The Survival, Implantation And Differentiation Of Exogenous BMSCs In Mouse Condylar Cartilage Of Experimental Osteoarthritic TMJ

Osteoarthritis （OA） is a kind of joint disease with high incidence rate, which ischaracterized by the degradation of articular cartilage and the abnormal rebuilding ofsubchondral bone. Temporomandibular joint （TMJ） is one of the predilection site ofOA.The defect of articular cartilage caused by osteoarthritis cannot be repaired perfectlyby traditionaI treatment．Recent research show that bone mesenchymal stem cells （BMSCs）have the potential of self-renewaI and multiple differentiation, and differentiate intochondrocytes under certain conditions．In this experiment the mouse model with condylecartilage degradation induced by malocclusion was used to evaluate migration to lesion,implantation and differentiation of BMSCs in mouse osteoarthrihritic TMJ. Methods and results1. Establishment of OA-like lesion in rat’s TMJ cartilage by experimentallycreated unilateral anterior crossbite prosthesis60female C57BL6mice （weigh16-18g）,6weeks of age, were provided by theanimal center of the Fourth Military Medical University. Animals were randomly andequally divided into an experimental group （Exp） or a sham-operated control group （Con）.In the experimental group, the left maxillary and mandibular incisors were bonded withmetal tube in order to create a crossbite relationship between the left incisors. Mouse inthe control group received no treatment but the same standardized diet throughout theexperimental procedure. Both experimental animals and controls were sacrificed after3weeks. The left TMJ tissues were collected and prepared for HE staining, toluidine bluestaining and morphological measurement. In the control groups, the surface of thecondylar cartilage was intact and smooth. Chondrocytes arranged orderly in the cartilage.In the experimental groups, the cartilages showed serious degradation, characterized asdecreased thickness of cartilage and the cell number, irregularity of cellular arrangementand some cell-free areas. Toluidine blue staining indicated the degradation of cartilagematrix. In addition, bone histomorphometry showed decreased BV/TV,Tb.Th, andincreased Tb.Sp compared with the control groups.2. Efforts of TMJ cartilage on inducing the differentiation of BMSCs intochondrocytesTranswell dishes with8m pore size were used for GFP-BMSCs differentiation assays.Condyle cartilage tissue of experimental group and control group were seeded into theupper chamber, and GFP-BMSCs were added to the lower chamber. After3,7,14and21days’ coculturing, the total RNA of GFP-BMSCs cocultured or non-concultured wereextracted with TRIZOL and COLII were analysed quantitively with Real Time PCR.The GFP-BMSCs in coculture system, noncocultured and primary chondrocyte weregrowed on glass coverslips and then stained by tuoidine blue and immunofluorescencechemistry method.The resuls showed that there is no significant different expression of COLII inGFP-BMSCs cocultured with condyle cartilage between experiment and control group （P>0.05）,but the COLII expression level increased signifcanly in cocultured groupcomparing to non-concultured group.3. Efforts of TMJ cartilage on inducing the migration of GFP-BMSCs.Transwell dishes with8m pore size were used for GFP-BMSCs migration. Condylecartilage tissue of experimental group and control group were seeded into the lowerchamber, and GFP-BMSCs were added to the upper chamber. Samples were prepared forDAPY staining after24hours, toluidine blue staining after7days. The condyle cartilagewere collected for HE, histochemistry and immunofluorescence chemistry staining. Theresults indicated that much more DAPY staining positive cells were observed after24hours in experimental group comparing to control group（P>0.05）. After7days, theGFP-BMSCs in or close to the condyle cartilage appeared blue-purple color with toluidineblue staining, but no color appeared in GFP-BMSCs far away from the condyle cartilage.After21days, the HE staining data showed there were abundant heterological nuclearimmersed in the mouse condyle cartilage in experimental group, but no heterologicalnuclear were observed in control group. With immunohistochemistry staining andimmunofluorenscence staining for GFP, positive expression were observed in condylecartilage experiment group not in control group.4. GFP-BMSCs were transfected with luciferase lentivirus to express luciferase stably.25l GFP-BMSCs with concentration of1x10⁸/ml were injected around one side ofcondyle in3-week mouse models, the equal volume of saline were injected around theother side. The signaling were observed with IVIS imaging system for28days. Thetransfected GFP-BMSCs expressed luciferase stably and the expression was closelycorrelated to number of the cells. The signaling intensity in the area of TMJ increasedfrom1.537×10P·s^-1·cm^-1·sr^-1to3.997×10^P·s^-1·cm^-1·sr^-1during the period of7days,and then decreaed after reaching the peak..After28days, the singaling intensity decreasedto1.797×10⁴P·s^-1·cm^-1·sr^-1.5.60C57BL6mice were equally divided into experimental group and control group.25l GFP-BMSCs with concentration of1x108/ml were injected around right side ofTMJ in3-week mouse models, the equal volume of saline were injected around the other side as control. The mice were sacrificed after4w,8w and12w injection. The both sides ofcondyle wer collected and prepared for HE staining and immunohistochemistry staining.The results indicated that compared with the left side, the thickness of cartilage in the rightside was significantly higher in the all experimental groups. After12weeks, there was nodifference in the thickness of cartilage between the experimental group and the controlgroup. Through the way of immunohistochemistry staining, GFP positive cell wereobserved in the condylar cartilage of experimental group, which was not obvious in thecontrol group.ConclusionThe degenerative pathological changes could be induced by Experimentally createdunilateral anterior crossbite prosthesis in TMJ. BMSCs could migrate directional andinplant in the degenerative cartilage to play a reparative role by differentiating intochondrocytes.