Tissue & Organ Mechanobiology, Institute of Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland.
Spine J. 2013 Mar;13(3):273-83. doi: 10.1016/j.spinee.2012.12.007. Epub 2013 Jan 23.
Proteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc.
To characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM.
Disc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model.
Phosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy.
A large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc.
By varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.
通过对椎间盘中的蛋白酶进行消化，可以模拟椎间盘退化的情况，从而对退行性椎间盘的细胞-支架构建体进行研究。
用木瓜蛋白酶来描述不同严重程度糖胺聚糖（GAG）和水分流失的体外椎间盘退化模型（DDM），并确定引入该 DDM 后人类间充质干细胞（MSCs）的初始反应。
通过向牛尾椎间盘样本的终板内注射不同浓度的木瓜蛋白酶，诱导椎间盘退化模型。之后，将标记的 MSCs 引入该模型中。
向牛尾椎间盘样本中注射磷酸盐缓冲盐水（PBS 对照）或不同浓度的木瓜蛋白酶（3、15、30、60 和 150 U/mL）。注射后 10 天，通过二甲亚甲基蓝测定法评估椎间盘的 GAG 含量，并通过活/死染色和共聚焦显微镜确定细胞活力。通过组织学评估总体基质组成，并通过磁共振成像可视化水含量。还记录 DDM 的压缩和扭转刚度。在第二部分，将 MSCs 用荧光细胞膜示踪剂标记，并注入 DDM 核或 PBS 对照中。通过共聚焦显微镜评估间充质干细胞的活力和分布。
向椎间盘内注射 >30 U/mL 的木瓜蛋白酶可使牛椎间盘的 GAG 和水分大量流失。向椎间盘内注射 30、60 和 150 U/mL 的木瓜蛋白酶，磁共振成像显示为 II 级、III 级和 IV 级椎间盘退变。椎间盘中心的空洞有利于随后注射髓核组织工程构建体，同时保留完整的纤维环。剩余椎间盘细胞活力不受影响。注入蛋白酶处理后的 DDM 椎间盘的 MSCs 显示出比注入 PBS 注射对照椎间盘更高的细胞活力。
通过改变注射用木瓜蛋白酶的浓度，可以诱导出越来越多的 GAG 和水分流失，从而模拟不同严重程度的椎间盘退化。注入椎间盘的 MSC 悬液短期存活非常低。然而，应该清楚的是，这种牛椎间盘 DDM 并不能反映临床情况，但为测试新的组织工程方案提供了令人兴奋的可能性。
