Alterations in Articular Cartilage of the Rabbit Mandibular Condyle Following Surgical Induction of Anterior Disc Displacement: Light and Electron Microscopic Immunocytochemistry Using Colloidal Gold Conjugates

Abstract

The purpose of this study was to test the hypothesis that surgical induction of anterior disc displacement (ADD) in the rabbit craniomandibular joints (CMJ) will lead to degenerative osteoarthritic changes detectable a t the molecular, subcellular and cellular levels in the articular cartilage of the rabbit mandibular condyle. Ultrastructural features of the normal rabbit mandibular condyle were compared to those of experimental condyles a t two weeks following induction of ADD. The quantities of type-VI and -IX collagens, as well as the components of proteoglycans, such as chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), k e ra tan sulfate (KS) and link protein (LP) were measured using immunogold labeling technique at the light and the electron microscopic levels. The right joint of each of 20 rabbits was exposed surgically, and all discal attachments were severed except for th e posterior attachment. The disc was then displaced anteriorly and sutured to the zygomatic arch. The left joint served as a sham -operated control. Ten additional joints were used as non­ operated controls. Deeply anesthetized rabbits were perfused with 2% buffered formalin two weeks after surgery. The mandibular condyles were excised and decalcified in ethylenediam inetetraacetic acid (EDTA). Paraffin embedded tissues were sectioned a t 5 (im for light microscopic study, while water-soluble plastic embedded sections were used for electron microscopy. Sections were incubated in monoclonal antibodies directed against C4S, C6S, KS and LP, and in polyclonal antibodies against type-VI and -IX collagens. After incubation in the appropriate colloidal gold conjugated secondary antibodies, tissue sections were studied with light and electron microscopes. In addition, immunostaining for proliferating cell nuclear antigen (PCNA) was performed using paraffin sections, and the PCNA indices of control and experimental condyles were determined. Pathological alterations were obvious in the experimental condyles, and appeared to be characteristic osteoarthritic changes. These include cartilage neovascularization, chondrocyte clustering, vacuolation, loss of extracellular matrix next to the membranes of chondrocytes, and an increase in num ber of apoptotic chondrocytes. Increased num bers of PCNA-positive cells in the osteoarthritic cartilage of the experimental group indicated a n active chondrocytic proliferation. Ultrastructural changes in injured chondrocytes included increased amounts of RER and Golgi, suggesting an increase in the synthesis and secretion of possibly degradative enzymes with a decrease in the normal secretory products. The results of th e immunocytochemistry using colloidal gold conjugates both a t the light and electron microscopic levels showed statistically significant depletion of C4S, C6S, KS, LP, type-VI collagen and type-IX collagen in the osteoarthritic cartilage (P < 0.05). The reduction of binding molecules such as LP, type-VI and type-IX collagens suggest a possible mechanism for the observed loss of integrity of the extracellular matrix. It is concluded that surgical induction of ADD in the rabbit CMJ leads to molecular, cellular and extracellular alterations in the articular cartilage of the mandibular condyle similar to those described previously in hum an ADD and in osteoarthritis of other synovial joints. The results of this study provide evidence that the loss of the shock absorber function of the disc, and the exposure of the condyles to overloading may cause the injured chondrocytes to secrete degenerative cytokines as indicated by the loss of proteoglycans, binding collagens and LP. These molecular changes are expressed a t the subcellular and cellular levels as osteoarthritis or degenerative joint disease.