Department of Biomedical Engineering, Rensselaer Polytechnic Institute , Troy, New York.
Tissue Eng Part A. 2013 Nov;19(21-22):2452-63. doi: 10.1089/ten.TEA.2012.0644. Epub 2013 Jul 11.
The use of hydrogels for bone regeneration has been limited due to their inherent low modulus to support cell adhesion and proliferation as well as their susceptibility to bacterial infections at the wound site. To overcome these limitations, we evaluated multifunctional polysaccharide hydrogels of varying stiffness to obtain the optimum stiffness at which the gels (1) induce proliferation of human dermal fibroblasts, human umbilical vascular endothelial cells (HUVECs), and murine preosteoblasts (MC3T3-E1), (2) induce osteoblast differentiation and mineralization, and (3) exhibit an antibacterial activity. Rheological studies demonstrated that the stiffness of hydrogels made of a polysaccharide blend of methylcellulose, chitosan, and agarose was increased by crosslinking the chitosan component to different extents with increasing amounts of genipin. The gelation time decreased (from 210 to 60 min) with increasing genipin concentrations. Proliferation of HUVECs decreased by 10.7 times with increasing gel stiffness, in contrast to fibroblasts and osteoblasts, where it increased with gel stiffness by 6.37 and 7.8 times, respectively. At day 14 up to day 24, osteoblast expression of differentiation markers-osteocalcin, osteopontin-and early mineralization marker-alkaline phosphatase, were significantly enhanced in the 0.5% (w/v) crosslinked gel, which also demonstrated enhanced mineralization by day 25. The antibacterial efficacy of the hydrogels decreased with the increasing degree of crosslinking as demonstrated by biofilm formation experiments, but gels crosslinked with 0.5% (w/v) genipin still demonstrated significant bacterial inhibition. Based on these results, gels crosslinked with 0.5% (w/v) genipin, where 33% of available groups on chitosan were crosslinked, exhibited a stiffness of 502±64.5 Pa and demonstrated the optimal characteristics to support bone regeneration.
水凝胶在骨再生中的应用受到限制，因为它们的固有低模量难以支持细胞黏附和增殖，并且在伤口部位容易受到细菌感染。为了克服这些限制，我们评估了不同硬度的多功能多糖水凝胶，以获得凝胶（1）诱导人真皮成纤维细胞、人脐静脉内皮细胞（HUVEC）和鼠前成骨细胞（MC3T3-E1）增殖的最佳硬度，（2）诱导成骨细胞分化和矿化，以及（3）表现出抗菌活性的最佳硬度。流变学研究表明，通过交联壳聚糖成分，用不同量的京尼平交联甲基纤维素、壳聚糖和琼脂糖的多糖混合物制成的水凝胶的硬度增加。随着京尼平浓度的增加，凝胶的凝胶时间从 210 分钟减少到 60 分钟。与成纤维细胞和成骨细胞相反，随着凝胶硬度的增加，HUVECs 的增殖减少了 10.7 倍，而分别增加了 6.37 和 7.8 倍。在第 14 天到第 24 天，0.5%（w/v）交联凝胶中骨钙素、骨桥蛋白和早期矿化标志物碱性磷酸酶的成骨细胞分化标志物表达显著增强，第 25 天也显示出增强的矿化。通过生物膜形成实验证明，水凝胶的抗菌功效随交联程度的增加而降低，但用 0.5%（w/v）京尼平交联的凝胶仍显示出显著的抑菌作用。基于这些结果，用 0.5%（w/v）京尼平交联的凝胶（其中壳聚糖上 33%的可用基团交联）表现出 502±64.5 Pa 的硬度，并表现出支持骨再生的最佳特性。
