Osteoarthrosis (OA), a local inflammatory disease that affects the articular cartilage and subchondral bone, is a major pathology in temporomandibular joint (TMJ) disorders . Abnormal mechanical stress and biochemical factors produced from the joint tissues are involved in the progression of OA. Osteoarthrotic changes in the joints are characterized by fibrillation and erosion of cartilage, chondrocyte proliferation and osteophyte formation at the joint margins and sclerosis of subchondral bone . Imbalance occurs between the synthetic and degenerative process within the chondrocytes that leads to the net loss of cartilage tissue and the subsequent pathological condition . These changes result from poorly understood events occurring over a long period, and are characterized by cartilage matrix degradation and inhibition of matrix component synthesis . At late stages of the disease, articular damage leads to joint impairment and pain.

Many experimental models mimicking OA lesions have been developed to describe the pathophysiology of the disease and to evaluate the putative chondroprotective properties of antirheumatic drugs. Significant effort has been made to develop animal models of OA that mimic the human disease, and these have led to the development of several ‘chemical’ or ‘surgical’ models. Chemical models involve intra-articular injection of compounds that can have a number of effects on joint physiology including inhibition of chondrocyte metabolism by papain or iodoacetate, damage to ligaments and tendons with collagenase , or selective joint denervation with immunotoxins . Surgical models induce joint instability by partial meniscectomy, usually in combination with transection of collateral and/or cruciate ligaments or myectomy . A series of experimental studies with surgical induction of anterior disc displacement in the rabbit showed that disc displacement led to degenerative changes in the condylar cartilage but they induced mechanical alteration and artificial surgical damage to the joint structures. OA lesions similar to those observed in humans are generated by intra-articular injection of drugs targeting articular cells, such as sodium mono-iodoacetate , interleukin-1 or tumour necrosis factor-α or extracellular matrix components (collagenase , papaine and fibronectine ).

The advantage of intra-articular injection into the joints is that it is easy to modulate the progression and severity of the articular lesions by modifying the concentration of the substances. Chondrocyte metabolism inhibitor (MIA) has been reported to induce the disruption of glycolysis and subsequently cell death and the loss of chondrocytes. When used in rodents, the model reproduces cartilage lesions with loss of proteoglycan matrix and functional joint impairment similar to human OA . In cartilage, lesions are characterized by chondrocyte necrosis, cell cloning (chondrones), fibrillation, loss of stainable proteoglycan matrix and erosion with exposure of subchondral bone. Reported bone lesions include remodelling and sclerosis of subchondral bone with osteophyte formation .

MIA injection induces various histological changes in the knee joints that closely resemble human OA . Although the biological action of this drug has been well documented in the knee joints , only one animal study reported the injection of 1.5 mg MIA into the joint without disc derangement via joint capsule opening . The study showed that the injection of this substance created advanced osteoarthritic lesions within 30 days and allowed the observation of every osteoarthritic stage describe by D ijgraaf et al. within 40 days. The authors suggested that the severity of damage was time dependent and the use of MIA allowed observation, within 20–30 days, of every osteoarthritic stage .

Regarding the imaging of OA, CT has been found to be superior to magnetic resonance imaging in the assessment of bony TMJ components. In the literature, the grading systems of osseous changes vary greatly, making comparison of different findings difficult . Since OA may appear in different parts of the TMJ, the grading systems on CT introduced by M oystad et al. , give a more standardized and detailed description of the radiographic findings through the whole joints and indicate the severity of disease .

The aim of this study was to assess MIA-induced OA in rabbit TMJ using different doses and time intervals. The null hypothesis is to find the most appropriate dose of MIA and the shortest time to induce OA in TMJ without surgery and joint capsule opening, similar to the clinical use of arthrocentesis in humans. The authors investigated four doses of MIA (1.5, 2, 2.5, 3 mg/ml) for inducing osteoarthrotic changes in rabbit TMJ within three time intervals (2, 4 and 6 weeks) using histological and histomorphometric evaluations. The results of this study will be used to determine the efficacy of high molecular weight sodium hyaluronate on OA of rabbit TMJ.

