| Objective:To observe the growth character of rabbit adipose derived stem cells rabbit adipose derived stem cells and to test the its stem cell features; To research the experimental result of early osteonecrosis of rabbit femoral head induced by a comibination of methylprednisolone and lipopolysaceharide by histopathologic technique, MRI and Micro-CT imaging; To explore a therapeutic effect of autologous adipose derived stem cells with calcium alginate for steroid induced osteonecrosis of the femoral head (ONFH) in rabbit. Methods:1) from28weeks old New Zealand white rabbits were obtained by means of, obtaining and isolating the subcutaneous fat tissue from the scapular area, then digesting with collagenase type I. In vitro primary culture and passage culture, observe the biological characteristics as proliferation and rabbit adipose stem cells (cluster differentiation, CD) surface antigens were detected by flow cytometry, taking third passage of rADSCs to conduct adipogenic, chondrogenic and osteogenic induction, observed. pluripotential differentiation ability.2) Fifty male New Zealand white rabbits (age28weeks, weighing3-4kg) were intravenously injected once with10μg/kg body weight of lipopolysaccharide on day0. After24hours,3injections of20mg/kg body weight of methylprednisolone were given intramuscu-larly every24hours. On day0(preinduction/baseline) and at6weeks postinduction, in vivo MRI, Micro-CT imaging and Hematoxylin and Eosin staining was performed to valuate the ONFH model.3)6weeks after the last injection of methylprednisolone, the model were identified by MRI. Micro-CT imaging and HE staining; Animal models were made and divided into four groups randomly.Group A was used as control, while animals of group B, C and D were received core decompression and implantation with calcium alginate, core decompression and autografting rADSCs (not osteogenic induced) with calcium alginate, core decompression and autografting rADSCs (osteogenic induced) with calcium alginate. After two weeks osteoinduction in vitro, the rADSC with lginate Calcium complex injected into the osteonecrotic lesions under the X ray guidance, implantation of,4weeks and8weeks after surgery, rabbits in each group were randomly selected and first undergone MRI and Micro-CT scanning then the animals were killed and the bilateral femoral head were removed for the routine histology and immunohistochemical detection of osteocalcin (OCN) protein expression respectively. Results:1) We get the cells in vitro has a strong expansion, no significant change has seen after long-term passage; flow cytometry results show that the rADSCs positive to CD13, CD34, CD44, CD90, CD105, CD166, and CD49. Meanwhile, showed negative expression to CD14, CD45and CD31, excluding the hematopoietic system and endothelial cell contamination. Two weeks after the osteogenic induction of third passage rADSCs in vitro, positive staining of ALP and alizarin red staining; Two weeks after adipogenic induction, oil red staining is positive; two weeks after chondrogenic induction by micromass culture, immunofluorescence staining showed that the expression of collagen type Ⅱ positive.2) The MRI show that ONFH group SE T1WI sequence shown as points, thin line, sheet and intermediate signal intensity, T2WI manifested as points, thin line, like high signal intensity lesions TlWI showed a sheet on the middle signal intensity, T2WI show flaky low signal portion and joint effusion. HE staining show that the femoral head cartilage became thinning, trabecular bones have apparent difference in sparse and disorganized, part of the trabecular bone has fracture, diffuse presence of empty lacunae wre apparent, hematopoietic marrow cells within the femoral head were significantly reduced, shows little intravascular thrombosis; Micro-CT scanning show that the experimental group showing that the number of trabecular bone marrow cavity was significantly reduced, thinning, osteoporosis, some trabecular bone has been broken, missing, there low-density areas, three-dimensional reconstruction showed that three-dimensional bone structural damage of trabecular bone, the direction was poor, disorganized, and the surrounding trabecular bone connectivity is poor, showed a low-density areas;immunohistochemistry showed that the experimental group showing active osteogenesis within the trabecular bone. Conclusion:The differentiation potential of rADSCs used in three experiments was confirmed, these results show that the adipose derived stem cells we have isolated stem cells; a comibination of methylprednisolone and lipopolysaccharide could induce the early osteoneerosis of rabbit femoral head safely; adipose-derived stem cells with calcium alginate is new therapeutic approach to femoral head necrosis. |
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