Cell death and cell proliferation in cartilage layers in human anterior cruciate ligament tibial insertions after rupture

Abstract

The purpose of this study is to investigate cellular responses and histological changes of cartilaginous layers in human anterior cruciate ligament (ACL) tibial insertions after rupture compared with those in normal insertions. Fully 16 tibial insertions of ruptured ACLs were obtained during primary ACL reconstructions. We also obtained 16 normal ACL tibial insertions from cadavers. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) to detect apoptosis, proliferating cell nuclear antigen (PCNA) staining, and histological examination were performed. The percentage of TUNEL-positive chondrocytes in ruptured ACL insertions (30.2 +/- 15.6%) was higher than that in normal insertions (9.6 +/- 5.8%). The percentage of PCNA-positive chondrocytes was significantly different between ruptured ACL insertions (19.9 +/- 15.0%) and normal insertions (12.3 +/- 7.3%). The average thickness of the cartilage layer, the glycosaminoglycan-stained area, and the number of chondrocytes per millimeter in ruptured ACL insertions was smaller than those in normal insertions. The decrease in the number of chondrocytes owing to an imbalance between cell death and cell proliferation in the ACL insertions after rupture, as compared with normal insertions, may lead to histological changes of the cartilage layer in the insertions. An in-depth understanding of injured ACL insertion may help elucidate the etiology of histological changes and the function and significance of the existence of the cartilage layer of insertion. This understanding may help in developing optimal treatment protocols for ACL injuries if apoptosis and cell proliferation are controlled.

Influence of Gradual Elongation to the Patella Tendon Insertion in Rabbits

“…In humans, ACL tibial insertions after rupture result in an increase in the chondrocyte apoptosis rate and a decrease in average thicknesses of the stained GAG areas; this is also well replicated in ACL tibial insertions after ACL resection in rabbit models of repair in our previous studies [5,6,7,8]. On the other hand, in the ACL partial resection animal model, the average thicknesses of the stained GAGs areas in the remaining ligament area increased up until 4 weeks, and then gradually decreased until 8 weeks owing to an imbalance between chondrocyte apoptosis and chondrocyte proliferation [9].…”

“…Sections were then incubated with terminal deoxynucleotidyl transferase (TdT) and digoxigenin-labelled nucleotides for 60 min at 37 °C in a humid chamber, followed by peroxidase-conjugated digoxigenin antibody for 30 min at room temperature in the humid chamber. The immunoreaction product was developed in diaminobenzidine, and the sections were counterstained with Mayer’s haematoxylin for 30 s. TUNEL-positive nuclei stained dark brown and TUNEL-negative nuclei stained blue [5,6,7,8,9]. …”

“…As a negative control, Antibody Diluent (Code No. S0809, DAKO, Carpinteria, CA, USA) was used in place of the primary antibody [7,9]. …”

“…Histomorphometrical analyses were as previously described [6,7,8,9]. Since the tidemark was not clear by HE staining and Safranin-O staining, we used Weigert’s iron hematoxylin solution in Safranin-O staining for staining the tidemark.…”

Influence of Knee Immobilization on Chondrocyte Apoptosis and Histological Features of the Anterior Cruciate Ligament Insertion and Articular Cartilage in Rabbits

“…We have previously reported an increased chondrocyte apoptosis rate and histological changes in the cartilage layers in the tibial insertions of human ACLs post-rupture [5,6]. An increased chondrocyte apoptosis rate also preceded a decrease in thickness of GAG layer in the ACL insertion in an ACL resection animal model [7,8].…”