Effect Of Mesenchymal Stem Cells Into Osteoblasts And Cbfα1 Gene Expression By Extracorporeal Shock Wave On Patients Of Avascular Necrosis Of The Femoral Head

Objective: avascular necrosis of the femoral head (ANFH) has been one of the thorny problem of orthopedic sector. It has been confirmed that systemic bone mesenchymal activity decreased and mesenchymal stem cells (MSCs) into fat cell increase in patients, corresponding lead to mesenchymal stem cells into osteoblasts and the bone formation of bone marrow decreased. Currently, the clinical doctors have not particularly effective method to treat ANFH of Ficatâ… -â…¢period. It has been reported that extracorporeal shock wave therapy (ESWT) treat ANFH achieved more satisfactory effectin recent years. Core binding factorα1 (Cbfα1) is not only osteoblast-specific transcription factor, but the cartilage cells mature, osteoclast differentiation and the development of angiogenesis invasion of cartilage tissue also plays an important regulatory role. Currently, domestic and international no news reports that Cbfα1mRNA how to express after extracorporeal shock wave (ESW) intervention MSCs of the patients of ANFH, the experiment carried out around the center, with a view to explain the effectiveness of ESWT treats ANFH.Methods: This study isolated and cultured ANFH MSCs in vitro by density gradient centrifugation with adherent; According to the principle of random implanted into Group A (ESW intervention group), Group B(blank control group); After the optimal energy density (5kv/500 frequency) interference, CCK-8 method detected the OD value of the two Groups at different days, compared differences in cell proliferation; modified Gomori's Ca-Co method staining and Alizarin red staining cells were detected ALP and the extracellular matrix mineralization, observation and comparison cell differentiation into osteoblasts between the two Groups. using RT-PCR combinate OD semi-quantitative to analyzes the expression differences of Cbfα1mRNA and OCmRNA between the two Groups at different days. Using a statistical method to analyse the corresponding part of the data.Results:1. The primary cells were round, sizes, numbers large, refraction strong of soma, nuclear oval; it was observed that some cells adherent after 24 h, the adherent cells increased and there are clonal after 48 h, the adherent cells were clonal growth at the 5rd-7th d, the morphology of cells changes a long spindle partly; it can be passaged when primary cultured cells integration and achieved 85% of the growth surface after two weeks;2. The passaged cells showed colony-like growth early; the cells enter the peak of proliferation at the 4-5th d, nuclear mitotic phase was increased, and symmetrical division, cell were arranged in the direction rules, the overwhelming majority of elongate spindle, the late vortex-like mesh cells were growing and into spacious flat-zoster or multilateral by the spindle, showing osteogenic differentiation trend; Group B enter to the peak of proliferation period than Group A, the division asymmetry, various separatist, the sizes of cell morphology, cell division in non-contour and the performance of the cell differentiation into other types;3. The cell activity of Group A was detected by live/dead viability/cytotoxicity kit (L-3224) staining after ESW intervention is (95±3%) which has not significant differences compared with Group B. Growth curve manifests that the OD value of Group A increased at the 2nd d gradually, it reached the peak at the 6th d and has begun to enter the platform at 7th d; the OD values of Group B increased significantly at the 3th d and appearance the peak at the 7th d and has begun to enter the platform at the 8th d. the two groups revealed that no significant difference at the 1st d(P>0.05) and the rest were significantly different (P<0.01); 4. The two groups cells was detected by modified Gomori's Ca-Co method staining. Results manifests that the ALP staining positive after ESW intervention can be found at the 6th d, but there was no significant difference of masculine rate(P>0.05); With the extension of incubation time, ALP staining intensity of the two groups was increased significantly, Group A is more obvious which reached peak and a lot of dense black precipitation within the extracellular at the 24th d; the positive rate of Group A declined slightly at the 30th and 36th d and Group B positive rate increased, but Group A staining intensity is still stronger than the Group B. The ALP staining of Group B is positive, but weaker strength than Group B, which only can be seen scattered on the gray brown sediments in the cytoplasm and has not large tracts of black precipitation. The sizes and diversity of Group B is diverse. The average positive of the cell counting of ALP staining were significantly different (P<0.05)at each time points (except for 6th d ) between the two groups. The peak value of average positive rate of Group A up to 90% at the 24th d. The peak value of average positive rate of Group B increased to 50% at the 30th d.5. The P₇ cells of Group A was stained by Alizarin red which showed that there are a large number orange red calcified nodules at the 20th d,then select three vision randomiy and counted mineralized nodule under 100 times light microscope and the results is 7.0±1.3 in Group A, Group B were not significant mineralized nodules and always negative;6. PCR products revealed that Cbfα1mRNA expression of THE two groups can be detected at the 3rd d after ESW intervention, the subsequent expression increase gradually, The Group A was stronger than the Group B significantly. The OCmRNA expression of the Group A appears at the 12th d and Group B appears until the 18th d, the subsequent expression of the two groups strengthened gradually. The semi-quantitative analysis of the OD manifests that Cbfα1 mRNA sustained expression in the whole process of ESW induced osteoblast differentiation which appears peak at the 12th d and weakened expressed at the 15th d, apart from the 3th d were no significant differences (P<0.05). The rest of the Group a was significant difference compared with the Group B (P<0.01); OCmRNA expression earlier and OD of Group A was significantly stronger than the Group B, there was a significant different(P<0.01).Conclusion:1. The appropriate intensity ESW can promote and induct MSCs proliferation and osteoblastic differentiation on patients of ANFH; 2. The secretion increased of ALP and extracellular matrix mineralization of the MSCs on patients of ANFH are the key steps of ESW promote osteogenic differentiation ;3. Cbfα1mRNA expression is earlier than the OCmRNA expression which is early expression products of MSCs on patients of ANFH osteogenic differentiation, and which sustained in the entire process of ESW induced osteogenic differentiation; and which sustained expression in the entire process of ESW induced osteoblast differention; OCmRNA expression in the mineralization period of MSCs on patients of ANFH which is the key factor of matrix mineralization;4. Promoting MSCs on patients of ANFH proliferation, induced osteoblast differentiation and strengthening Cbfα1mRNA expression is one of the effectiveness of ESWT treats ANFH;5. ESWT combinates MSCs transplanting may be a positive and effective way of treats ANFH and other bone repair capacity constrained diseases in clinical in the future.