School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore.
ACS Appl Mater Interfaces. 2010 Jul;2(7):2012-25. doi: 10.1021/am1002876.
Ultraviolet (UV) photo-cross-linkable hydrogels have been commonly used for three-dimensional (3D) encapsulation of cells. Previous UV cross-linkable hydrogels have employed one-shot hardening of mixtures of hydrogels and cells. Here we propose an alternative method of making hydrogel-encapsulated cell constructs through layer by layer (LBL) buildup of alternating layers of cells and hydrogel. The LBL method potentially permits better spatial control of different cell types and control of cell orientation. Each hydrogel layer must be hardened before deposition of the next layer of cells. A UV-curable gel precursor that can also be gelled at physiological temperature is desirable to avoid repeated UV exposure of cells after deposition of each successive hydrogel layer. We designed, synthesized, and applied such a precursor, dual-curable-both thermoresponsive and UV-curable-chitosan-graft-polyethylene glycol-graft-methacrylate (CEGx-MA) copolymer (x is the PEG molecular weight in Daltons). We found that CEG350-MA copolymer solutions (5 wt % polymer) formed physical gels at approximately 37 degrees C and could be further photopolymerized to form thermally stable dual-cured hydrogels. This material was applied to the creation of a two-layer LBL smooth muscle cell (SMC)/hydrogel construct using temperature elevation to approximately 37 degrees C to gel each hydrogel layer. The physically gelled two-layered hydrogel/cell construct was finally exposed to a single UV shot to improve its mechanical properties and render it thermally stable. CEG350-MA solution and gel are nontoxic to SMCs. Cells remained mostly viable when they were encapsulated inside both physically gelled and dual-cured CEG350-MA and suffered little damage from the single brief UV exposure. The combination of LBL tissue engineering with a dual curable hydrogel precursor such as CEG350-MA permits the buildup of viable thick and complex tissues in a stable, biocompatible, and biodegradable matrix.
紫外（UV）光交联水凝胶常用于三维（3D）细胞封装。以前的 UV 可交联水凝胶采用的是水凝胶和细胞混合物的一次性硬化方法。在这里，我们提出了一种通过交替层的细胞和水凝胶的层层（LBL）构建来制造水凝胶包封细胞结构的替代方法。LBL 方法有可能更好地控制不同细胞类型的空间位置，并控制细胞的方向。在沉积下一层细胞之前，必须使 LBL 方法中的每一层水凝胶变硬。需要一种可在生理温度下凝胶化的紫外光可固化凝胶前体，以避免在沉积每一层连续水凝胶后对细胞进行重复的紫外光暴露。我们设计、合成并应用了这种前体，即双固化——既对热响应又对紫外光响应的壳聚糖接枝聚乙二醇接枝甲基丙烯酸酯（CEGx-MA）共聚物（x 是道尔顿的 PEG 分子量）。我们发现，CEG350-MA 共聚物溶液（5wt%聚合物）在大约 37°C 时形成物理凝胶，并可进一步光聚合形成热稳定的双固化水凝胶。该材料用于创建双层 LBL 平滑肌细胞（SMC）/水凝胶结构，通过升温至约 37°C 使每一层水凝胶凝胶化。最后，将物理凝胶的双层水凝胶/细胞结构暴露于单次 UV 照射下，以提高其机械性能并使其热稳定。CEG350-MA 溶液和凝胶对 SMC 无毒。当细胞被包裹在物理凝胶和双固化的 CEG350-MA 中时，细胞大多保持存活，并且很少受到单次短暂的 UV 照射的损伤。LBL 组织工程与 CEG350-MA 等双固化水凝胶前体的结合，允许在稳定、生物相容和可生物降解的基质中构建有活力的厚而复杂的组织。
