| Objective1. Establishment of the rabbit model of femoral head necrosis:rabbit model of femoral head necrosis was established by liquid nitrogen refrigeration, pathology change process of rabbit femoral head necrosis was discussed, the results of this study could provide more building methods for the research of femoral head necrosis therapy.2. Icariin concentration screening:By detecting the in vitro effect of different concentrations icariin on promotion of bone mesenchymal stem cell proliferation and differentiation, selecting optimal concentration and the treatment time point.3.Study of rabbit femoral head necrosis:on the basis of the successful establishment of the rabbit model of the femoral head necrosis frozen by liquid nitrogen, combining with pith drilling decompression, bone mesenchymal stem cells with appropriate activity and number which in vitro induced by icariin were then transplanted into reduced pressure zone of femoral head necrosis to prevent bone destruction and prompt new bone formation, and strengthen repair osteogenesis ability, it can improve the therapeutic effects and provide theoretical support for clinical research of the treatment of femoral head necrosis.Methods1. Experimental research of rabbit bone marrow mesenchymal stem cells inducedin vitro byicariin:the rabbit primitive mesenchymal stem cells which extracted from one eligible one week old New Zealand white rabbit were cultured ex vivo. The second generation rabbit bone mesenchymal stem cellswith high fecundityis were induced by icariin in vitro for 24h,48h and 72h. The concentration of icariin was 0,0.01μMol,0.1μMol,1μMol, 10μMol respectively. The samples of cells extracts in different time points were determined by MTT, ALP activity, ALP staining and alizarin red staining. Combined with real-time fluorescent quantitative-pcr and western blot technology, the expression of BMP2, COL-1, OPN levels were determined,The data was treated statistically.The optimal icariin concentration and treatment time point were determined,the cells in vitro induced at this condition were then mixed with BD Matrigel.BD Matrigel as a carrier can provide nutrition, keep the cell activity, and change rapidly from liquid to glue when it is heated to 22-35 degrees centigrade.2. The study of rabbit bone mesenchymal stem cells induced in vitro by icariin transplantated to repair osteonecrosis of the femeral head:60 healthy adult rabbits selected into the experiment were divided into group A (control) group B (model group), group C (treatment group with pulp core decompression 12), group D (treatment group with pulp core decompression combine with transplantation rabbit bone mesenchymal stem cell 12), group E (treatment group with pulp core decompression combine with transplantation rabbit bone marrow mesenchymal stem cell induced in vitro by icariin 12). First rabbit model of the femoral head necrosis was established by liquid nitrogen refrigeration.3 experimental rabbit were taken randomly at two weeks after the X-ray operation, spiral CT examination under anesthesia, then the rabbits executing ear venous air embolism were dissected to take the samples of femoral head and throughmicro-CT examination and detection of pathological tissue morphology. A number of testing to ensure establishsuccessfully rabbit model of osteonecrosis of femoral head induced byliquid nitrogen, group C were treated with pulp core decompression, group D were treated with pulp core decompression combine with transplantation rabbit bone mesenchymal stem cell, and group E were treated with pulp core decompression combine with transplantation rabbit bone mesenchymal stem cell induced in vitro byicariin.3 experimental rabbit were taken randomly in every group at two weeks, four weeks and eight weeks after the X-ray operation, spiral CT examination under anesthesia, then the rabbits executing ear venous air embolism were dissected to take the sampleof femoral head and throughmicro-CT examination, detection of pathological tissue morphologyand immunofluorescence. The data has been treated statistically to probe into the pros and cons of each treatment.Results1. rabbit model of the femoral head necrosis was established successfully by liquid nitrogen refrigeration. A typical phenotype of rabbit femoral head necrosis was confirmed after two weeks of liquid nitrogen freezing by imaging and pathological histomorphology tests. The objective data of experimental femoral head necrosis from pathological evolution process was also obtained.2. Successfully isolated BMSCs, the optimal concentration and treatment time point of cultivate bone marrow mesenchymal stem cells in vitro induced by icariin were confirmed. BD Matrigel as a carrier can provide nutrition, keep the cell activity.3. The original data of groups of rabbit femoral head necrosis measurements at each time point were obtained successfully, There were significant differences between groups, it provides theoretical supports for the clinical research of the necrosis of the femoral head.Conclusion1. rabbit model of the femoral head necrosis is established by liquid nitrogen refrigeration. after two weeks postoperatively by imaging and pathological femoral trabecular bone tissue morphology detection, in sparse array chaos and has some faults, decrease hematopoietic cells in the marrow cavity, modified cell death, appear a large number of empty pit of bone, bone cells within the bone pit profile disappeared, the nucleus pycnosis, hyperaemia of medullary cavity, blood coagulation, full of necrotic tissue fragments, femoral head weight bearing area of cartilage cells within the bone pit atrophy, the nucleus pycnosis, to the early performance of typical femoral head necrosis. Confirmed after two weeks of liquid nitrogen freezing to the preparation of rabbit model of the femoral head necrosis.2.the second generation rabbit bone mesenchymal stem cells are inducted by icariin in vitro of 24h,48h and 72h.The concentration of icariin is 0, 0.01μMol,0.1μMol, 1μMol, 10μMol. After 48h induced by icariin in vitro, bone mesenchymal stem cells number was dose dependently increased, within 48h, has proliferation effect on bone marrow mesenchymal stem cell 1μMol icariin has the optimal proliferation activity. Bone marrow mesenchymal stem cells proliferation ability and differentiation ability were enhanced by inducing for 72h. Icariin can promote proliferation of bone marrow mesenchymal stem cells and the optimum concentration of proliferation and differentiation is 1μMol, best time is 48h.3. Through the micro-CT internal microstructure observation of femoral head, model group within the femoral bone trabecular structure serious disorder, there are serious trabecular bone fracture, bone trabecular number decreased significantly, the bone trabecular spacing increases, the femoral bone volume and surface area decreased significantly, bone mineral salt density decreased significantly. Between the group after treatment in the pulp core decompression and bone marrow mesenchymal stem cell group, icariin groups of femoral head necrosis lesions are improved, and the effect of icariin group best. Above results showed that icariin of joint between the bone marrow mesenchymal stem cells to treat femoral head necrosis effect is obvious, icariin by inducing bone marrow mesenchymal stem cell proliferation and mature to give play to the role of treatment. Statistics micro -CT scans of bone structure main parameters, pathological morphology, immunofluorescence technique analysis, core decompression combined icariin induced formed between bone marrow mesenchymal stem cells to the curative effect of treatment group was superior to that of pulp core decompression joint between bone marrow mesenchymal stem cell transplantation in treatment group, pulp core decompression in treatment group and model group. Preliminary confirmed that the pith in the process of decompression injection by icariin between bone marrow mesenchymal stem cells induced by in vitro significantly promote the restoration of femoral head necrosis. |
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