Vickers Louise, Thorpe Abbey A, Snuggs Joseph, Sammon Christopher, Le Maitre Christine L
Biomolecular Sciences Research Centre Sheffield Hallam University Sheffield UK.
Materials and Engineering Research Institute Sheffield Hallam University Sheffield UK.
JOR Spine. 2019 Jun 26;2(2):e1055. doi: 10.1002/jsp2.1055. eCollection 2019 Jun.
We have previously reported a synthetic Laponite crosslinked poly N-isopropylacrylamide-co-N, N'-dimethylacrylamide (NPgel) hydrogel, which induces nucleus pulposus (NP) cell differentiation of human mesenchymal stem cells (hMSCs) without the need for additional growth factors. Furthermore NP gel supports integration following injection into the disc and restores mechanical function to the disc. However, translation of this treatment strategy into clinical application is dependent on the survival and differentiation of hMSC to the correct cell phenotype within the degenerate intervertebral disc (IVD). Here, we investigated the viability and differentiation of hMSCs within NP gel within a catabolic microenvironment. hMSCs were encapsulated in NPgel and cultured for 4 weeks under hypoxia (5% O) with ± calcium, interleukin-1β (IL-1β), and tumor necrosis factor alpha (TNFα) either individually or in combination to mimic the degenerate environment. Cell viability and cellular phenotype were investigated. Stem cell viability was maintained within hydrogel systems for the 4 weeks investigated under all degenerate conditions. NP matrix markers: Agg and Col II and NP phenotypic markers: HIF-1α, FOXF1, and PAX1 were expressed within the NPgel cultures and expression was not affected by culture within degenerate conditions. Alizarin red staining demonstrated increased calcium deposition under cultures containing CaCl indicating calcification of the matrix. Interestingly matrix metalloproteinases (MMPs), ADAMTS 4, and Col I expression by hMSCs cultured in NPgel was upregulated by calcium but not by proinflammatory cytokines IL-1β and TNFα. Importantly IL-1β and TNFα, regarded as key contributors to disc degeneration, were not shown to affect the NP cell differentiation of mesenchymal stem cells (MSCs) in the NPgel. In agreement with our previous findings, NPgel alone was sufficient to induce NP cell differentiation of MSCs, with expression of both aggrecan and collagen type II, under both standard and degenerate culture conditions; thus could provide a therapeutic option for the repair of the NP during IVD degeneration.
我们之前报道过一种合成的锂皂石交联聚N-异丙基丙烯酰胺-co-N，N'-二甲基丙烯酰胺（NP凝胶）水凝胶，它能诱导人间充质干细胞（hMSCs）向髓核（NP）细胞分化，无需额外的生长因子。此外，NP凝胶在注入椎间盘后能支持整合，并恢复椎间盘的机械功能。然而，将这种治疗策略转化为临床应用取决于hMSC在退变的椎间盘（IVD）内能否存活并分化为正确的细胞表型。在此，我们研究了在分解代谢微环境中hMSC在NP凝胶内的活力和分化情况。将hMSC封装在NP凝胶中，在缺氧（5% O）条件下，分别或联合添加钙、白细胞介素-1β（IL-1β）和肿瘤坏死因子α（TNFα）培养4周，以模拟退变环境。研究细胞活力和细胞表型。在所有退变条件下研究的4周内，水凝胶系统中干细胞活力得以维持。NP基质标志物：聚集蛋白聚糖（Agg）和II型胶原（Col II）以及NP表型标志物：缺氧诱导因子-1α（HIF-1α）、叉头框蛋白F1（FOXF1）和配对盒蛋白1（PAX1）在NP凝胶培养物中表达，且表达不受退变条件下培养的影响。茜素红染色显示，在含有氯化钙的培养物中钙沉积增加，表明基质钙化。有趣的是，在NP凝胶中培养的hMSC所表达的基质金属蛋白酶（MMPs）、含血小板反应蛋白基序的解聚蛋白样金属蛋白酶4（ADAMTS 4）和I型胶原受钙上调，但不受促炎细胞因子IL-1β和TNFα上调。重要的是，被认为是椎间盘退变关键因素的IL-1β和TNFα并未显示会影响NP凝胶中骨髓间充质干细胞（MSCs）向NP细胞的分化。与我们之前的研究结果一致，单独的NP凝胶足以在标准和退变培养条件下诱导MSCs向NP细胞分化，并表达聚集蛋白聚糖和II型胶原；因此可为IVD退变期间NP的修复提供一种治疗选择。
