Experimental And Clinical Study Of Sheng Mai Cheng Gu Capsule With Hip Abduction Orthosis For The Treatment Of Necrosis Of Children Femoral Head

1.Objective:Ischemic necrosis of children femoral head,also called Legg-Calve-Perthes disease was describled by Legg,Calve,Perthes three professors in 1910.Many experts have studied much about every aspect of this disease since then,they have make much progress.There are various kinds of etiologies about this disease,many treatments based on etiologies had been reported during the study process,however the operation based on the possible etiologies had brought a lot of controversy such as the following questions: is it necessary ? is it effective? Do it have side effect?.these debate need to be considered seriously,to be supported by long-term follow-up of a lot of cases and scientific effect evaluation,nowadays most scholars oppose that complex and traumatic operations would not be used untill the etiology is clear.Chinese medicine consider this disease is caused by deficiency of the kidney and liver,bone marrow is so few that it can not nourish the bone,if the external pathogen invade,it will cause the condition of the stagnation of qi and stasis of blood,meridian obstruction.The pathogenesis of necrosis of femoral head is lack of blood.Deficiency of kidney and liver is the common pathogenesis,we should invigorate the kidney and liver,activate blood circulation to dissipate blood stasis,move the qi to stop the pain.Sheng Mai Cheng Gu Capsule is a Chinese medicine for necrosis of the femoral head,there are many experiments and clinical application reports that have indicated its effectiveness.More and more scholars have pay more attention to the treatments based on the discipline of pathological change.The methods that prevent the femoral head deformity such as hip abduction orthosis and osteotomy containment treatment should be undertaken when it is waiting for the natural recovery to reach the matching state of hip and acetabular according to the biological shape,the method have the good effect and application future.Bone marrow mesenchymal stem cells study is important and meaningful in recovery and traumatic study field because it can proliferate again and again,can differentiate into the osteolbast.This study is planning to isolate and culture the bone marrow mesenchymal stem cells,then use the Chinese medicine Sheng Mai Cheng Gu Capsule that can invigorate the kidney and liver,activate blood circulation to dissipate blood stasis to culture the cells to observe whether the medicine can promote the cell proliferation and osteoblast or not,and We shall analyze the result,ln the clinical study,we compared and analyzed the outcomes of different treatments such as no systematic treatment,hip abduction orthosis,chinese medicine with orthosis to explore the medicine mechanism and clinical application of chinese medicine combined with hip abduction orthosis.2.MethodsStudy one:isolation and culture of MSCs and its osteoblast identification.1.bone marrow mesenchymal stem cells were isolated from Rat lower extremity, cultured in the condition of 37℃,5g/LCO₂,saturated humidity.The medium was changed to renew the metabolic environment and discard the non-adherent cells every three days.When the cell convergence were about 80%,it was digested with 2.5g/L trypsin(0.4g/L EDTA) at the ratio of 1:2.2.Cells shape was observed through inverted microscope.P2 and P3 cells were digested with 2.5g/L trypsion,vaccinated in twenty four hole culture plate with 1×10⁴/mL density,the number of cells were calculated every day for seven days and the growth curve was made.3.The CD34,CD44,CD54 antibody immunosorbent assay,calcium-cobalt method,mineralized nodules method were used to evaluate the osteoblast effect of MSCs.Study two:Experimental study of effect of Sheng Mai Cheng Gu Capsule serum on MSCs proliferation and osteoblast differentiation.1.Production of Sheng Mai Cheng Gu Capsule serum:The rats were feed with 1 g / kg Sheng Mai Cheng Gu Capsule,feed them in two hours.The blood was collected through Abdominal aorta in one hour,Centrifugal 3000 r / min,20min, the upper layer of liquid was collected,Filtered for sterilization,the 100 g / L medicine serum medium with L-DMEM were produced,the other group used the saline.2.The appropriate density bone marrow mesenchymal stem cells was choosed, then the Sheng