Laboratoire de Biologie Tissulaire et Ingénierie thérapeutique, UMR5305 CNRS/Université Lyon 1, UMS3444 BioSciences Gerland-Lyon Sud, Lyon, France; Université de Lyon, Université Lyon 1, Faculté d'Odontologie, Lyon, France; Hospices Civils de Lyon, Service d'Odontologie, Lyon, France.
Université de Lyon, Université Lyon 1, CNRS, Ingénierie des Matériaux Polymères (IMP), UMR5223, Villeurbanne, France.
Dent Mater. 2019 Apr;35(4):523-533. doi: 10.1016/j.dental.2019.01.018. Epub 2019 Jan 31.
Regenerating a functional dental pulp in the pulpectomized root canal has been recently proposed as a novel therapeutic strategy in dentistry. To reach this goal, designing an appropriate scaffold able to prevent the growth of residual endodontic bacteria, while supporting dental pulp tissue neoformation, is needed. Our aim was to create an innovative cellularized fibrin hydrogel supplemented with chitosan to confer this hydrogel antibacterial property.
Several fibrin-chitosan formulations were first screened by rheological analyses, and the most appropriate for clinical use was then studied in terms of microstructure (by scanning electron microscopy), antimicrobial effect (analysis of Enterococcus fæcalis growth), dental pulp-mesenchymal stem/stromal cell (DP-MSC) viability and spreading after 7 days of culture (LiveDead test), DP-MSC ultrastructure and extracellular matrix deposition (transmission electron microscopy), and DP-MSC proliferation and collagen production (RT-qPCR and immunohistochemistry).
A formulation associating 10mg/mL fibrinogen and 0.5% (w/w), 40% degree of acetylation, medium molar mass chitosan was found to be relevant in order to forming a fibrin-chitosan hydrogel at cytocompatible pH (# 7.2). Comparative analysis of fibrin-alone and fibrin-chitosan hydrogels revealed a potent antibacterial effect of the chitosan in the fibrin network, and similar DP-MSC viability, fibroblast-like morphology, proliferation rate and type I/III collagen production capacity.
These results indicate that incorporating chitosan within a fibrin hydrogel would be beneficial to promote human DP tissue neoformation thanks to chitosan antibacterial effect and the absence of significant detrimental effect of chitosan on dental pulp cell morphology, viability, proliferation and collagenous matrix production.
在根管治疗后的根管内再生功能性牙髓已被提出作为一种新的治疗策略。为了实现这一目标，需要设计一种合适的支架，既能防止残留牙髓细菌的生长，又能支持牙髓组织的新生。我们的目的是创造一种创新的细胞化纤维蛋白水凝胶，并用壳聚糖来赋予这种水凝胶抗菌性能。
首先通过流变学分析筛选了几种纤维蛋白-壳聚糖配方，并进一步研究了最适合临床应用的纤维蛋白-壳聚糖配方，包括微观结构（扫描电子显微镜）、抗菌效果（粪肠球菌生长分析）、牙髓间充质干细胞/基质细胞（DP-MSC）活力和培养 7 天后的扩散（LiveDead 试验）、DP-MSC 超微结构和细胞外基质沉积（透射电子显微镜）以及 DP-MSC 增殖和胶原蛋白产生（RT-qPCR 和免疫组织化学）。
发现将 10mg/mL 纤维蛋白原与 0.5%（w/w）、40%乙酰化度、中等摩尔质量壳聚糖结合，可以形成一种在细胞相容性 pH 值（#7.2）下的纤维蛋白-壳聚糖水凝胶。纤维蛋白-壳聚糖水凝胶与纤维蛋白单独的比较分析表明，壳聚糖在纤维蛋白网络中具有很强的抗菌作用，同时对 DP-MSC 活力、成纤维细胞样形态、增殖率和 I/III 型胶原蛋白产生能力没有显著的不良影响。
这些结果表明，在纤维蛋白水凝胶中加入壳聚糖可以通过壳聚糖的抗菌作用和壳聚糖对牙髓细胞形态、活力、增殖和胶原基质产生没有显著的不良影响，有利于促进人牙髓组织的新生。
