1.Isolation and culture of rabbit nucleus pulposus cells in vitro by type Ⅱ collagenase digestion methods plus explant culture method
Shuwen LI ; Haijun WU ; Heping YIN ; Ming BAI ; Zhicai DU
Chinese Journal of Tissue Engineering Research 2013;(39):6861-6866
BACKGROUND:Intervertebral disc can bear load but lack vessels. Nucleus pulposus cel s have the problem of phenotype loss during in vitro culture that can lead to degenerative changes. The mechanism of intervertebral disc degeneration remains unclear.
OBJECTIVE:To explore the approaches of isolation, adherence culture, amplification and identification of the rabbit nucleus pulposus cel s in vitro, to observe the growth characteristics of nucleus pulposus cel s in different generations.
METHODS:Type Ⅱ col agenase digestion method plus explants culture method was used to isolate and purify nucleus pulposus cel s and then amplify in vitro. The morphology and growth of primary and passaged cel s was observed under the inverted microscope, the number of cel s was counted and the growth curve was draw. The morphology of the cel s was observed under light microscope after hematoxylin-eosin staining, and the expressions of col agen type Ⅱ and aggrecan were examined by immunocytochemistry.
RESULTS AND CONCLUSION:Nucleus pulposus cel s of rabbit were isolated, cultured and amplified in vitro successful y. Growth activity was observed, and found that the 1-3 generation nucleus pulposus cel s proliferated more rapidly and vigorously. The proliferation of nucleus pulposus cel s was decreased while the cel passaged more generations. These isolated and cultured nucleus pulposus cel s could positively express the col agen type Ⅱ and aggrecan. In vitro combination of type Ⅱ col agenase digestion method and explants culture method could obtain high purity nucleus pulposus cel s, and the cultured nucleus pulposus cel s were grew in round or polygonal. The 1-3 generation of cel s had strong activity.
2.Bone filling mesh container for osteoporotic vertebral fractures with posterior vertebral wall breakage:it is effective for prevention of bone cement leakage
Ming BAI ; Heping YIN ; Shuwen LI ; Zhicai DU
Chinese Journal of Tissue Engineering Research 2014;(47):7545-7549
BACKGROUND:Percutaneous kyphoplasty for osteoporotic vertebral fractures with posterior vertebral wal breakage can relieve pain rapidly, but there is a risk of leakage of bone cement. OBJECTIVE: To observe the effect of bone filing mesh container in percutaneous kyphoplasty for osteoporotic vertebral fractures with posterior vertebral wal breakage. METHODS: Forty senile patients with osteoporotic vertebral compression fractures were enroled, whose CT and MRI showed different degrees of posterior vertebral wal breakage, including 19 males (21 vertebral bodies) and 21 females (28 vertebral bodies), aged 50-87 years. These 40 patients were subjected to percutaneous kyphoplasty, and bone filing mesh container was used to deliver bone cement. Then, changes in visual analogue scale score, vertebral height and leakage of bone cement were observed in patients before and after treatment. RESULTS AND CONCLUSION:The surgery was successful in al the 40 patients, and no pulmonary embolism, cement leakage, and spinal cord and nerve root injuries appeared. Al the patients were folowed for 10-12 months. The visual analogue scale scores and vertebral height were improved significantly at both 1 week and 3 months after treatment compared with those before treatment, but there was no difference in the visual analogue scale scores and vertebral height at 1 week and 3 months after treatment. These findings indicate that percutaneous kyphoplasty with bone filing mesh container is effective to prevent bone cement leakage.
3.The application of automatic nerve retractor in micro-endoscopic discectomy
Heping YIN ; Shuwen LI ; Ming BAI ; Zhicai DU ; Zhenhua CAO ; Haijun WU ; Gedong MENG
Chinese Journal of Orthopaedics 2013;(1):39-43
Objective To explore the function and clinical effect of automatic nerve retractor in micro-endoscopic discectomy.Methods From August 2009 to December 2010,350 patients with lumbar disc hemiation were treated by micro-endoscopic discectomy,including 196 males and 154 females,aged from 17 to 68 years (average,42 years).Three cases were at L1-2,8 at L2-3,12 at L3-4,186 at L4-5 and 141 at L5S1.The automatic nerve retractor was used in all micro-endoscopic discectomy.The visual analogue scale (VAS) and Oswestry disability index (ODI) were used to evaluate clinical outcomes.Results All patients were followed up for 6 to 16 months (average,9 months).The mean VAS score decreased from preoperative 8.79±1.15 to 3.80±1.14 3 months after operation and 3.65±1.14 6 months after operation.The mean ODI score decreased from preoperative 78%±1.71% to 28%±1.72% 3 months after operation and 28%±1.88% 6 months after operation.Postoperative VAS and ODI scores decreased significantly compared with those before operation.The VAS and ODI scores 6 months after operation were not significantly improved compared with those 3 months after operation.No spinal cord and nerve root injury and epidural hematoma formation occurred in all cases.Conclusion In micro-endoscopic discectomy,the automatic nerve retractor can help the operator obtain effective exposure,protect nerves from injury,alleviate workload of the operators,therefore it has a great clinical application value.
4.Differentiation of SOX-9 and GDF-5 co-transfected bone marrow mesenchymal stem cells into nucleus pulposus cells
Zhicai DU ; Heping YIN ; Shuwen LI ; Haijun WU ; Ming BAI ; Zhenhua CAO ; Gedong MENG
Chinese Journal of Tissue Engineering Research 2015;19(19):2953-2958
BACKGROUND:Transplantation of mesenchymal stem cels to prevent and treat degeneration of the intervertebral disc is a feasible method. Mesenchymal stem cels co-transfected by SRY-related high mobility group-box gene 9 (SOX-9) and growth differentiation factor-5 (GDF-5) can differentiate into nucleus pulposus cels, in order to obtain greater effect of induction and proliferation of nucleus pulposus cels. OBJECTIVE:To investigate the effect of SOX-9 and GDF-5 co-transfection on the differentiation of rabbit bone marrow mesenchymal stem cels into nucleus pulposus cels. METHODS: We separated and cultured bone marrow mesenchymal stem cels from the bone marrow of rabbit aged 4 months. Passage 3 cels were divided into five groups andin vitro induced to differentiate into nucleus pulposus cels: non-transfected group, empty vector transfection group, SOX-9 transfection group, GDF-5 transfection group, SOX-9 and GDF-5 co-transfection group. At 14 days after transfection, RT-PCR was employed to assay SOX-9, GDF-5 and colagen type II mRNA expressions in bone marrow mesenchymal stem cels. The marker of nucleus pulposus cels-KRT19 expression was also detected by immunohistochemical staining. RESULTS AND CONCLUSION:In the co-transfection group, the mRNA expressions of SOX-9, GDF-5, and colagen type II were significantly higher than those in the SOX-9 transfection group, GDF-5 transfection group, and both these two groups, respectively (P < 0.05). Cels were positive for KRT19 in the SOX-9 and GDF-5 groups, and strongly positive for KRT19 in the co-transfection group. These findings indicate that double gene-transfected bone marrow mesenchymal stem cels are better than single gene-transfected cels with regard to differentiation into nucleus pulposus cels and secretion of extracelular matrix.