| Temporomandibular joint disorder (TMD) is the fourth largest orofacialdisease, and the abnormal mechanical stress on the temporomandibular joint(TMJ) caused by the abnormal molar occlusion is considered a possiblepathogenic factors in TMD. Osteoarthritis (OA) is one of the severe pathologicalchanges that often occurs in the TMJ of patients with severe statement of TMD.It is characterized by the progressive articular cartilage degradation andsubchondral bone changes. The current understanding of the pathologicalessence and the pathogenesis of TMJOA is still unclear, resulting in the poorclinical outcomes. Therefore, in order to further clarify the pathogenesis of TMJOA, it is necessary to establish a TMJOA animal models which largelymimicing the natural occurrence of TMJOA. Articular cartilage, an avascularand alymphatic tissue, has long been considered to be inert tissue, immune privilege area. Although the in vivo and in vitro studies have showed that thechondrocyte possess the abilities of migration and phagocytosis, whether thecartilage-specific phagocytes express the specific surface maker of macrophage,and what a role they play in the cartilage degradation is still unknown. Thepresent study firstly revealed age-related changes of the rat TMJ condyle,providing baseline data for subsequent studies of TMJOA animal models.Secondly, using young rats as the research object, different types of abnormalmolar occlusion were established by orthodontics method to demonstrate thatthe abnormal molar occlusion could be considered as an independent factorcausing the OA-like change of rat condylar cartilage. Thirdly, the TMJOA ratmodel was established by the sequential movement of the first and third molars,through which the pathological essence and pathogenesis of cartilage andsubchondral bone lesions in TMJ OA were investigated. Finally, the existence ofcartilage-specific phagocytes and its role in cartilage degradation were furtherexplored. Collectively, these results will provide new strategy for the clinicaltreatment TMJ OA.Firstly, using gross measurement, histomorphometry and Micro-CT analysisto investigate the age-related changes of the thickness of TMJ condylar cartilageand bone mineral density and microstructure of subchondral bone from2to7month old rats, in order to provide baseline information for the followingpathological studies of madibular condylar cartilage and its subchondral bone.Secondly,8weeks old rats were used in the present studies, and differentabnormal molar occlusion were experimentally established by orthodonticsmethods, that is mesially pushing the left maxillary and right mandibular firstmolar (M1)(Exp_I), distally pushing the left maxillary and right mandibularthird molar (M3)(Exp_II), distally pushing the two mandibular M3(Exp_III) and distally pushing the two maxillary M3(Exp_IV), and no operation weredone in the control group. Quantitative analysis of the HE and toluidine bluestaining were used to investigate the effect of abnormal molar occlusion on themorphology of TMJ condyle cartilage. Thirdly, using above method, theabnormal M1and M3occlusion (EMO) were created accordingly to establishthe TMJ OA rat model. Immunohistochemical staining, TUNEL andrealtime-PCR were used to investigate the balance of cell proliferation and deathof degraded cartilage in the8and12-wk experimental groups and the relatedsignal transduction mechanism. Fourthly, using Micro-CT analysis,histomorphometry, dynamic labeling of bone formation, TRAP andimmunohistochemical staining, and realtime-PCR to investigate the effect ofchondrocyte-secreted regulatory factors within the degraded cartilage onsubchondral bone changes in the progression of OA. Lastly, adopting degradedand normal TMJ condylar cartilage of EMO and control rats, and knee OA andnormal cartilage, the roles of cartilage-specific phagocytes in OA cartilagedegeneration were investigated by in vivo and in vitro experiments usingelectron microscopy, western blot, flow cytometry, cell sorting, live cellsworkstations, et al.The main results:1. The age-related changes of TMJ condyle of rat primarily occurred between2and5month. Rapid decrease of cartilage thickness but increase of subchondralbone density occurred respectively from2to3and3to4months old in femaleand2to4and3to5months old in male (P <0.05), whereas rapid changes ofsubchondral bone architecture occurred from3to4months old in both sexes (P<0.05). The significant enlargement of condyle size occurred at4or5monthsold in female but at5or6months in male (P <0.05). 2. Cartilage degenerative remodeling was observed in the experimental groups,typically characterized by a homogeneous eosinophilic mass and the local lossof proteoglycans. These changes were most often located in the central andposterior regions of the condyle. These regions of degenerative remodeling wereobserved in7,4, and2of the10condyles from female Exp II, III, and IV,respectively, and in2,1, and1of the10condyles from male Exp II, III, and IV,respectively. No degenerative remodeling regions were observed in the controland Exp I animals. Proliferative changes took the form of cartilage protrusionsthat penetrated into the subchondral bone in the central and posterior parts of thecondylar cartilage. These were observed in2and5of the10condyles fromfemale Exp III and IV, and in2,2,3,and1of the10condyles from male Exp I,II, III, and IV, respectively. Proliferative changes were also observed in1of the10condyles in the male control group but were not seen in female Exp I and IIor the female control group. In female rats, the percentage areas of cartilagedegenerative remodeling in Exp II (P=0.003) and Exp III (P=0.018) weresignificantly higher than those of the control group, whereas those in Exp I andIV were identical to those of the control (both, P <0.05). The percentage area ofcartilage degenerative remodeling in Exp II (P=0.012), but not in Exp III (P=0.116), was significantly higher than that of Exp IV. There was no differencebetween Exp II and Exp III (P=0.112). In male rats, the percentage areas ofcartilage degenerative remodeling were not statistically different between thecontrol and the experimental groups, or between any2experimental groups (all,P>0.05).The percentage area of cartilage degenerative remodeling in femalerats was significantly higher than that in male rats in Exp II (P=0.012) but wasidentical in all other experimental groups (P>0.05). 