Mwale Fackson, Wang Hong Tian, Roughley Peter, Antoniou John, Haglund Lisbet
1 Division of Orthopaedic Surgery, McGill University , Montreal, Canada .
Tissue Eng Part A. 2014 Nov;20(21-22):2942-9. doi: 10.1089/ten.TEA.2013.0749. Epub 2014 Jun 25.
Link N is a naturally occurring peptide that can stimulate proteoglycan synthesis in intervertebral disc (IVD) cells. IVD repair can also potentially be enhanced by mesenchymal stem cell (MSC) supplementation to maximize extracellular matrix (ECM) production. In a previous study, we have shown that Link N can inhibit osteogenesis and increase the chondrogenesis of MSCs in vitro. The aim of the present study was to determine the potential of MSCs and Link N alone or in combination with regard to tissue repair in the degenerate disc. Bovine IVDs with trypsin-induced degeneration were treated with MSCs, Link N, or a combination of MSCs and Link N. Trypsin-treated discs were also injected with phosphate-buffered saline to serve as a degeneration control. The ECM proteins and proteoglycans were extracted from the inner nucleus pulposus (NP) of the discs, and sulfated glycosaminoglycans (GAGs) were analyzed by the dimethyl methylene blue dye-binding assay. The expression of type II collagen was analyzed by western blot. To track the MSCs after injection, MSCs were labeled with PKH67 and observed under confocal microscopy after the 2 week culture period. The GAG content significantly increased compared with the degeneration control when degenerate discs were treated with MSCs, Link N, or a combination of both Link N and MSCs. Histological analysis revealed that the newly synthesized proteoglycan was able to diffuse throughout the ECM and restore tissue content even in areas remote from the cells. The quantity of extractable type II collagen was also increased when the degenerate discs were treated with MSCs and Link N, either alone or together. MSCs survived, integrated in the tissue, and were found distributed throughout the NP after the 2 week culture period. MSCs and Link N can restore GAG content in degenerate discs, when administered separately or together. Treatment with MSCs and Link N can also increase the expression of type II collagen. The results support the concept that biological repair of disc degeneration is feasible, and that the administration of either MSCs or Link N has therapeutic potential in early stages of the disease.
连接蛋白N是一种天然存在的肽，能够刺激椎间盘（IVD）细胞中蛋白聚糖的合成。通过补充间充质干细胞（MSC）以最大化细胞外基质（ECM）的产生，也可能增强IVD修复。在先前的一项研究中，我们已经表明连接蛋白N在体外可抑制MSC的成骨作用并增加其软骨形成。本研究的目的是确定MSC和连接蛋白N单独或联合应用在退变椎间盘中进行组织修复的潜力。将胰蛋白酶诱导退变的牛IVD用MSC、连接蛋白N或MSC与连接蛋白N的组合进行处理。还用磷酸盐缓冲盐水注射经胰蛋白酶处理的椎间盘作为退变对照。从椎间盘的髓核（NP）内部提取ECM蛋白和蛋白聚糖，并通过二甲基亚甲基蓝染料结合试验分析硫酸化糖胺聚糖（GAG）。通过蛋白质印迹法分析II型胶原蛋白的表达。为了在注射后追踪MSC，用PKH67标记MSC，并在培养2周后在共聚焦显微镜下观察。当退变椎间盘用MSC、连接蛋白N或连接蛋白N与MSC两者的组合处理时，GAG含量与退变对照相比显著增加。组织学分析显示，新合成的蛋白聚糖能够扩散到整个ECM中，甚至在远离细胞的区域也能恢复组织含量。当退变椎间盘单独或一起用MSC和连接蛋白N处理时，可提取的II型胶原蛋白的量也增加。培养2周后，MSC存活、整合到组织中，并发现分布在整个NP中。单独或联合给予MSC和连接蛋白N均可恢复退变椎间盘中的GAG含量。用MSC和连接蛋白N处理还可增加II型胶原蛋白的表达。这些结果支持椎间盘退变的生物修复是可行的这一概念，并且在疾病的早期阶段给予MSC或连接蛋白N具有治疗潜力。
