SDF/CXCR Axis And OPG/RANKL Changes In The Experimentally Created Disordered Occlusion Of Rats

Temporomandibular joint osteoarthritis (TMJ-OA) is a degenerativedisease characterized by the condylar cartilage damage, subchondral boneremodeling, and synovial inflammation. In a series of previous studies, wereported a rat model with a specially designed occlusal abnormality that highlyresembled clinical occlusal problems. Typical TMJ OA-like changes wereinduced at8-and12weeks, which is a sign of the enhanced remodeling of TMJwhen the balance between anabolism and catabolism in osteochondral junctionis lost.In the present study, we used this rat model for a24weeks observation, toinvestigate the histological changes, and the distribution pattern of themultifunctional chemokine of SDF-1and its receptor CXCR4, two markersrelated to cartilage destruction (IL6and MMP-9), and two markers related toendochondral ossification (OPG and RANKL) in the mandibular condyle, tobetter understand the features of the long-term condylar adaptation to the disordered occlusion.Experiment one: SDF/CXCR axis and its downstream factors in theTMJ-OA rat modelObjective: To study the expression changes and effects of SDF/CXCRaxis and its downstream factors in the TMJ-OA rat model. Methods: Twelve8-week-old female Sprague–Dawley rats were allotted randomly intoexperimental group and sham-operated control group (N=6). The first and thirdmolars of the left upper and right lower jaw were moved mesially and distally,respectively, in the experimental group. The sham-control rats received the sameprocedure but without disturbing the occlusion. The rats were sacrificed at theend of the24th week after the beginning of the experiment. The right TMJ tissueblocks in each group were dissected out for histological and immuno-histochemical staining. And the left TMJ cartilage was dissected out for RNAextraction. Immunohistochemistry and Real-Time PCR were used to detect theSDF/CXCR, IL-6and MMP9expression levels in the condylar cartilage.Results: Experimental group showed local pathological changes in the middleand posterior thirds of condylar cartilage, such as uneven distribution of cellulardisposition, loss of cartilage surface integrity, cell-free area and the conjunctiveinvaginations penetrating into the subchondral bone. The percentage ofimmunopositive cell areas showed the protein expressions of CXCR, MMP9,and IL-6in experimental group significantly enhanced (P<0.05). The mRNAlevels of SDF, CXCR, MMP9, and IL-6also increased with significantdifferences (P<0.05). Conclusions: This study found that experimental occlusaldisorders in a long time could still lead to degenerative changes in the ratmandibular condylar cartilage. SDF-1/CXCR4axis and its downstream factorsmay play an important role in the development of TMJ-OA, but the exact mechanisms still need further study.Experiment two: OPG/RANKL expression changes in the TMJ-OA ratmodelObjective: To study the expression changes and effects of OPG/RANKL inthe TMJ-OA rat model. Methods: Twelve8-week-old female Sprague–Dawleyrats were allotted randomly into experimental group and sham-operated controlgroup (N=6). The rats were sacrificed at the end of the24th week after thebeginning of the experiment. The thickness of the calcified cartilage layer wasmeasured at the quartering points of the center and posterior thirds with acomputer-assisted image analyzing system. Osteoclasts were stained by thetartrate-resistant acid phosphatase (TRAP). Immunohistochemistry andReal-Time PCR were used to detect OPG/RANKL expression levels in thecondylar cartilage. Results: The thickness of the calcified cartilage layer in theexperimental group was significantly thicker compared with that in the controlgroup (P<0.05). TRAP staining showed that the distributions and numbers ofosteoclasts were similar between experimental and control group. Comparedwith the control group, the protein and mRNA levels of OPG were significantlyincreased (P<0.05). However, the protein and mRNA levels of RANKL were notsignificantly different from those in the control group (P>0.05). Compared withthe control group, the ratio of OPG/RANKL mRNA levels in experimentalgroup increased, approaching the significant difference (P=0.056). Conclusions:The enhanced OPG expression may be a compensatory response of thechondrocytes to the cartilage destruction, and that may not stop the OAprogression, but to some extent exacerbate the disease progresses.Experiment three: The expression changes of CXCR, IL6and collagen X inthe ATDC5cell line stimulated by the cyclic tensile strain and SDF Objective: To further explore SDF/CXCR signaling axis mechanisms inTMJ-OA by detect the expression changes of CXCR, IL6and collagen X in theATDC5cell line stimulated by the cyclic tensile strain and SDF. Methods:Using Insulin-Transferrin-Selenium to induce ATDC5cells to differentiate intochondrocyte-like cells. Three weeks later, the cells were divided into with orwithout cyclic tensile strain groups. And these two groups were further dividedinto negative control and SDF stimulation groups. The strain force applied was20%and at a rate of12cycles/minute (0.2Hz).12hours later, the total proteinswere extracted from the cells of the four groups. And western blot was used todetect the expression changes of CXCR, IL6and collagen X. Results: SDFcould enhance the CXCR, IL6and collagen X expressions in the chondrocytes.And20%tensile strain force could further up-regulate the three factors.Conclusions: Under the abnormal tensile force, SDF could up-regulate itsspecific receptor CXCR and thus increase its binding efficiency, finally leadingto the activation of CXCR/SDF axis, which on one hand enhanced theexpressions of IL6and other inflammatory factors, causing direct damages tothe cartilage tissue, and on the other hand directly promoted the chondrocyteshypertrophy, causing endochondral ossification.