3．The EMO groups showed time-and sex-related progressive degraded changes,typically taking the forms of obvious pit-lesions of TMJ cartilage surface, ahomogeneous eosinophilic mass and local loss of proteoglycans, decreasedchondrocytes number, and pyknotic nuclei of chondrocytes. Some chondrocytesaccumulated locally and formed the “island”. The normal structure of cartilagewas disrupted, showing papillary protruding into the subchondral bone. Thefiber layer of the cartilage posterior region became obviously hyperplasia andhypertrophy, with disrupted hypertrophic chondrocytes distribution. Theextensive regions of fibrosis cell-free region were often found beneath theposterior disk attachment.4. There were incrased degraded chondrocytes with pyknotic, homogeneous,and eosinophilic nuclei in the middle and posterior parts of the condylarcartilage in EMO groups. The ultrastructural observation showed increasednumber of apoptotic chondrocytes within the degraded cartilage of8-wkexperimental group, showing obvious cytoplasmic process disappearance,peripheral chromatin margination and the increased disorganised chromatincondensation. There were increased necrotic cells distributing locally within thedegraded cartilage of12-wk experimental group, accompanied with diminishedchondrocyte proliferation in the female12-week ECDO subgroup (P <0.05). Anincrease in the number of apoptotic chondrocytes was seen in both the female8-and12-week ECDO subgroups and in the male ECDO12-week subgroup (all P<0.05), but not in the male ECDO8-week subgroup (P>0.05). The mRNAexpression of caspase-3、caspase-9、MAP4K3and bax/bcl-2significantlyincreased in the experimental groups comparing to their age-matched controls (P<0.05), while those of caspase-8and P53were identical between experimentaland control groups (P>0.05). 5．Increased degraded cartilage areas and obvious cartilage calcification wereobserved in8-and12-week treated (EXP) groups compared to the age-matchedcontrols. Subchondral bone loss, characterized as decreased bone mineraldensity (BMD), bone volume fraction (BV/TV) and trabecular thickness (Tb.Th),but increased trabecular separation (Tb.Sp), was observed in the12-week butnot the8-week EXP group, respectively, versus their age-matched controls. Thesubchondral bone loss in the12-week EXP group was accompanied withdecreased new bone formation rate, but increased serum carboxy terminaltelopeptides (CTXs), and increased osteoclast numbers and proportion ofsurface area in the subchondral bone regions (All P <0.05). Increased mRNAand protein levels of M-CSF, VEGF, RUNX and RANKL/OPG ratio, butdecreased OPG, were found in condylar cartilage in the12-week EXP groupversus its age-matched controls, and those of RANKL/OPG ratios weresignificantly higher in the12-week EXP group than the8-week EXP (All P <0.05). In addition, increased mRNA levels of VEGF, RUNX and RANKL/OPGratio, but decreased OPG, were also found in condylar cartilage in the8-weekEXP group versus its agematched controls (All P <0.05).6. CD163+cells were identified in cartilage mid-zone, respectively occupying3.3%and2.6%in Col-II+cells sorted from knee and TMJ cartilage, amongwhich about70%possessed phagocytic activity. Phagocytes engulfingneighboring apoptotic and necrotic cells were found within theexperimentally-induced degraded TMJ cartilage, within which there wereincreased expression of CD163, TNF-α and MMPs (All P <0.05), butunchanged levels of ACP-1, NO and ROS relating to cellular digestingcapability. Increased number of CD163+cells with enhanced phagocytic activitywere verified in Col-II+cells sorted from degraded TMJ cartilage of8-wk experimental group versus controls, and also in the isolated Col-II+cellsstimulated by TNF-α (P <0.05). Mid-zone distribution of CD163+cellsaccompanied with increased expression of CD163and TNF-α were furtherconfirmed in the isolated Col-II+cells from human OA knee cartilage versuscontrols (P <0.05).Conclusions:1．Rapid age-related changes of rat condylar cartilage and subchondral boneprimarily occurred before4months of age, resulting in thinner cartilage butlarger and thicker subchondral bone. The present results provided baselineinformation for the following pathological studies of madibular condylarcartilage and its subchondral bone, within which the false results caused byage-related changes should be carefully avoided.2．The mandibular condylar cartilage of female rats responded variously todifferent combinations of molar movement; the most obvious remodeling wasobserved in groups in which the maxillary left and mandibular right third molarswere moved.3. EMO could induce obvious degradation in the female rat condylar cartilage.This animal model will be used in the pathogenesis study of TMJ OA in thefollowing studies.4. EMO could induce decreased chondrocytes proliferation but increased deathwithin degraded cartilage of female rat condyle. The MAP4K3-mediatedendogenous apoptotic pathway may play a role in the excessive apoptosis ofchondrocyte in the degraded cartilage.5. Obvious subchondral bone loss followed cartilage degradation in themandibular condyles in the present rat models and suggested that the imbalanceof chondrocyte-secreted regulatory factors within the degraded cartilage may play a role in the osteoclastogenesis, and thus leading to the subchondral boneloss in OA.6. CD163+phagocytes were identified within joint cartilage. Their increasednumber with enhanced phagocytic activity in degraded cartilage indicate anactive capability of eliminating degraded tissues. |
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