| Temporomandibular joint disorder (TMD) is a common disease in dental clinic.Temporomandibular joint (TMJ) osteoarthrosis (OA) is the most serious subgroup ofTMD. The main manifestions of OA involve cartilage degeneration and abnormalsubchondral bone remodeling. Subchondral bone remodeling participates in thepathological process of OA. The increased subchondral bone turnover caused by aberrantosteoblast and osteoclast activities is one of the hallmarks of OA. Since it is difficult todiagnose OA in the early stage, animal experiments provide the possibility to study theearly pathological changes of OA. Our group generated an animal model of unilateralanterior crossbite (UAC), which caused OA-like changes in TMJ condyles.In our previous studies, we found that UAC caused bone loss in rats TMJ condyles,with increased osteoclast activity. Osteoblast activity was firstly decreased at2weeks andthen increased at4weeks. In the present study, we detected the numbers of osteoclast andosteoblast in the subchondral bone of rats TMJ condyles by TRAP andimmunohistochemical stainings. The results showed that UAC caused the decreased number of osteoclast at2weeks (P<0.05) but increased number of osteoclast at4and8weeks (P<0.05) in the subchondral bone of rats TMJ condyles. The number of osteoblastwas decreased at2,4and8weeks (P<0.05). The mismatching of osteoclast and osteoblastnumbers and activities resulted in abnormal subchondral bone remodeling in rats TMJcondyles. The increased osteoblast activity is insufficient to overcome the bone resorptioncaused by robustly enhanced osteoclast activity, resulting in subchondral bone losseventually.Osteoclast activity in the subchondral bone of UAC rats TMJ condyles began toincrease at2weeks and osteoblast activity started to enhance at4weeks. Therefore, wefurther studied the2and4week groups at celluar and molecular levels.Bone marrow stromal cells (BMSCs) possess the differentiation potential towardostoblasts and play a critical role in bone repair. Then, whether UAC caused the number ofosteoblast in subchondral bone of rats TMJ condyles to decrease by reducing the numberof BMSCs? The flow cytometry analysis showed that UAC induced the percentage ofBMSCs to subchondral bone marrow cells of rats TMJ condyles to decrease at4weeks(P<0.001), which may result in the decreased number of osteoblast in subchondral bone ofrats TMJ condyles. Furthermore, whether UAC mediated osteoblast activity insubchondral bone of rats TMJ condyles by altering the bone formation capacity of BMSCs?We cultured BMSCs from subchondral bone of rats TMJ condyles in both2and4weeksgroups in vitro and further detected the proliferation, osteoblast differentiation andmigration capacities of these BMSCs. The results showed that the proliferation, osteoblastdifferentiation and migration capacities of BMSCs were decreased at2weeks, with thedown-regulated mRNA level of Runx2and the Rankl/Opg ratio, which may account forthe decreased number and activity of osteoblast in subchondral bone of rats TMJ condylesat2weeks. However, the proliferation, osteoblast differentiation and migration capacitiesof BMSCs were increased at4weeks, with the up-regulated mRNA level of Runx2and theRankl/Opg ratio, which may explain why osteoblast activity was enhanced in subchondralbone of rats TMJ condyles at4weeks. The above results suggest that UAC mediates boneformation in subchondral bone of rats TMJ condyles by altering the bone formation capacity of the local BMSCs in subchondral bone.Cell-to-cell contact between osteoclast precursors and osteoblast lineage cells isrequired in osteoclast formation. BMSCs are more important than mature osteoblasts inosteoclast precursors differentiation in their proximity. Moreover, BMSCs can induceosteoclast precursors migration toward bone surface. We detected the role of BMSCsisolated from the subchondral bone of rats TMJ condyles in osteoclast precursorsmigration and differentiation. The results showed that UAC stimuli inhibitedBMSCs-induced osteoclast precursors migration at2weeks (P<0.001), which mayaccount for the decreased osteoclast number in the subchondral bone of rats TMJ condylesat2weeks. And UAC stimuli enhanced BMSCs-induced osteoclast precursors migration(P<0.001) and differentiation (P<0.05) at4weeks, with the up-regulated CXCL12proteinlevel and the Rankl/Opg mRNA ratio in BMSCs, which may result in the increasednumber and activity of osteoclast in the subchondral bone of rats TMJ condyles at4weeks.The above results suggest that UAC mediates bone resorption of subchondral bone of ratsTMJ condyles through altering the local BMSCs-induced osteoclast precursors migrationand differentiation.Real-time PCR analysis showed that UAC up-regulated the expressions of Wnt5a andRor2in BMSCs isolated from the subchondral bone of rats TMJ condyles. It was reportedthat Wnt5a plays an important role in controlling cell functions, including cell migration,formation of osteoblasts and osteoclasts. These suggest us that Wnt5a/Ror2signaling mayparticipate in controlling the BMSCs-mediated subchondral bone remodeling. The resultsshowed that ectogenesis Wnt5a promoted the osteoblast differentiation of GFP-BMSCsand the GFP-BMSCs-induced RAW264.7cells migration and differentiation. Inhibition ofRor2in GFP-BMSCs decreased the expressions of Runx2, Cxcl12and Rankl/Opg ratio,and suppressed the osteoblast differentiation of GFP-BMSCs (P<0.001) and the migrationof the co-cultured RAW264.7cells (P<0.01). Furthermore, JNK inhibitor SP600125decreased the Wnt5a induced osteoblast differentiation of GFP-BMSCs to the lowest levelamong the four groups (JNK、Ca2+/NFAT、TGF-β and NF-κB inhibitory groups)(P<0.001).It was reported that JNK signaling enhanced the osteoblast differentiation of BMSCs by increasing the activity of Runx2. Ca2+/NFAT inhibitor Cyclosporin A suppressed theWnt5a-mediated BMSCs-induced osteoclast precursors migration below the control level(P<0.001), and inhibited the osteoclast precursors differentiation (P<0.05). It was reportedthat Ca2+/NFAT signaling in osteoblasts promoted osteoclast precursors recruitment bycontrolling the expression of chemokines, and that RANKL activated Ca2+/NFAT signalingin osteoclast precursors and then promoted osteoclast formation. These results suggest thatWnt5a/Ror2signaling in BMSCs promotes osteoblast differentiation of BMSCs byup-regulating the expression of Runx2, and enhance BMSCs-induced osteoclast precursorsmigration and differentiation by up-regulating the expression of Cxcl12and Rankl,resulting in the increased bone turnover of TMJ condyles subchondral bone and theoverall bone loss.To conclude, UAC causes changes in the bone formation capacity of BMSCs isolatedfrom subchondral bone of rats TMJ condyles and in the BMSCs-induced osteoclastprecursors migration and differentiation, resulting in the alterations of cellular behaviourof osteoblasts and osteoclasts. Wnt5a/Ror2signaling in BMSCs may involve in thisprocess. The aberrant subchondral bone remodeling of rats TMJ condyles caused by UACcontributes to the insufficient bone formation by osteoblasts to overcome the bonerepsortion by osteoclasts, leading to an overall subchondral bone resorption phenotype inearly stage of TMJOA. This study clarified the mechanisms of aberrant subchondral boneremodeling of rats TMJ condyles caused by abnormal biological force and provided a newexplanation for pathogenesis of TMJOA. |
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