The Changes Of The Nucleus Pulposus After The Whole Intervertebral Discs Were Cultivated

Intervertebral disc degeneration is the important reason of low back pain and the damage of spine. Intervertebral disc degeneration could lead to disc herniation,spinal stenosis and spondylolishesis, etc. More than 90% spine operations are concerned with disc degeneration.The etiology of disc degeneration is very complex, and our research on this topic is moving forward gradually. Many methods and models were used for the experiments including in vivo system and/or in vitro system. Disc degeneration could be induced by mechanical or chemical method, but it could not be used for the test about cell metabolism and signal conduction. Cell culture is a method of in vitro experiment, but the cell without matrix could not present its phenotype.A system of organ culture should be built to make sure the integrity of intervertebral disc and the interaction between cells and matrix. It has advantage in cell culture and signal conduct. Part 1 Organ culture of the whole intervertebral disc in vitroObjective:To find a practical method of culturing discs organ system by observing the changes of the nucleus pulposus after the whole intervertebral discs, including cartilage eng-plate nucleus,pulposus and anulus fibrosus, were cultivated.Methods:1,A total of 350 intervertebral discs were taken out completely from 60 healthy SD rats (about 150g) aged 5-6 weeks of clear grade and divided into 5 groups randomly. The whole intervertebral disc includes nucleus pulposus. annular fibrosus. cartilagineus end plate and little cancellated bone adhering end plate;2,The discs were rinsed by high osmotic saline solution containing heparin for 3 times and trimmed with microscopic spectacles, then put into the culture plate. The whole intervertebral discs were cultured with high osmotic culture medium and this medium was changed once every day. The high osmotic culture medium was made by adding 3.72g NaCL to 1000ml DMEM/F12 culture medium. Culture condition is 37℃,5%CO2. The concentration of fetal bovine serum is 20%;3,10 discs were selected randomly and divided into 5 groups randomly, and the 5 discs of control group were soaked in liquid nitrogen for 3 times(1 minute per time).240μl NBT was added into the culture medium. The discs were taken out after 8 hours, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, The discs were embedded in paraffin, then sectioned for observation.4.75 discs were selected randomly and divided into 5 groups randomly and cultured in the medium. Each group has 15 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin, sectioned for observation. 240μl NBT was added into the culture medium 8 hours before the discs were taken out.5,10 discs were selected randomly and divided into 5 groups randomly, then cultured in the medium. Each group has 2 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin and sectioned to 4-6μm for immunohistochemistry test;6,75 discs were selected randomly and divided into 5 groups randomly, then cultured in the medium. Each group has 15 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin and sectioned to 4-6μm for HE stain;7,75 discs were selected randomly and divided into 5 groups randomly, then cultured in the medium. Each group has 15 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin and sectioned to 4-6μm for safranineO stain;8,100 discs were selected randomly and divided into 4 groups randomly, then cultured in the medium. Each group has 25 discs. The discs of one group were taken out respectively after 0,3,7 and 14 days, and rinsed by high osmotic saline solution for 3 times.250μg nucleus pulposus were taken out from 5 discs, then decomposed by Trizol for PCR. Result:1,The whole intervertebral disc organ could be dissected from the spine of SD rat except for little cancellated bone adhering end plate;2,NBT could stain live disc cells, but couldn't stain dead cells;3,The cell viability was not changed significantly at 14 day (P> 0.05) and was significantly lower at 21 days than at other time points (P< 0.01).4,The immunohistochemistry staining results for collageⅡwere positive in nucleus pulposus cells at every time point.5,HE staining showed that the tissue integrity and morphology of the whole intervertebral discs were not changed at 14 days6,SafranineO staining showed no significant differences in the matrix grey scale (P> 0.05) at 0-14 days and significant differences between at 21 day and at 0-14 days (P< 0.05).7,RT-PCR results showed that the mRNA of CollageⅠincreased with time, but the expressions of CollageⅡ, aggrecan and decorin decreased, showing statistically significant differences in the mRNA expressions of the matrix protein at each time point (P<0.05).Conclusion:1,NBT could be used to detect live disc cells;2,Our system could be used to cultivate the whole intervertebral discs, it is an ideal model for further studies on physiology and pathology of intervertebral discs. Part 2 Organ culture of the whole intervertebral disc in subcutaneous tissue of SD ratObjective:Is subcutaneous tissue of SD rat a good area for Organ culture of the whole intervertebral disc?Methods:1,A total of 160 intervertebral discs were taken out completely from 32 healthy SD rats (about 150g) aged 5-6 weeks of clear grade and divided into 6 groups randomly. The whole intervertebral disc includes nucleus pulposus,annular fibrosus,cartilagineus end plate and little cancellated bone adhering end plate;2,The discs were rinsed by high osmotic saline solution containing heparin for 3 times and trimmed with microscopic spectacles, then put into the culture plate in culture condition for using later;3,The discs were imbedded in the subcutaneous tissue of 16 healthy SD rats anesthetized by 10% Chloral Hydrate. And the SD rats were fed commonly after analepsia;4,15 discs were taken out respectively from 3 SD rats after 3 and 7days randomly, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last the discs were embedded in paraffin and sectioned to 4-6μm for safranineO stain;5,25 discs were taken out respectively after 3 and 7days randomly, and rinsed by high osmotic saline solution for 3 times.250ug nucleus pulposus were taken out from 5 discs, then decomposed by Trizol for PCR. Result:1,The architectonic of matrix was decomposed in safranineO stain at 3 and 7 day. The result of image analysis was different statistically at 0,3 and 7day (P< 0.01);2,RT-PCR results showed that the mRNA of Collage I increased with time, but the expressions of Collage II, aggrecan and decorin decreased, showing statistically significant differences in the mRNA expressions of the matrix protein at each time point (P<0.05).Conclusion:The method and the location of culturing the whole intervertebral disc in the body should be discussed carefully later.