Mai Cheng Gu Capsule serum medium and saline medium were used to culture cells,the number of cells were measureed each day by counting to observe the proliferation of stem cells.3.Medium was removed after seven days,washed three times with PBS,one minute each time,neutral formalin for fixation for ten minutes,washed with distilled water for three times,added fresh incubation medium for six hours,washed with distilled water,,then 20g/L Cobalt nitrate water was used for immersion for five minutes,washed with distilled water,then used lOg/L Ammonium sulfide water for staining for five minutes,washed with distilled water,neutral gum was used for fixation,and observed through microscope. the medium was removed after twelve days,washed three times with PBS,950g/h alcohol was used for immersion for thirty minutes,washed three times with PBS,then the 1g/L Alizarin red staining was used,neutral gum was used for fixation and observed through microscope.Study three:Clinical study of Perthes disease.1.A retrospective study of children femoral head necrosis from August, 2004 to Jaunary,2007,in the First Affiliated Hospital of GuangZhou Unive rstiy of Chinese Medicine was made.There were three groups:no systematic treatment,hip abduction orthosis,Chinese medicine with orthosis,we colle cted and sorted out imformation,analyzed the effectiveness rate, excellent and good rate according to the Stulberg,Mose circle criteria, epiphyseal extrusion,clinical symptoms,then analyzed with the Chi-squar e test.3.Results: Study one:isolation and culture of MSCs and its osteoblast identification.1.Bone marrow mesenchymal stem cell could be got through the all bone marrow culture,the cells have excellent ability to proliferation in vitro,the cell can finish the original culture in four-seven days.F2 cells growed rapidly, passed at the ratio of 1:3,the cells can cover the bottom of bottle in three days and form the good mono-layer cells.The cells can be induced into osteoblast by induction medium.2.The study indicate the growth state of MSCs is stable,there is no difference between P2 and P3 in shape and growth curve.The cells are even and fibroblast-like.Potential adaptation period are 1～2 days,logarithmic phase are the third or the fifth day,then come to plateau.In the logarithmic phase,colony multipliy for thirty four hours,passage period is about six days, the number of cells will double through each passage.3.immunosorbent assay indicated membrane of MSCs is dark red,opaque in CD44(+)CD54(+),but don't showed the feature of hematopoietic stem cell.After induction,ALP staining is obvious that grey and black particles or massive Precipitation within the Cytoplasmic.Alizarin red staining can show red Nodular particles indicated mineralized matrix deposition.Study two:Experimental study of effect of Sheng Mai Cheng Gu Capsule serum on MSCs proliferation and osteoblast differentiation.1.Sheng Mai Cheng Gu Capsule serum can get more cells through proliferation than the saline group.2.After the Chinese medicine serum medium culture,ALP staining was cle ar,there were grey and black particles or massive Precipitation within th e Cytoplasmic.Alizarin red staining can show red Nodular particles.Study three:Clinical study of Perthes disease.1.In the clinical study,we had seventy five cases(seventy eight hips) at last.No systematic treatment group had eighteen cases(eighteen hips),hip abduction orthosis group had eighteen cases(nineteen hips),Chinese medicine combining orthosis group had thirty nine cases(fourty one hips).After three years follow-up,there was difference in VAS hip pain and outcome(P＜0.05),the Chinese medicine combining orthosis group hava a better outcome.The clinical results showed there was significant difference in three groups,the Sheng Mai Cheng Gu Capsule combined with abduction orthosis gourp had the best results.4.Conclusions:4.1 We can have the BMSCs by using the all bone marrow culture method.The cells can be induced into osteoblast by induction medium. 4.2 Sheng Mai Cheng Gu Capsule serum can promote the stem cells proliferation and induce them into osteoblasts in vitro compared to saline group,this may be one mechanism of this medicine that can treat the necrosis of children femoral head.4.3 Applying the the Sheng Mai Cheng Gu Capsule combined with hip abduction orthosis to the treatment of Perthes disease is a good and effective method,...