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BACKGROUND: There are different methods to isolate and culture human nucleus pulposus cells, and the differences in digestive enzymes components and digestion time quite are significant. So how to rapidly and efficiently harvest human nucleus pulposus cells has become a research hotspot. OBJECTIVE: To optimize the digestive enzymes components and digestion methods for the preparation of human nucleus pulposus cells. METHODS: Nucleus pulposus tissue specimens were selected from three adult discs in the Department of Orthopedics, China-Japan Union Hospital of Jilin University. The acute traumatic disc tissues that outstanding to the spinal canal were taken under aseptic conditions, and then the peripheral white annulus and jel y-like nucleus pulposus in the center could be seen. According to different mixed enzyme concentration ratio, the samples were divided into two groups. The enzyme Ⅰ group was treated with 0.2% Ⅱ col agenase; and the mixed enzymeⅡ group was digested with 0.25% trypsin for 30 minutes, and then treated with 0.2% Ⅱ col agenase. According to digestion time, each group was divided into three subgroups: 2 hours group, 4 hours group, and overnight group. Final y, suspended cel volume was decided as 2 mL to count cells. Dulbecco’s modified Eagle’s medium containing fetal bovine serum was used for cel culture in vitro. Trypan blue staining was performed to count total cel number and ratio of living cells. Methylthiazolyldiphenyl-tetrazolium bromide assay was used to detect the growth curve of nucleus pulposus cells. RESULTS AND CONCLUSION: Based on the two digestion enzyme concentration, the number of digested cells in the enzyme Ⅰ group was larger than that in the enzyme Ⅱ group after digested for 2 and 4 hours, but the difference was not significant (P > 0.05). Overnight, cellsurvival rate was decreased in the enzyme Ⅰ group after digested for 2 and 4 hours when compared with the enzyme Ⅱ group, and the difference was significant (P < 0.05). After digested for 4 hours, tissue blocks disappeared, and the number of cells reached maximum. The results indicate that enzyme Ⅰgroup composite with Ⅱ col agenase is benefit for the separation of nucleus pulposus cells, and the digestion time is appropriate to 4 hours. This condition has the advantages of simple operation, high efficiency and low cost, and it considered that digestion of nucleus pulposus tissues with 0.2% Ⅱ col agenase for 4 hours is the best condition to obtain nucleus pulposus cells.
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10.3969/j.issn.2095-4344.2013.24.001
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BACKGROUND:Insulin-like growth factor 1 is the key factor during cartilage development, which is involved in the growth and reconstruction of condylar cartilage. OBJECTIVE:To study the effect of insulin-like growth factor 1 on cel apoptosis and the apopotosis-associated factors of Bcl-2, Bax mRNA and protein expressions of rat condylar chondrocytes. METHODS:The 1-day-old and 28-day-old rat condylar chondrocytes were cultured and identified in vitro. The condylar chondrocytes with different ages were divided into experimental group and control group. After being starved for 24 hours, chondrocytes in the experimental group were incubated with 100μg/L recombined rat insulin-like growth factor 1 for 48 hours, while the chondrocytes in the control group were incubated normal y. RESULTS AND CONCLUSION:Compared with the control group, after being incubated with recombined insulin-like growth factor 1, the number of condylar chondrocytes was increased with high speed proliferation (Pproliferation and reduce cel apoptosis of newborn and adolescent rat condylar chondrocytes, which may be mediated by Bcl-2 and Bax.
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BACKGROUND:Many studies have shown that matrix metal oproteinases 1, 3, 9 and 13 play an important role in articular cartilage degeneration and destruction, but there is less special research on the articular synovium. OBJECTIVE:To observe the effect of long-distance running on the expressions of matrix metal oproteinases 1, 3, 9 and 13 in the synovium. METHODS:Fifteen male Wistar rats were divided into three groups:control group, tablet group and uphil group. Rats in the control group received ordinary captivity;rats in the tablet group ran on the horizontal treadmil (0°) at the speed of 1 km/h for 1 hour daily, and lasted for 45 days;rats in the uphil group daily ran on the horizontal treadmil (0°) at the speed of 1 km/h for 1 hour, and lasted for 15 days, and then the rats ran on the uphil treadmil (+20°) at the speed of 1 km/h for 1 hour daily and lasted for 30 days. The knee joint synovium injury models with varying degrees were established. The dual hind knee joints were obtained after modeling for paraffin-embedded. Then the overal sagittal slices were obtained for hematoxylin-eosin staining and immunohistochemical staining, and the experimental results were observed and analyzed. RESULTS AND CONCLUSION:After long-distance running, the expression of matrix metal oproteinases 1 in synovium of the tablet group and uphil group was increased when compared with that of the control group (P0.05). There was no significant difference in matrix metal oproteinases 3 expression (P>0.05). The expressions of matrix metal oproteinase 9 and matrix metal oproteinase 13 in synovium were in gradient increasing state (Pexercise can influence the normal physiological structure of rat knee joint synovium by changing the expression of matrix metal oproteinases.
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BACKGROUND: Ischemia/reperfusion can induce degenerative alterations in articular cartilage. However, the precise mechanism remains poorly understood. OBJECTIVE: To observe the morphological changes and the apoptosis of articular cartilage of femoral head epiphyses with ischemia/reperfusion. METHODS: A total of 80 Sprague-Dawley rats were randomly assigned to two groups: ischemia/reperfusion (model of ischemia/reperfusion in hip joint) and sham-surgery (exposure of abdominal aorta for 5 minutes) groups, with 40 animals in each group. Articular cartilages of femoral head epiphysis were col ected in 6, 12, 24, and 48 hours, 5 days, and 2 and 4 weeks after operation. Morphology of articular cartilage of femoral head epiphyses was examined by light microscope, and cel apoptosis was detected by TUNEL method. RESULTS AND CONCLUSION: Light microscopy showed chondrocytes degeneration and reduction, as wel as fibrosis in matrix of cartilage in the ischemia/reperfusion group. Chondrocyte apoptosis was observed in both groups by TUNEL. Several apoptotic cells, less than five, were observed in the sham-surgery, while 10-30 apoptotic cells were found in ischemia/reperfusion group at 48 hours. Results indicated that ischemia/reperfusion can induce degenerative changes in articular cartilage of femoral head epiphyses, and cel apoptosis in developing hip joint may participate in damage of articular cartilage. Inhibition of chondrocyte apoptosis in articular cartilage may be useful for the prevention and cure of early osteoarthritis.