Swiss Paraplegic Research, 6207, Nottwil, Switzerland.
Eur Spine J. 2012 Aug;21 Suppl 6(Suppl 6):S826-38. doi: 10.1007/s00586-011-1975-3. Epub 2011 Aug 24.
Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells.
Four types matrixes approved as medical devices for other applications were tested as scaffolds for MSCs: two made of equine or porcine collagen, one of gelatin and one of chitosan. Bone marrow-derived human MSCs were seeded in these scaffolds or embedded in alginate, as a three-dimensional control. After five weeks in culture, NP-like differentiation of the cell-scaffold constructs was analyzed by qRT-PCR, histology, total DNA quantification, proteoglycan accumulation and immunohistochemistry.
MSCs in collagen matrixes and gelatin produced more mRNA and proteins of the chondrogenic markers collagen type I, collagen type II (COL2) and aggrecan (ACAN), when compared with cells embedded in alginate or chitosan. Proteoglycan accumulation and cell survival were also higher in collagen and gelatin matrixes. Gene expression results were also confirmed by histological and immunohistochemical staining. In contrast to alginate control, the gene expression of the undesired bone marker osteopontin was lower in all tested groups. In porcine collagen supports, MSC expression ratio between COL2/ACAN closely resembled the expression of nucleus pulposus cells, but gene expression of recently described NP markers keratin19, PAX1 and FOXF1 was lower.
Collagen supports provide a readily available, medically approved and effective scaffold for chondrogenic differentiation in vitro, but the phenotype of differentiated MSCs is not yet completely equivalent to that of NP cells.
基于细胞的疗法可用于退化的椎间盘（IVD）再生，是目前手术干预的替代方法。间充质干细胞（MSCs）与支架结合，可能是再生椎间盘核（NP）的理想候选物，NP 是 IVD 的压力分布部分。虽然目前人们对 MSCs 分化的生长因子的应用给予了极大的关注，但在本研究中，我们比较了海绵样基质在支持细胞分化为 NP 样细胞方面的性能。
测试了四种类型的已批准用于其他应用的医疗器械基质作为 MSC 的支架：两种由马或猪胶原制成，一种由明胶制成，一种由壳聚糖制成。骨髓来源的人 MSC 被接种在这些支架或藻酸盐中，作为三维对照。培养五周后，通过 qRT-PCR、组织学、总 DNA 定量、糖胺聚糖积累和免疫组织化学分析细胞-支架构建物的 NP 样分化。
与嵌入藻酸盐或壳聚糖的细胞相比，在胶原基质和明胶中的 MSC 产生更多的软骨形成标志物胶原 I、胶原 II（COL2）和聚集蛋白聚糖（ACAN）的 mRNA 和蛋白质。胶原和明胶基质中的糖胺聚糖积累和细胞存活率也更高。基因表达结果也通过组织学和免疫组织化学染色得到证实。与藻酸盐对照相比，所有测试组中不期望的骨标志物骨桥蛋白的基因表达都较低。在猪胶原支架中，MSC 表达的 COL2/ACAN 比值与核髓细胞的表达非常相似，但最近描述的 NP 标志物角蛋白 19、PAX1 和 FOXF1 的基因表达较低。
胶原支架为体外软骨分化提供了一种现成的、医学上可接受的有效支架，但分化的 MSC 的表型尚未完全等同于 NP 细胞。
