| Object:Femoral head necrosis includes the necrosis of osteocytes, bone marrow tissue, and fat cells in femoral head. When it comes to the later period, the femoral head will collapse and become deformed, with the joint space decreases, even disappears. In this period, pain and restriction of the movement of hip joint is the most common clinical symptom. Till today there is no valid or widely applied therapy in its early period, consequently, it is not possible to efficiently stop or postpone the deterioration of the disease. In the late period, total hip arthroplasty is the only efficient way to restore the function of hip joint. The duration of artificial hip joint widely in use today is around20years; revision arthroplasty is inevitable if the patients receive total hip arthroplasty in their youth. Not only the pain and economic pressure faced by the patients, the effect of revision operation is also far inferior to that of first time replacement. As is indicated above, in the early period of the disease, it is of great importance to explore an efficient way to stop or postpone the deterioration of femoral head necrosis. Among the causes of femoral head necrosis, steroid-induced femoral head necrosis has the highest incidence and obvious drug history, together with the widely used magnetic resonance imaging (MRI), detection of the necrosis in its early stage is much easier than those caused by other factors. The disease deteriorates rapidly and many of the patients receive total hip arthroplasty in their youth, for them, revision operation is inevitable. In this research we select steroid-induced femoral head necrosis as the point of penetration to explore the therapy for femoral head necrosis in its early period. Bone marrow mesenchymal stem cells (BMSCs), which are capable of differentiating to variety types of mature cells, play a role of great importance in tissue engineering, its position in tissue regeneration and repair are widely accepted by researchers and doctors. Also the application of BMSCs has been regarded as an effective therapy for early stage femoral head necrosis, several problems such as the source of BMSCs, the method of collection and cultivation of BMSCs and the pathway of auto-transplantation need further research to make it more facilitating to operate. Vascular endothelial growth factor (VEGF) locates in the center of the regulation of the formation of bone and vascular. There are five isoforms of VEGF, among which VEGF-a plays the most important role. Most of the researches about the level of VEGF-a in bone are qualitative. In this research, we adopt the method of real time polymerase chain reaction (RT-PCR) to evaluate the expression of VEGF-a, to get a more accurate view of the physiological condition of bone after the transplantation of BMSCs. In order to explore the therapy for steroid-induced femoral head necrosis, we select rabbit to construct animal model, then transplant autologous BMSCs collected from tibial bone marrow cavity and cultivated in laboratory, pathological test and real time polymerase chain reaction are used to evaluate the efficiency of the therapy.Materials and methodExperiment I1.20New Zealand white rabbits,3months’old,2.0-2.5kg, are involved in this experiment. The animals are randomly divided into two groups, animal model group and control group, each of them has10rabbits. Animals of the model group are injected intramuscularly one time methylprednisolone sodium succinate20mg/kg, while the control group is exposed to the same volume physiological saline. In order to prevent infection, all of the animals are injected penicillin sodium4×105IU two times per week.2.6weeks after the injection of methylprednisolone sodium succinate, all the animals are killed by aeroembolism. Every femoral head is collected under aseptic condition. Femoral heads of the left side are immersed in10%formalin for pathological test, those of the other side are frozen in-80℃circumstance for real time polymerase chain reaction test(RT-PCR) to evaluate the expression of vascular endothelial growth factor-a(VEGF-a).Experiment Ⅱ1.30New Zealand white rabbits,3months’old, are randomly divided into3groups, each group has10rabbits. Group A:animal model group; Group B:physiological saline group; Group C:BMSCs therapy group. All the animals are injected methylprednisolone sodium succinate once20mg/kg intramuscularly to establish animal model of steroid-induced femoral head necrosis. All the animals are injected penicillin sodium4×105IU twice a week to prevent infection.2.4weeks after the exposure to methylprednisolone sodium succinate, autologous bone marrow is collected to purify BMSCs. Rabbits of Group C are intravenous anaesthetized by3%pentobarbital sodium1ml/kg. Through tibial plateau penetrating into tibial bone marrow cavity with16#medullo-puncture needle, then about10ml bone marrow are collected. BMSCs are extracted by simple centrifugation, then cultivated in laboratory, after one passage, BMSCs suspensions are prepared for auto-transplantation.3. Animals of Group C are intravenously anaesthetized by3%pentobarbital sodium. Then drilling a whole on the left femur inferior to the greater trochanter, through which the BMSCs suspension is injected into the femoral bone marrow cavity. Animals of Group B are exposed to physiological saline of the same dose through the same pathway. After the operation, all the animals are intramuscularly injected penicillin sodium4×105IU once every day for5days, then twice a week.4.4weeks after the operation of transplantation, all the animals are killed by aeroembolism. Femoral heads of the left side are collected under strictly aseptic operation, and then every specimen is divided into two along coronal plane, one is immersed into10%formalin for pathological test, the other one is frozen in-80℃circumstance for real time polymerase chain reaction test(RT-PCR). ResultExperiment IPathological test (HE staining):in animal model group, empty osseous lacuna possess a large proportion of the lacunas, the arrangement and size of fat cells are disorderly. The number of hematopoietic cells in bone marrow tissues decreases, with the pyknosis of nucleus. In some specimen, regions of necrosis stained homogenous red and rupture of bone trabecula can be detected. According to pathological diagnostic standard,7of all the9specimen of animal model group are diagnosed as femoral head necrosis, while in the control group, there is no specimen meet the diagnostic standard of osteonecrosis.Real time polymerase chain reaction test (RT-PCR):the expression of VEGF-a of animal model group is obviously lower than that of control group.Experiment IIPathological test (HE staining):the proportion of empty bone lacuna of Group C is obviously lower than the other two groups. Arrangement and size of fat cells in the specimen of Group C is more regular than those of the other two groups. The number of hematopoietic cells in bone marrow tissues decreases in Group A and B, and in the two groups, ruptured bone trabecula and necrotic region stained homogenous red can be detected. According to pathological diagnostic standard,2specimen of Group C are diagnosed as femoral head necrosis, while in Group A and Group B, the number is7, respectively. Statistically, the incidence of femoral head necrosis of Group C is obviously lower than the other two groups, while between Group A and Group B, there is no obvious difference.Real time polymerase chain reaction test (RT-PCR):In Group C, the expression of VEGF-a is obvious higher than the other two groups, while between Group A and Group B, there is no statistical difference.Conclusion1. Short-term high-dose corticosteroid therapy is able to downgrade the expression of VEGF-a. 2. Tibial bone marrow of rabbit is a reliable resource of autologous bone marrow mesenchymal stem cells (BMSCs). Cultivated BMSCs extracted by simple centrifuge is able to meet the need of therapy.3. Autologous BMSCs transplantation, which is capable of upgrading the expression of VEGF-a, is an effective therapy for steroid-induced femoral head necrosis in rabbit.4. One time intramuscularly injection of methylprednisolone sodium succinate20mg/kg is capable of effectively establishing animal model of steroid-induced femoral head necrosis in rabbit. |
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