| Objective:To establish a method of isolation and cultivation of the rat bone marrow mesenchymal stem cells(BMSCs); To investigate the optimal dosage and timing for5’-bromo-2’-deoxyuridine (BrdU),4_,6-Diamidino-2-phenylindole dihydrochloride (DAPI) and hoechst33342labeling of rat bone marrow mesenchymal stem cells in vitro and compare these three methods; To investigate the influence of autologous bone marrow mesenchymal stem cells transplantation on fracture healing combined with central nerve injuries in rats.Methods:BMSCs were isolated from the marrow of the tibia and the femur of male Sprague Daw-ley rat, the specific surface antigens (CD34, CD44, CD45) of the3rd generation BMSCs were identified by flow cytometry and BMSCs were labelled with BrdU, DAPI and hoechst33342respectively. The label of efficiency for BrdU was aseessed with immunocytochemistry and that of DAPI and hoechst33342were observed under fluorescence microscope. Cell proliferation was evaluated by using MTT. The advantages and disadvantages of the three methods were analyzed. Then48healthy adult SD rats (male or female) were randomly divided into6groups (8rats in each group):group A, simple(left) tibial fracture; group B, left tibial fracture combined with T10spinal cord transection (SCT); group C, left tibial fracture combined with T10spinal cord transection (SCT)+autologous mesenchymal stem cell transplantation; group D, left tibial fractures+DMEM; group E, left tibial fracture combined with T10spinal cord transection (SCT)+DMEM, group F, left tibial fracture combined with autologous mesenchymal stem cell transplantation. The tibial fracture stabilized it with a modular intramedullary nail and all of the operated hind limbs were further immobilized in a plaster cast to avoid unequal load bearing. BMSCs were taken15days before operation, then BMSCs were labeled with Brdu and implanted into the fractures of C> F groups when the model were made well after2days. B, C, E groups of animals were evaluated by using TARLOR score postoperation; The fractured tibiae were radioactivity (X-ray) evaluated separately at1w,2w,3w and4w after injury to assess fracture healing.Result:Flow cytometry showed that BMSCs expressed CD44but not CD34or CD45and there was no significant difference in the expression of stem cell specific surface antigen between before labeling and after labeling. The optimal concentration and timing of the BMSCs with BrdU labeling were respectively10μmol/L and48hours and that of DAPI labeling were20/μg/ml and30min and5μg/ml,1h with hoechst33342. All of B, C, E groups animals TARLOR score reaches the requirement of experiment. Postoperative1,2,3,4weeks the tibia callus width:1,2,3weeks after operation, the tibia callus width of B, C, E, groups are statistically significant (P<0.05) than the rest of the groups;4weeks the tibia callus width of group C decreased compared B and E groups, but B, C, and E groups are still statistical significance (P<0.05) than the rest of the group.Conclusions:High-purified BMSCs can be obtained by adherent method; the results suggest that the three different ways of labeling (BrdU, DAPI and hoechst33342) provides a stable, reliable and feasible means for a dynamic observation of the implanted BMSCs in vivo. The bony callus was more rapid and abundant in transversal spinal cord lesion than in controls, however, it was a defective organization of the large callus. The denervation influenced the function of fracture healing state, We conclude that neural regulation plays a important role in the type of fracture healing, primary or secondary, and in the amount and quality of the callus. Our study that treatment with bone marrow mesenchymal stem cells had positive effect on the defective callus in spinal cord transection (SCT) rats. |
PDF Full Text Request