3B's Research Group-Biomaterials, Biodegradables and Biomimetics, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, Guimarães, Portugal.
Tissue Eng Part C Methods. 2011 Oct;17(10):961-72. doi: 10.1089/ten.TEC.2011.0115. Epub 2011 Jul 7.
Low back pain is one of the most reported medical conditions associated to intervertebral disc (IVD) degeneration. Nucleus pulposus (NP) is often regarded as the structure where IVD degeneration begins. Gellan gum (GG)-based hydrogels for acellular and cellular tissue engineering strategies have been developed for finding applications as NP substitutes. The innovative strategy is based on the reinforcement of the hydrogel matrix with biocompatible and biodegradable GG microparticles (MPs), which are expected to improve the mechanical properties, while allowing to tailor its degradation rate. In this study, several GG MP/hydrogel disc formulations were prepared by means of mixing high acyl GG (0.75% (w/v)) and low acyl GG (2% (w/v)) GG aqueous solutions at different ratios, namely, 75%:25% (v/v), 50%:50% (v/v), and 25%:75% (v/v), respectively. The GG MP size was measured using a stereo microscope, and their dispersion within the hydrogel matrix was evaluated by means of staining the MPs with Toluidine Blue-O. The developed GG MPs/hydrogel discs were physicochemically characterized by Fourier-transform infrared spectroscopy and (1)H-nuclear magnetic resonance spectroscopy. The swelling behavior and degradation rate were assessed by immersion in a phosphate buffer saline for 14 days. The morphology and mechanical behavior were investigated by scanning electron microscopy and dynamic mechanical analysis, respectively. The mechanical properties of the hydrogel disc were improved by mixing the gels with the MPs. In addition, the possible cytotoxicity of the leachables released by MPs/hydrogel discs was screened in vitro, using a mouse lung fibroblast cell line (L929 cells). To investigate the encapsulation efficacy of L929 cells into the GG MPs/hydrogel discs, cells were stained with DAPI blue/Texas Red-Phalloidin and observed by confocal microscopy, after 24, 48, and 72 h of culturing. A cell viability assay was also performed using Calcein AM staining. The cell culture studies demonstrated that MPs/hydrogel discs are noncytotoxic over L929 cells. It was also demonstrated that L929 cells can be successfully encapsulated into the GG MPs of different formulations, remaining viable after 72 h of culturing. This study showed that GG hydrogel matrices reinforced with cell-loaded MPs could be a candidate strategy for NP regeneration.
下腰痛是与椎间盘（IVD）退变相关的最常见医学病症之一。髓核（NP）通常被认为是 IVD 退变开始的结构。已开发出基于胶凝多糖（GG）的水凝胶用于无细胞和细胞组织工程策略，以寻找作为 NP 替代品的应用。这项创新策略基于用生物相容性和可生物降解的 GG 微球（MPs）增强水凝胶基质，这有望改善机械性能，同时允许调整其降解率。在这项研究中，通过以不同比例混合高酰基 GG（0.75%（w/v））和低酰基 GG（2%（w/v））GG 水溶液，制备了几种 GG MP/水凝胶盘制剂，分别为 75%:25%（v/v）、50%:50%（v/v）和 25%:75%（v/v）。使用立体显微镜测量 GG MP 的尺寸，并通过用甲苯胺蓝-O 染色 MPs 来评估 MPs 在水凝胶基质中的分散情况。通过傅里叶变换红外光谱和（1）H 核磁共振光谱对所开发的 GG MPs/水凝胶盘进行了物理化学表征。通过将水凝胶盘在磷酸盐缓冲盐水（PBS）中浸泡 14 天来评估其溶胀行为和降解率。通过扫描电子显微镜和动态机械分析分别研究了形态和力学性能。通过混合凝胶和 MPs 来改善水凝胶盘的力学性能。此外，通过使用小鼠肺成纤维细胞系（L929 细胞），在体外筛选了由 MPs/水凝胶盘释放的浸出物的潜在细胞毒性。为了研究 L929 细胞包封到 GG MPs/水凝胶盘中的包封效率，在培养 24、48 和 72 h 后，用 DAPI 蓝色/Texas Red-Phalloidin 对细胞进行染色，并通过共聚焦显微镜进行观察。还进行了使用 Calcein AM 染色的细胞活力测定。细胞培养研究表明，MPs/水凝胶盘对 L929 细胞无细胞毒性。还证明 L929 细胞可以成功包封到不同配方的 GG MPs 中，在培养 72 h 后仍保持存活。这项研究表明，用负载细胞的 MPs 增强的 GG 水凝胶基质可能是 NP 再生的候选策略。
