摘要
目的 探讨髓核球型细胞簇在椎间盘退变过程中的修复作用及其潜在的治疗应用.方法 对SupraGel水凝胶进行降解及流变性能表征,通过活死细胞学染色对SupraGel水凝胶中的球型髓核细胞簇进行生物相容性检测.对20只SD大鼠构建尾椎间盘穿刺退变模型,分组及干预措施:穿刺退变节段但不进行干预(阳性对照组)、穿刺退变节段注射SupraGel水凝胶(水凝胶组)、穿刺退变节段注射球型细胞簇(细胞簇组)、穿刺退变节段注射负载球型细胞簇的SupraGel水凝胶(水凝胶+细胞簇组)和健康节段(阴性对照组).干预后的第4周和第8周进行X线片及MR检查,根据椎间盘高度指数及Pfirrmann分级评估椎间盘退变情况.通过HE染色、番红O-固绿染色及Ⅱ型胶原免疫组化染色对椎间盘组成结构和细胞外基质的变化情况进行评估.结果 SupraGel水凝胶10h降解剩余率为73.7％,具有优异的剪切变稀和自恢复特性.髓核细胞能够在SupraGel水凝胶中自发聚集形成稳定球形细胞簇,在第7天依旧保持活性.体内实验中,干预后第4周和第8周水凝胶+细胞簇组椎间高度指数分别为88.12％±4.55％、74.13％±4.09％,高于阳性对照组和水凝胶组,低于阴性对照组,差异有统计学意义(P＜0.05);Pfirrmann分级分别为2.40±0.55、2.60±0.89,第4周时低于阳性对照组和水凝胶组,第8周时低于阳性对照组、水凝胶组和细胞簇组,高于阴性对照组,差异有统计学意义(P＜0.05);形态学评分分别为(6.60±0.55)、(9.40±1.14)分,第4周时低于阳性对照组、水凝胶组及细胞簇组,第8周时低于阳性对照组、水凝胶组及细胞簇组,高于阴性对照组,差异有统计学意义(P＜0.05);Ⅱ型胶原光密度分别为0.12±0.01、0.10±0.01,第4周时高于阳性对照组、水凝胶组和细胞簇组,第8周时高于阳性对照组和水凝胶组,差异有统计学意义(P＜0.05).结论 髓核球型细胞簇可成功构建,并证实在大鼠椎间盘退变模型中有延缓退变的作用.
Abstract
Objective To explore the repair effect of nucleus pulposus spherical cell clusters in the process of interverte-bral disc degeneration and its potential therapeutic application.Methods The degradation and rheological properties of Supra-Gel hydrogel were characterized,and the biocompatibility of spherical nucleus pulposus cell clusters in SupraGel hydrogel was tested by live-dead cytological staining.A caudal disc puncture degeneration model was established in 20 SD rats,and the rats were divided into groups and intervention measures:puncture of degenerated segment without intervention(positive control group),puncture of degenerated segment injection of SupraGel hydrogel(hydrogel group),puncture of degenerated segment injection of spherical cell clusters(cell cluster group),puncture of degenerated segment injection of SupraGel hydrogel loaded with spherical cell clusters(hydrogel+cell cluster group)and healthy segment(negative control group).X-rays and MRI examinations were per-formed at the 4th and 8th weeks after intervention,and the disc degeneration was evaluated according to the disc height index and Pfirrmann grading.HE staining,safranin O-fast green staining and COL-2 immunohistochemical staining were used to evaluate the changes in the disc structure and extracellular matrix.Results The degradation residual rate of SupraGel hydrogel was 73.7％af-ter 10 h,which showed excellent shear thinning and self-recovery properties.Nucleus pulposus cells could spontaneously aggre-gate in SupraGel hydrogel to form stable spherical cell clusters,which remained active on the 7th day.In the in vivo experiment,the intervertebral height index of the hydrogel+cell cluster group was 88.12％±4.55％and 74.13％±4.09％at 4 and 8 weeks after intervention,which were higher than those of the positive control group and hydrogel group,and lower than those of the negative control group,with statistically significant differences(P＜0.05);the Pfirrmann grades were 2.40±0.55 and 2.60±0.89,which were lower than those of the positive control group and hydrogel group at 4 weeks,and lower than those of the positive control group,hy-drogel group and cell cluster group at 8 weeks,and higher than those of the negative control group,with statistically significant dif-ferences(P＜0.05);the morphological scores were 6.60±0.55 and 9.40±1.14,which were lower than those of the positive control group,hydrogel group and cell cluster group at 4 weeks,at the 8th week,the levels of collagen type Ⅱ were lower than those of the positive control group,hydrogel group and cell cluster group,but higher than those of the negative control group,and the differ-ence was statistically significant(P＜0.05).The optical density of type Ⅱ collagen was 0.12±0.01 and 0.10±0.01,respectively,which were higher than those of the positive control group,hydrogel group and cell cluster group at the 4th week,and higher than those of the positive control group and hydrogel group at the 8th week,and the difference was statistically significant(P＜0.05).Conclusion Nucleus pulposus spherical cell clusters can be successfully constructed and have been shown to have the effect of delaying degeneration in the rat intervertebral disc degeneration model.
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