of injury (p 50 weeks.
Surgical procedure Intra-operatively, residual ACL tissue was harvested using arthroscopic scissors by cutting the stump as close to the attachment site as possible. In cases where the residual stump was adherent to the PCL, the attachment was carefully dissected out before the remainder of the ACL was cut from its insertion site. All tissues were processed and assessed using transmission electron microscopy.
Transmission electron microscopy The tissue samples were fixed in a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4 for 2 h at 4 °C. The tissues were then post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h, followed by dehydration in graded ethanol (30, 50, 70, 90, 95 and 100%), and subsequent transfer to propylene oxide. The tissues were embedded in Araldite CY212 and polymerized in an oven at 60 °C for 72 h. Thick sections (1 µm) were cut with an ultramicrotome and mounted on to glass slides and stained with toluidine blue. Thin sections (60–70 nm thick) were stained with aqueous uranyl acetate and lead citrate and observed under a Tecnai G 2- 20 S-twin transmission electron microscope (Fei Company, The Netherlands) at a suitable magnification (4000×–10,000×).
Medical Molecular Morphology
Ultrastructural evaluation To study the ultra-morphol
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