The Function Of Chemotactic Factors SDF-1and RANTES In The Therapeutic Role Of Exogenous BMSCs In TMJOA

Osteoarthritis (OA) is the most common joint disease, which is characterized by jointcartilage degradation and ultimate joint destruction. OA brings the patients pain andlimitation of movement, and causes great pressure to the patients. The pathologicalmechanism of OA is not clear, and several factors are related to the progression of OA,including injury, genetic factor, abnormal position of joint, age, malnutrition and abnormalbiological stress. OA occurs frequently in the knee, elbow and other weight-bearing joints,and temporomandibular joint (TMJ) is one of the predilection site. Because it is hard to getthe early-stage OA samples of patients, research based on animal models is now becomingan effective way to study OA. The self-repairing ability of joint cartilage is limited, and theregenerated cartilage is predominantly fibrocartilage, whose mechanical property is worsethan normal hyaline cartilage. Till now, medicine which can effectively reverse or terminatethe progression of OA is still lacking, and the clinical treatment strategies are mainly focusedon stopping pain, physiotherapy, controlling inflammation and slowing down the progressionof OA. Stem cell therapy has gained a lot attention these years. Clinical and animal studieson stem cell therapy of OA mainly used radiological, functional and morphological indices to simply evaluate the curative effect, but systematic studies on the efficacy and mechanismof stem cell treatment are still lacking. The key point in stem cell therapy is that the stemcells can be attracted by and migrate to the target tissue, and the chemotactic factorsexpressed by the target tissue play significant role in the migration of stem cells.Chemotactic factors stromal cell-derived factor-1(SDF-1) and RANTES are main regulatorsof stem cell migration, but there is still little information on the chemotactic role of SDF-1and RANTES in the stem cell therapy of OA. The present study developed a mal-occlusioninduced TMJ OA mouse model which has definite pathogenic role, systematically proved thetherapeutic role of exogenous BMSCs local injection, and investigated the mechanism of thistherapeutic role from the aspects of stem cell survival, differentiation and chemotaxis. Wefound that local injection of exogenous BMSCs had positive therapeutic function on themal-occlusion induced TMJ OA, and the key mechanism of the function was that theover-expressed SDF-1and RANTES in the degraded cartilage could attract exogenousBMSCs to migrate to and implant in it. The whole study can be divided into the followingthree parts:Part1: Unilateral anterior crossbite induced TMJ OA in mice and the mechanisms in itBackground：Generally, temporomandibular joint disorder (TMD) is believed to havemultiple etiological factors, among which occlusal factor has attracted the most attention. Upto now, there is still a strong controversy over the effect of abnormal occlusion on TMJ. OAis a severe pathological change of TMD, which is characterized by cartilage degradation andsubchondral bone changes. Semaphorin4D (Sema4D) and Plexin-B, has been shown toinhibit bone formation. However, the expression pattern of Sema4D/Plexin-B1during theremodeling of TMJ has never been investigated before.Aims: To develop a new animal model by changing the occlusal relationship in mice，andinvestigate the changes in condylar cartilage and subchondral bone of the TMJ and thechanges in the expression of Sema4D and Plexin-B1.Methods: By bonding a single (single group) or a pair (pair group) of metal tube(s) to left incisor(s), a crossbite-like relationship was created between left-side incisors in mouse. Themorphological changes in TMJ condyle were examined by hematoxylin&eosin andtoluidine blue staining. Indices of osteoclastic activity, including tartrate-resistant acidphosphatase (TRAP) staining and macrophage-colony stimulating factor (M-CSF) wereinvestigated by histochemistry or realtime-PCR. The osteoblastic activity was indexed byosteocalcin expression. Expression of Semaphorin4D and its receptor, Plexin-B1, weredetected by realtime-PCR. Two-way analysis of variance was used to assess the differencesbetween groups.Results: One week and three weeks after bonding metal tube(s), cartilage degradation andsubchondral bone loss were noticed histologically. Both indices of osteoclastic activity(TRAP and M-CSF) were increased in cartilage and subchondral bone after bonding metaltube(s). Osteocalcin expression in cartilage was increased at week three, while its expressionin subchondral bone was increased at week one but decreased at week three. The Sema4Dexpression in cartilage and subchondral bone was decreased at week one but increased atweek three. For