The Establishment Of Unilateral Anterior Crossbite-induced TMJ OA Rat Model

Temporomandibular joint(TMJ) is one of the most common sites of occurrence of OA(ostearthritis), and it is the severe situation of temporomandibular disorders(TMD).The functional movement and biomechanical conditions of TMJ are closely related todental occlusion, hereafter abnormalities in dental occlusion are considered as one of the potential etiological factors for TMJ OA. Recently,our group reported a unilateral anterior crossbite prosthesis in rats and mice, which induced TMJ OA-like lesions. Based on our previous results, the current study aims to comprehensively assess our rat animal model by terms of gross morphology observation with dissecting microscope, radiographical analysis with micro CT analysis, histopathological assessment with scoring system of TMJ OA, ultrastructural observation with transmission electron microscope, and m RNA detection with real-time PCR assay.Part 1. The establishment of unilateral anterior crossbite(UAC) and the gross observation of the TMJ cartilageObjective: To establish the UAC rat model and observe the condylar surface with dissecting microscope.Methods: One-hundred and twenty 6-week-old Sprague-Dawley(SD)rats with eqaual sex distribution,female(weight,140-160g)and male(weighted,150-170g)were provided by the animal center of the Fourth Military Medical University. All procedures and the care administered to the animals were approved by the University Ethics Committee. The rats were randomly divided into 4-, 12- and 20-week groups, and each group was divided into UAC experimental subgroup and sham-operated control subgroup(Con)(n=10). In UAC groups, the unilateral anterior crossbite was created by bonding two metal tubes on left maxillary and mandibular incisors. At the end of the tube bonded to the left mandibular incisor, a 135 degree angle leaned to labial side was performed to make the two tube installed incisors bite with cross relation as described preciously.With this method, a scissor bite was created, that is, the left side incisors crossbite with each other while the right side incisors bite in normal. The gross observation method: Take the condyle head off the joint capsule, strip the surrounding soft tissues, cut off the condyle head from the condylar neck and put it into the physiological saline, number it, observe and take photos with dissecting microscope, and then assess them.Results: In most age-matched controls of both female and male rats, the surface of the TMJ condyle is continuous and smooth, plumply spindle. At 4 weeks, the mandibular condylar surface was gangly long spindle but reddened in UAC group vs. the age-matched control. At 12 weeks, the joint condyle of UAC rats showed slight deformation and sunk in the middle of the condyle. The deforming and the sunk got more severe, appearring as“lageniform”or “splayed” at 20 weeks.Conclusions: UAC leaded to the deformation of rat TMJ condyle, and the deformation became more and more severe with time.Part 2. The imageology study on subchondral bone of mandibular condyle withmicro-CTObjective:To scan and assess the longitudinal changes of subchondral bone of mandibular condyle.Methods:Two cylinder regions of intrest(length 0.5mm, radius 0.25mm) were selected at the middle ofthe center and posterior of condylar subchondral bone as described previously. Within the selected regions, BV/TV(the ratio of bone volume to tissue volume), BS/BV(the ratio of bone surface area to bone volume), Tb.Th(trabecular thickness), Tb.Sp(trabecularseparation), Tb.N(trabecular number) were determined by Mimics 16.0 software. And the final statistical data was the mean of data of the two cylinders.Results: The values of BV/TV decreased and the 4-, 12- and 20-week UAC groups in both female and male compared to their age-matched controls.The values of TB.SP increased in the 4-, 12- week UAC groups in both female and male compared to their age-matched controls.The values of BS/BV increased in the 4-, 12-week UAC groups of female rats while only increased in 4-week UAC group of male rats compared to their age-matched controls.The values of Tb.Th decreased in 12- and 20-week UAC group in female rats but showed no significant differences in 4-week UAC groups of female rats;while in male rats, the value of Tb.Th decresed only in 20-week UAC groups compared to their age-matched controls.The values