Materials and methods

All experimental procedures were approved by Istanbul University Intuitional Animal Care and Use Committee (2007/25). 13 New Zealand white rabbits (male, on average 18 weeks old and 3.5 kg) were used in this study. All rabbits were housed with a 12 h light–12 h dark cycle (artificial lighting), free access to water and food and at an ambient temperature of 21 °C and 50% humidity. One rabbit served as a control for detecting the histologically normal appearance of the condyle. After 2 weeks, all rabbits were anaesthetized with an intramuscular injection of a mixture of 5% ketamine (20 mg/kg) and 2% xyladine (0.1 mg/kg). 50 μl doses of 1.5, 2, 2.5, 3 mg/ml concentrations of MIA were injected into a single joint of each group using an arthrocentesis technique. These concentrations were calculated according to G uincamp et al. . The study group consisted of 24 joints. The number of each concentrations of MIA injected into joints was six, and the numbers of joints in the three time intervals (2, 4 and 6 weeks) was two for each concentrations of MIA. Each MIA concentration of 1.5 and 2 mg/ml was injected into each joint of one rabbit and the 2.5 and 3 mg/ml injections were injected into each joint of one rabbit to prevent the effects of loading. The injection of different MIA concentrations was performed blindly and the assessments of the condyle and histology were made blindly by one of the authors.

The animals were killed with an overdose of pentobarbital after each follow-up period (2, 4 and 6 weeks). All joints were removed and fixed in buffered formalin. They were prepared for undecalcified histology. The specimens were dehydrated in a graded series of ethanol and embedded in methyl methacrylate-based resin (Technovit 7200 VLC, Kulzer & Co., Wehrheim, Germany). Undecalcified ground sections were made according to the method described by Donath and Breuner. Sections of each experimental site were taken through the longitudinal axis of each sample reduced to a thickness of 40 μm. Two sections were prepared from each block. The sections were stained with toluidine blue. All sections were used for histological and histomorphometric evaluations. The digital images of the sections were obtained using a digital camera (Olympus ^® DP 70, Tokyo, Japan) attached to a microscope (Olympus ^® BX50, Tokyo, Japan) at a magnification of 4×. The images were transferred to a PC and Bioquant Osteo II image analysis software (Bioquant Image Analysis Corporation, Nashville, TN, USA) was used to assess them.

The normal histological appearance of the condyle displayed four distinct regions in the articular cartilage (fibrous, proliferating, mature and hypertrophic) and characteristic zonal cellular arrangements ( Fig. 1 ). The bony changes on articular cartilage ( Fig. 2 ), the appearance of chondrocytes (normal, hypocellularity or clusters) ( Fig. 3 a and b), invaginations in osteochondral junction and changes in the trabecular structure of subchondral bone in joints injected with all concentrations of MIA were recorded.

Normal histologic appearance of rabbit condyle (toluidine blue 20×).

Thickening of cartilage (toluidine blue 20×).

(a and b) Hypocellularity as the appearance of chondrocytes (toluidine blue 20×).

CT scans were obtained pre- and post-injection at 2, 4 and 6 weeks. The evaluation system described by M oystad et al. was used for CT evaluation of OA. The total score for each TMJ was used to grade the joint for severity. The progression or regression of osseous changes between the CT examinations before and after TMJ injections was expressed as the numerical differences in scores. The radiographic signs of OA (erosions, sclerosis, osteophytes, and flattening of the condyle) were localized and registered in the medial, central and lateral parts of the mandibular condyle. The observations were scored using a scoring system, where minor erosion/sclerosis was defined as erosion/sclerosis confined to one-third of the upper half of the condyle, moderate erosion/sclerosis confined to two-thirds of the upper half of the condyle, and severe erosion/sclerosis confined to the whole condyle. The total score for each TMJ was used to grade the joint for severity: normal, 0; minor abnormality, 1–5; moderate abnormality, 6–16; and severe abnormality, 17–28.