The Experimental Study Of Intervertebral Disc Tissue And Cell And The Histological Influence Of Collagenase On Them In Vitro Culture

[Objective] Collagenase is currently used as the critical drug for the treatment of discogenic pain. We aim to construct the intervertebral disc tissue and cell culture system in vitro and further observe the histological changes of intervertevral disc tissues and cells after injection of collagenase in different concentration and evaluate the effects of collagenase on disc tissues and cells by measuring wet weights and alive cells counting. MethodExperiment Ⅰ First, we respectively construct the culture systerm of human degenerated disc tissue and rabbit normal intervertebral disc in vitro. After acquiring the discs of human and rabbit, then cutting them and culture them in DMEM supplemented with 20% fetal calf serum, tranfoming growth factor- β(TGF-β),and with media changes being performed every 3 days.Experiment Ⅱ Part Ⅰ .Selcting Randomly 18 degenerated intevertebral disc specimens from patients who were orperated secondary to the herniation of cervical intervertevral disc(HCID). Culturing the disc tissues according to the method of experimenti. Then these tissues bedivided into trial group (Jk ? which respectively contains 200U/ml> 400U/ml collagenase 200H1 and control group in which same volume normal solution was given. Each group has 6 samples. Observing the morphological changes of disc tissues by discrepancy microscope at 24 hours, 48hours, 1 week after the enzyme and normal soulution was given, partll. 24 experiment rabbits Selected randomly were sacrificed by injecting 10ml air into the vein vessel. Acquiring the intervertebral disc in the asepsis state, then culture them according the method of experiment I. When the monolayer cells grow into their next generation. Divided these cells into trial group (TX ?, ?which were respectively given 200U/ml> 400U/mK 800U/ml collagenase 200W and control groups which were given equal volume of normal solution. Each group has 6 samples. Observing their morphological changes by microscope at different time points as before. Partlll. Selcting Randomly 16 intevertebral disc specimens from patients as before, cutting them into small size and culture them, then divived them into trial goups which were given 400U/ml collagenase 200m and equal volume of normal solution was added in control groups. Measuring the wet weights of the disc tissues at Oh, 12h, 24h, 36h time points after the enzyme was given. 2. Select randomly another 16 rabbits' intervertebral discs , cut and culture them as before, then counting alive disc cells which were disolved by collagenase and diffuse into the surrounding solution by 0. 4% trypan blue dye. SPSS13. 0 was used in this part for statistic analysis. To compare group difference, We use the method of repeated measure of analysis of variance (ANOVA) .One way ANOVA was used to compare different time points within group. T-test was used to conpare the wet weight and alivecounting at the same time point between groups. ResultsExperiment I Human degenerated intervertebral disc tissues in the culture state shows abundant extracelluar martrix. The collagen fiber grow fastly and the disc cells show good condition, we found that the monolayer cells of intervertebral disc appear as different morphology. Within one week , the disc cells lost their original shape and became shuttle, and their axises parallel to each other. Experiment IIParti:There was no significant morphological difference between all the trial group which contains 200U/ml and the 400U/ml collagenase. The histological appearance in all the control groups was no difference. The tissue strcture of disc became relax after 24 hours following collagenase was given. The disc cells in the extracelluar matrix (ECM) distribution was parallelized with the collagen fibers in disc tissues. The ECM was abundant in the control groups. The disk cells distribution were regularly paralized with the collagen fibers. The ECM in the disc tissues was decreased after 48 hours following the collagenase was given and the ECM was further decreased after one week, Meanwhile, the disk cells tends to necrosis gradually with time changes. The collagen fibers of disc tissue in the control group grows in their good condition.Partll Neither the 200U/ml nor 400U/ml collagenase group shows difference in morphological appearance. The disc cell grows normally as monolayer apparence in the 200U/mU 400U/ml collagenase group and control group. The disk cells appears as different shapes in these groups, while the disc cells in the 800U/ml collagenase group grow...