| Objective:â‘ To explore the feasibility of setting up animal model of steroid-induced avascular necrosis of femoral head by combining low-dosage methylprednisolone (MPS) with equine serum (ES);â‘¡Adipose stem cells were obtained by the method of collagenase I digestion, inoculated,cultured,prolificated and identified. To observe the basic biological properties and explore optimal collagenase duration and concentration for rabbit adipose stem cells(ASCs) cultured in vitro;â‘¢To observe the effect of long-term culture in vitro on capacity of ASCs osteogenic, Further investigating the future of ASCs in tissue engineering and regenerative engineering.Methods:â‘ Twenty healthy Japanese white rabbits were divided into experimental group (Group A) and control group (Group B) randomly. Group A consisted of fourteen animals that received 10ml/kg of Equine serum (ES) intravenously two times at two weeks intervals, the second injection were divided into two times for consecutive days, ES doses were 5mg/ml a day. After the first ES be injected 24 hours, 4mg/kg/week of methylprednisolone (MPS) acetate was injected by gluteus medius muscle for consecutive eight weeks. Group B was made up of twelve animals that were treated with isotonic saline, neither ES nor MPS. All animals were raised in cages on a standard diet and water. Twelve rabbits were killed at 4 weeks, respectively, including group A and group B. 10 at eight weeks. Hematologic studies were performed, time ranged from ahead of the first injection of ES to after 2 weeks, 4 weeks, 8 weeks of MPS be first injected respectively. Meanwhile, else examination was carried out including X-ray, Magnetic Resonance Imaging (MRI), histopathologic, and Transmission Electron Microscope (TEM) at 4 weeks or 8 weeks respectively.â‘¡The subcutaneous fat tissue was harvested from posterior cervical region of rabbit with the typeâ… collagenase. Dulbecco's Modified Eagle Medium (DMEM) with fetal bovine serum(FBS)was used for cell culture.The effect of different Collagenase concentrations and durations on biological characteristics and capacity of amplification was to be observed for adipose stem cells cultured in vitro. Immunofluorescence technic be used to check the existence of CD44. ASCs were induced into osteoblasts byosteogenic inducing fluid and into adipocytes by adipogenic inducing fluid. The osteogenic phenotypes were examined by Von Kossa staining or cell alkaline phosphatase staining and the adipocytes by Oil Red O staining.â‘¢The third, sixth, ninth, twelfth and fifteenth passage ASCs were differentiate into osteoblasts by rhBMP-2 respectively. To identified the osteogenic differentiation by Von Kossa staining or cell AKP staining. Meanwhile, Osteoblasts were identified by ALP activity and calcium concentration staining one or two weeks later. The effect of long-term culture of adipose stem cells on osteogenic capacity was studied.Results:â‘ Two animals died after the second injection of ES in group A, other animals was survived and passed the experiment successfully. Serum level of Cholesterol and triglyceride were significantly higher at 2, 4 and 4 weeks after corticosteroid treatment in rabbits with ES and relative to control group rabbits receiving isotonic saline. There were significant difference between two groups (P<0.05),There was also prominent difference in APTT(P<0.05), nevertheless, it was sharply decreased. There was no apparent changes between two group at two weeks, the density of bone in Metaphysis were decreased at the fourth weeks, it seems that lies in area of the pouch changes, The joint space was no clear and the bone structure was unclear 8 weeks after steroid treatment, changes of local signal were seen in part of animals with MRI, sign of Subcortical hemorrhage was seen in 8 week. The rate of empty lacunae significantly increased in 4 weeks and 8 weeks by Hematoxylin and eosin staining, there was significantly different between group A and group B(.P<0.01), a few osteocyte structure was unclear under Transmission Electron Microscope 4 weeks, part of osteocyte has caryorrhexis and caryolysis sign, Apoptosis cell was appeared abundantly and structure of bone collagen was confused.â‘¡The ASCs that was resected from posterior cervical region of rabbits was rapidly expand and can proliferate stably, there was no significant decrease to passage eighteen, Immunofluorescence technic showed ASCs were positive for CD44 and can maintained undifferentiated stage. Calcium nodes characteristic of osteoblasts were observed in the ADSCs on Von Kossa staining after induction with medium, and red-stained fats characteristic of adipocytes were noted in the cytoplasm on Oil Red O staining, according to the collagenase concentration,cells were divided into three groups, with concentrations of 1.0,1.5 and 2.0mg/ml.At a duration of 0.5 hour and 1 hour,the 2.0mg/ml group had the highest total cells number(TCN)and the total cell rate was statistically different from the other groups.when the duration was 1.5 hours.Meanwhile ,TCN increased in every group and there was no statistical differences among the 1.0,1.5,2.0 mg/ml groups. Live cell rate was the highest in the 1.0 mg/ml groups, but there was no significant difference among these groups.To observe the cell growth characteristics of the third passage. with concentrations of 1.0 mg/ml and take 1.5 hour have the best proliferation capability.â‘¢Different passage ASCs were differentiate into osteoblasts by rhBMP-2 for 1 weeks and 2 weeks, Compared with the control group, AKP activity and calcium concentration was significantly different (P<0.01), there was no obvious difference before passage 12 in AKP activity (P<0.05), and there were statistical difference between passage and passage 9 (P<0.05), the have no significant different before passage 12 in calcium concentration (P<0.05), yet, there were statistically different between passage 15 and passage 12 before(P<0.05), the osteogenic differentiation by Von Kossa staining or cell AKP staining were positive for calcium node and red-brown particle in kytoplasm. Conclusion:â‘ Combining ES and steroid significantly increased incidence of osteonecrosis in rabbits, maybe contributed to set up steroid-induced avascular necrosis of femoral head.â‘¡The adipose stem cells(ASCs)is a kind of multidifferentiation potential stem cells that could be isolated from adipose tissue. could grow and proliferate rapidly in vitro.â‘¢The optimal culture conditions for rabbit adipose stem cells are collagenase concentration at 1.0 mg/ml,with digestion duration of 1.5hours.â‘£There was no significant difference before passage 12 on osteogenic ability, and promising provided adequate active seed cells for bone tissue engineering. |
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