Plexin-B1expression, a significant increase was detected in subchondralbone at week three.Conclusions: Bonding a single or a pair of metal tube(s) to left incisor(s) is capable ofinducing remodeling in TMJ, which involved cartilage degradation, alteration of osteoclasticand osteoblastic activity, and role of Sema4D/Plexin-B1.Part2: The therapeutic role of exogenous BMSCs in TMJ OABackground: Stem cell therapy is a new treatment method for OA and is attracting muchattention. BMSCs have multilineage differentiation potential, and are the ideal seed cells forOA therapy. Although there are several clinical and animal studies reported the therapeuticeffect of BMSCs on OA, there are still limited information on the therapeutic effect ofBMSCs local injection in OA and the molecular expression during it.Aims: The present study aimed to systematically investigate the therapeutic function ofexogenous BMSCs on TMJ OA. Methods: To ensure that the cartilage degradation in the present study was induced bymal-occlusion, we removed the prosthetics and observed the changes in the TMJ condyle.Three weeks after bonding the metal tubes, exogenous GFP-BMSCs were injected into theTMJ region every week. The morphological changes of the cartilage and subchondral bonewere investigated by HE staining and micro-CT, the changes in the cartilage matrix wereidentified by safranin O staining, the osteoclastic activity in the subchondral bone wasinvestigated by TRAP staining, the implantation of exogenous GFP-BMSCs into thecondylar cartilage was investigated by immunohistochemical staining of GFP, thedifferentiation of GFP-BMSCs in the cartilage was observed by double immunofluorescenceof GFP with Col-II, Col-I or OCN, and the expression of related factors in the condylarcartilage was tested by realtime-PCR.Results: The lesion in the condyle could be partially reversed by removal of the prosthetics,suggesting the etiological role of mal-occlusion in the present TMJ OA. The thinning ofcondylar cartilage, loss of subchondral bone and cartilage matrix, and the increasedosteoclastic activity were reversed by local injection of GFP-BMSCs. The injectedGFP-BMSCs could implant into the degraded condylar cartilage and express Col-II, ratherthan Col-I and OCN. The decreased expression of Col-II, aggrecan and Col-X and increasedexpression of Col-I、OCN、MMP13、TNF-and IL1β were reversed by GFP-BMSCsinjection. The indices of fibrillation, Col-III and Tenascin-C, were down-regulated byGFP-BMSCs injection.Conclusion: Local injection of exogenous BMSCs had positive therapeutic effect onmal-occlusion induced TMJ OA, and this effect was based on the implantation andchondrogenic differentiation of BMSCs in the condylar cartilage.Part3: The mechanism of the therapeutic effect of exogenous BMSCs on TMJ OABackground: The survival, differentiation, migration and implantation of stem cell in thetarget tissue is the foundation and prerequisite of stem cell therapy, but the exact mechanismof the therapeutic effect of exogenous BMSCs on OA is still not clear. Aims: This study aimed to investigate the mechanism of the therapeutic effect of exogenousBMSCs on TMJ OA from the aspects of stem cell survival, differentiation, migration andimplantation.Methods: The ability of degraded condylar cartilage to induce the differentiation andimplantation of BMSCs was investigated by in-vitro co-culturing in a transwell system. Thesurvival situation of BMSCs was observed by bioluminescence technique. The expression ofSDF-1and RANTES in the degraded condylar cartilage was investigated byimmunohistochemistry and realtime-PCR, the role of chemotactic signal SDF-1/CXCR4andRANTES/CCR1in the migration of BMSCs was detected by in-vitro blocking of CXCR4and CCR1signaling, and the key function of SDF-1/CXCR4and RANTES/CCR1in thetreatment of TMJ OA with BMSCs was investigated by in-vivo blocking of CXCR4andCCR1.Results: Co-culturing experiment in the transwell system showed that either normal orexperimental condyle could induce BMSCs to express Col-II, but the experimental condylecould attact more implantation of BMSCs. The bioluminescence experiment showed that thesurvival capacity of exogenous BMSCs in the TMJ region reached a peak7days afterinjection, and the luminous intensity was lower in the experimental group than the controlgroup. The survival capacity of exogenous chondrocyte was very low in both theexperimental and control groups. The expression of SDF-1and RANTES was up-regulatedin the degraded condylar cartilage, the ability of the degraded condylar cartilage to induceBMSCs migration in vitro was inhibited by CXCR4and CCR1inhibitor, and the therapeuticeffect of BMSCs on TMJ OA could also be inhibited.Conclusion: The chemotactic signal SDF-1/CXCR4and RANTES/CCR1are key regulatorsin the treatment of TMJ OA by exogenous BMSCs.