BMSCs-壳聚糖凝胶复合体移植治疗椎间盘退变的实验研究

BMSCs-CHITOSAN HYDROGEL COMPLEX TRANSPLANTATION FOR TREATING INTERVERTEBRAL DISC DEGENERATION

目的探讨兔BMSCs-壳聚糖凝胶复合体移植治疗椎间盘髓核缺损退变的效果,为临床应用提供实验依据。方法6只健康1月龄新西兰白兔,雌雄不限,体重1.0～1.5 kg。取骨髓2 mL,分离培养BMSCs。取第3代BMSCs,5-BrdU活细胞示踪剂标记,与壳聚糖凝胶混匀,制备BMSCs-壳聚糖凝胶复合体。将6只动物建立兔椎间盘髓核缺损退变模型,并随机分为3组(n=2):正常对照组仅分离暴露椎间盘,不作任何处理;移植治疗组将30μL自体BMSCs-壳聚糖凝胶复合体注射入缺损椎间盘中心;缺损退变组仅注射入0.01 mol/L PBS液30μL。移植后4周处死动物,取出移植修复的椎间盘,行细胞5-BrdU标记检测、HE、aggrecan番红O染色及ColⅡ免疫组织化学染色;ColⅡ免疫组织化学染色切片行灰度值测定。结果细胞标记检测发现,自体BMSCs移植后继续存活并增殖,形成细胞克隆。正常对照组及移植组椎间盘HE染色示椎间盘结构清晰,髓核组织及外周纤维环分界清晰,细胞核及细胞浆染色明显;缺损退变组示椎间盘结构紊乱,髓核组织和外周纤维化分界不清。aggrecan番红O染色示正常对照组及移植治疗组椎间盘染色明显,椎间盘结构清晰;缺损退变组椎间盘结构紊乱,髓核组织和外周纤维化分界不清。ColⅡ免疫组织化学染色示正常对照组以中央髓核组织染色为主,呈黄褐色阳性反应,椎间盘结构清晰;移植治疗组中央髓核组织呈阳性反应,细胞间质可见明显黄褐色,大体结构仍保持完整;缺损退变组染色较前两组浅,且结构不清。3组ColⅡ免疫组织化学染色切片行灰度值测定,正常对照组为223.84±3.93,与移植治疗组(221.03±3.53)比较差异无统计学意义(P>0.05),但两组与缺损退变组(172.50±3.13)比较,差异均有统计学意义(P<0.05)。结论兔BMSCs-壳聚糖凝胶复合体可修复椎间盘缺损退变,为临床应用可注射式组织工程髓核移植治疗椎间盘退变奠定实验基础。

Objective To investigate the therapeutic effect of BMSCs- chitosan hydrogel complex transplantation on intervertebral disc degeneration and to provide experimental basis for its clinical application. Methods Two milliliter of bone marrow from 6 healthy one-month-old New Zealand rabbits were selected to isolate and culture BMSCs. Then, BMSCs at passage 3 were labeled by S-BrdU and mixed with chitosan hydrogel to prepare BMSCs- chitosan hydrogel complex. Six rabbits were selected to establish the model of intervertebral disc degeneration and randomized into 3 groups （n=2 per group）： control group in which intervertebral disc was separated and exposed but without further processing; transplantation group in which 30 uL of autogenous BMSCs- chitosan hydrogel complex was injected into the center of defected intervertebral disc; degeneration group in which only 30 uL of 0.01 mol/L PBS solution was injected. Animals were killed 4 weeks later and the repaired discs were obtained. Then cell 5-BrdU labeling detection, HE staining, aggrecan safranin O staining, Col II immunohistochemical staining and gray value detection were conducted. Results Cell labeling detection showed that autogenous BMSCs survived and proliferated after transplantation, forming cell clone. HE staining showed that in the control and transplantation groups, the intervertebral disc had a clear structure, a distinct boundary between the central nucleus pulposus and the outer anulus fibrosus, and the obviously stained cell nuclear and cytochylema; while the intervertebral disc in the degeneration group had a deranged structure and an indistinct division between the nucleus pulposus and the outer anulus fibrosus. Aggrecan safarine O stainning notified that intervertebral disc in the control and transplantation groups were stained obviously, with a clear structure; while the intervertebral disc in the degeneration group demonstrated a deranged structure with an indistinct division between the nucleus pulposus and the anulus fibrosus. Col II immunohistochemical staining showed that the tawny-stained region in the control group was located primarily in.the central nucleus pulposus with a clear structure of intervertebral disc, the central nucleus pulposus in the transplantation group was positive with obvious tawny-stained intercellular substances and a complete gross structure, while the stained color in the degeneration group was lighter than that of other two groups, with a indistinct structure. Gray value assay of Col II immunohistochemical staining section showed that the gray value of the control, the transplantation and the degeneration group was 223.84 ± 3.93, 221.03 ± 3.53 and 172.50± 3.13, respectively, indicating there was no significant difference between the control and the transplantation group （P 〉 0.05）, but a significant difference between the control and transplantation groups and the degeneration group （P 〈 0.05）. Condusion The rabbit BMSCs-chitosan hydrogel complex can repair intervertebral disc degeneration, providing an experimental foundation for the clinical application of injectable tissue engineered nucleus pulposus complex to treat intervertebral disc degeneration.