Research On The Role Of Endothelial Colony Forming Cells In Pathogenesis Of Glucocorticoid-induced Avascular Osteonecrosis Of The Femoral Head

Objectives:This study examined whether abnormalities of early EPCs and endothelial colony forming cells (ECFCs) are present and compared their functions in glucocorticoid (GC)-induced avascular osteonecrosis of the femoral head (ANFH).Methods:Early EPCs and endothelial colony forming cells (ECFCs) were obtained from33patients with glucocorticoid-induced ANFH and33age-and sex-matched control subjects. Cells were isolated, in vitro cultured and studied by Flow Cytometry and Immunofluorescence. Colony-forming unit counts were observed from33patients and33healthy controls. Growth kinetics, migratory capacity to multiple chemoattractants, in vitro tube formation capacity and cytokine (vascular endothelial growth factor and stromal cell-derived factor-1) levels in supernatants of two types of EPCs were assayed in ANFH patients and matched controls.Results:Mean numbers of colonies formed by both types of EPCs were decreased in ANFH patients (Early EPCs:2.42±1.46versus4.52±2.00,p<0.05; ECFCs:0.62±0.55versus1.12±0.82,p<0.05,). Early EPCs from ANFH patients showed impaired migratory capacity (63.8±11.7versus152.3±12.4,p<0.001) and VEGF secretion (50.8±7.2pg/ml versus62.8±10.1pg/ml, p<0.05). ECFCs from ANFH patients showed decreased tube formation capacity (7.1±2.7versus23.8±4.3,p<0.001) and proliferation.Discussion:Early EPCs and ECFCs were impaired in number and function in GC-induced ANFH, and their distinct reduced capacity profiles might reflect different roles they played in endothelial dysfunction of GC-induced ANFH. Objective:There is still uncertainty about in vitro culture and expansion of endothelial colony forming cells (ECFCs) and its application in tissue engineering. To elucidate a method for in vitro culture, identification and large-scale expansion of ECFCs and to investigate its biocompatibility with biological material. To explore the effect of Glucocorticoid on ECFC in vitro culture.Methods:Monocytes were isolated by Ficoll density gradient centrifugation from human umbilical cord blood, in vitro cultured and expanded in EGM-2medium on tissue culture plates precoated with type I rat tail collagen. Then cells were characterized by Immunofluorescence using Dil-Ac-LDL, FITC-UEA-1, and antibodies CD31, vWF, KDR, and Tie-2. The growth kinetics, tube formation capacity and biocompatibility with HA/TCP were observed. The concentration of SDF-1,CXCR4, KDR and Tie-2were determined by Western blot on ECFC under0,1uM,1nM Dexamethasone.Result:During culture, the cells displayed cobble-stone morphology with outgrowth, took up DiI-Ac-LDL, bound FITC-UEA-1, and stained positively for cell markers CD31, vWF, KDR and Tie-2. These cells demonstrated high proliferation rate after passaged, formed closed network structures on Matrigel and the bioactivity remained stable when co-cultured with HA/TCP. The concentration of SDF-1,CXCR4, KDR and Tie-2of ECFC were decreased under Dexamethasone.Conclusion Results indicate it is feasible to isolate and large-scale expand ECFCs from human umbilical cord blood in vitro and ECFCs may serve as seed cells in tissue engineering. The cytokine synthesis of ECFC is suppressed under Dexamethasone. Objective:To explore the roll of human umbilical cord blood derived ECFC on treatment of early rabbit Glucocorticoid induced femoral head necrosis.Methods:Monocytes were isolated by Ficoll density gradient centrifugation from human umbilical cord blood, in vitro cultured and expanded in EGM-2medium. Thirty-five new-Zealand rabbits were injected10μg/Kg Lipopolysaeeharide and three injections of Methylprednisolone. After4weeks, X-ray and MRI were observed in rabbits. Then femoral head necrosis rabbits were operated. We drilled2mm diameter tunnel under the greater trochanter to the femoral head and put ECFCs into the channel. Micro CT and histological evaluations were performed to observe the rabbit femoral heads after8weeks.Result:During culture, ECFC cells demonstrated high proliferation rate after passaged. X-ray and MIR showed nonuniformity of bone density of femoral head. Osteporosis,cystis were observed in subchondral bone in X-ray. Bone cell lacunae enlarged fat cells and microvascular thrombosis were observed in histological evaluations. After8weeks therapy of ECFC transplantation, we saw new trabeculae bone generation in histological evaluations and X-ray showed morphologically normal femoral heads and uniform bone mineral density.Conclusion A comibination of methylprednisolone and liPoPolysaccharideeould induce early osteonecrosis of rabbit femoral head safely. ECFCs could induced bone generation and bone generation in rabbit.