of Tb.N decreased in the 4- and 12-week UAC group of male ratswhile only decreased in the 4-week UAC group of female rats compared to their age-matched controls.Conclusions: UAC leaded to the bone loss of subchondral bone of the mandibular condyle, but the bone loss became less and less severe with time.Part 3. The histochemical stain and the histologic gradingObjective: To obseve and assess the mandibular condyle with histochemical stain.Method: The regular HE stainning and safranin O /fast green staining.Results: In the 4-, 12- and 20-week UAC group of both male and female rats, the histological score value was increased compared to the age-matched controls.The value of histological score in 4-week UAC group was identical to that of the 12-week controlgroup, and that of 12-week UAC group was identical to that of the 20-week control group in both male and female rats.Conclusions: UAC leaded to the increase of the histologic grading, and this increased tendency aggravated with time.Part 4. The ultrastructural observation with transmission electron microscopyObjective: To observe the ultrastructural changes of mandibular condyle induced by UAC stimulation.Methods: Cartilage samples were fixed in 2.5% glutaraldehyde and in 1% osmium tetroxide, rinsed with water, dehydrated in a graded series of ethanol followed by propylene oxide and kept overnight in Epon812. The samples were embedded in Epon812 and cured in an oven at 60Results: Disorganized collagen fibers, expanded collagen fibers spaces and vestige of mineralization around the chondrocytes in hypertrophic layer appeared in the mandibular condyle of both male and female rats, and this phenomenon were obvious and aggravated with time.Conclusions: UAC leaded to the decrease of collagen fibers, disorganized fibre, the enhancement of matrix mineralization in the mandibular cartilage of both female and male rats, autophagosomes in the mandibular cartilage of female UAC groups. In summary, the ultrastructural changes in the male UAC rats were not as severe as that of female rats.Part 5. The m RNA detection of genes in the mandibular condyle.Objective: To detect the changes of the related genes in the mandibular condyle of rats stimulated by UAC, and expore the pathlogical mechnism of TMJ OA.Methods: Extract the RNA of two condyle cartilage from two different mice(n=2) and detect and analysis with real-time PCR.Results: In each subgroup, every 2(n=2)out of 6 condyle cartilages were pooled to create a single sample, and 3 independent pooled samplesin both UAC and control groups wereprepared for real-time analysis and the mean values were derived.Conclusions: The m RNA levels of Aggrecan and Col2a1 decreased in the 4-, 12-, and 20-week UAC groups of both female and male rats compared to their age-matched controls.In female UAC group, Adamts-4 and Adamts-5, the principle enzymes responsible for degradation of Aggrecan, increased in 4-week groups but decreased in 12- and 20-week group compared to their age-matched controls, while in Male UAC Group, Adamts-4 and Adamts-5 increased in 4-week group, decreased in 12-week group, but was identical in 20-week group compared to their age-matched controls.Mmp3, among the most ubiquitously expessed Mmps, increased in each time point in both female and male rats compared to their age-matched controls.While Mmp13, the most potent peptidolytic enzyme among collagenases, preferentially cleaving Col2 and has a much higher catalytic velocity rate compared with other Mmps over Col2 and gelatin, increased in the 4-week UAC group but decreased in the 12-week UAC groupof both female and male rats compared to their age-matched controls. While in 20-week UAC groups, Mmp13 decreased in female rat but not in male rats compared to their age-matched controls.Col10 increased in 4-week,decreased in both 12 and 20-week groupsof both female and male UAC group compared to their age-matched controls.Alp increased in 4-week and decreased in 12- and 20-week UAC groups compared to their age-matched controls.Runx2 increased in 4-week but decreased in 12- and 20-weekcompared to their age-matched controls.Conclusion: UAC stimulation changed the m RNA level of Aggrecan、Col2a1、Adamts-4、Adamts-5、Mmp3、Mmp13、Col10、Alp、Runx2 at the three time points, suggesting UAC changed the anabolism, catabolism, and differentiation of the above genes, and thus may be the miniature of the molecular mechanism of TMJ OA.