Clinical And Experimental Research Of Comprehensive Conservative Treatment For Avascular Necrosis Of The Femoral Head

Objective Clinical and experimental researches were performed to summarize the treatment effect of comprehensive conservative methods for FicatⅠ-Ⅲstage of avascular necrosis of the femoral head(ANFH),in the mean time to search for one kind of suitable material for filling the bone defect after removal of necrotic lesions to promote bone repair.Further more,we try to investigate the tissue-engineered artificial bone material for treating advanced ANFH.The detail items were included as fellow.(1)Analyzing clinical data of the patients suffered from ANFH,who received comprehensive non-surgical treatment methods,to evaluate the efficacy of comprehensive conservative treatment.(2)Studying the clinical effect of nano-hydroxyapatete/collagen composite in Ficat StageⅢof ANFH,and the autologous tissue engineering composite of bone marrow in recurrent bone cyst of the femoral head and neck,to analyze the demands of selecting filler material for the repair of bone defect after the lesions removed.(3)A kind of composite material composed of calcium phosphate and collagen was made for the purpose of repairing bone defect and loading seed cells in tissue-engineered materials.(4)To study the effects of recombinant basic fibroblast growth factor(bFGF) gene transfection on biological characteristics of bone marrow-derived osteoblasts and angiogenesis to provide a basis for bone marrow-derived osteoblasts as seeds in tissue-engineered artificial bone.Methods(1)Clinical research:the clinical and image data of 135 patients of ANFH was analyzed retrospectively,and the comprehensive non-surgical treatment measures that the patients had received were composed of several kinds of methods,such as mitigating weight loading,functional exercise of hip joint, chirismus,hot fomentation,physical therapy and so on.Traditional Chinese Medicine and Western medicine were also taken orally to activate blood circulation for the purpose of dissipating blood stasis and osteogenic action. Among the 135 cases,80 cases were undergone the feeding artery perfusion in the suffered femoral head.(2)Under the monitor of X-ray imaging,three patients had undergone cutting open and drilling hole below the greater trochanter of femur,the necrotic tissues of four femoral heads were cleaned and implanted with the nano-hydroxyapatete/collagen composite.X-ray film was used for investigation during 6 to 11 months period of follow-up,among the three patients two cases were simultaneously reviewed by CT.One patient with recurrent bone cyst in the femoral head and neck also received the implanted treatment in the same way.Drilling into the cyst cavity of the bone,after the lesion was moved, the autologous bone marrow tissue engineering composite composed of autologous bone marrow,autogenous bone and methyl prednisolone were implanted into the cavity.X-ray or CT scan was reviewed every 2 to 3 months after operation.(3)Experimental studies:calcium phosphate and calcium hydrogen phosphate were mixed up with typeⅠcollagen according to quality ratio of 35%:35%:30%.The sponginess composite was made by drying technique of vacuum freeze and then dialyzed by semipermeable membrane.The bone marrow mesenchymal stem cells (BMSCs) of rabbit were isolated in vitro,with their number increased after culture and then osteogenically induced.The alkaline phosphatase in inductive culture medium supernatant was detected consecutively at 1,3 and 5 week,and the osteoblast differentiations was verified by alizarin bordeaux staining.The induced BMSCs were cultured in the dialyzed composite materials and then detected by pathological section.(4)The pcDNA3-bFGF eukaryotic expression plasmid of cow was transfected individually into the BMSCs and inducted bone marrow-derived osteoblast cells of rabbit by liposome-mediated method and G418 selection.The expression of bFGF in transfected cells was analyzed by RT-PCR and immunocytochemical staining.In addition,after stably expression of bFGF,the biological characteristics of BMSCs and bone marrow-derived osteoblasts were analyzed in the fields of morphology,proliferation,cell cycle,alkaline phosphatase, typeⅠcollagen and so on.After stably transfected of bFGF gene,the bone marrow-derived osteoblasts of rat were cultured with self-made calcium phosphate together,and then implanted into the intramuscular sacks of hind legs of rats.The rats were executed respectively after 2,4 weeks of normal feeding,and the composite materials were moved out to cut into pathological sections for observing the angiogenesis.Results(1)According to the evaluation criteria of the therapeutic effect of ANFH,the overall effective rate of 183 hips in the 135 cases was 85.8%(157/183),in which 20.8%(38/183)hips were excellent,48.1%(88/183) well,16.9%(31/183) stable and 14.2%(26/183) progressive.(2)Three patients received treatment of implanting nano-hydroxyapatite (nano-HA) artificial bone,in which two patients had good clinical effect 11 months later with three ANFH recovered well and the surface of the femoral head not collapsed further,another patient had bad clinical effect with the surface of the femoral head collapsed markedly owing to loading weight early and subsequently total hip replacement was performed.After 5,10 months,the hone cysts of one case treated by autologous bone marrow tissue engineering composite healed well with marginal sclerosis,but without recurrence or complications.The bone cyst healed in the degree of GradeⅣaccording to the Neer' s X-ray evaluation criteria of bone cyst.(3)In the experimental studies,after in vitro induced culture of BMSCs,the alkaline phosphatase(ALP) in the inductive culture medium supernatant was to increase gradually at 1,3 and 5 week(P＜0.05),and the alizarin bordeaux staining was strong positive.In the same time,the pathological section showed that the inductive BMSCs grew well in the composite material.(4)The pcDNA3-bFGF eukaryotic expression plasmid of cow was transfected into the BMSCs and bone marrow-derived osteoblasts of rabbit successfully and expressed stably.The expressions of bFGF mRNA and its protein in the transfected cells were demonstrated by RT-PCR and immunocytochemical staining. In addition,the growth speed of cells became faster after transfection of bFGF gene into the BMSCs and bone marrow-derived osteoblasts,and the cells rates in proliferating stage of the cell cycle increased,but the cell morphology and the secretary of ALP between transfected and non-transfected bone marrow-derived osteoblasts had not changed markedly(P＞0.05),however, the ALP secretion of transfected BMSCs were less than bone marrow-derived osteoblasts(P＜0.01).The mRNA expression of TypeⅠcollagen was seen in the bone marrow-derived osteoblasts whether it was transfected of bFGF or not, but it was not seen clearly in the BMSCs when transfected or not.After the composite materials containing transfected bFGF gene were removed out from rats,pathological section showed that the composite materials was surrounded partially by hyperplastic fiber connective tissues where there were a number of angiogenesis increasing with time.Conclusions(1)The combined non-surgical treatment has a definite therapeutic effect for ANFH,but the prognosis would be affected to a certain extent because of long period of conservative therapy and insufficient dependence.(2)Nano-hydroxyapatete/collagen composite bone can be used as repair material to treat ANFH,but it is lack of biological activity,which needs a longer time of treatment during this period the femoral head of necrosis may become badly collapsed easily because of premature load.The implantation of autologous bone marrow tissue engineering composite in the treatment of recurrent bone cyst is effective,it is worth promoting.(3)The self-made composite material has a good biocompatibility with the inductive BMSCs,which could be used as carrier for seed cells in repairing bone defect.(4)After stably expressed of bFGF,the proliferation of BMSCs and bone marrow-derived osteoblasts were faster,but the bone marrow-derived osteoblasts was more ideal than BMSCs when acting as the seeds of tissue engineering bone.After transfection,the stable expression of exogenous bFGF gene could prompt the growth of newborn blood vessels,which illustrated that bFGF can be used as a goal gene to transfect seed cells of tissue-engineered artificial bone.