Greene Tanja, Lin Chien-Chi
Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana, 46202 United States.
ACS Biomater Sci Eng. 2015 Dec 14;1(12):1314-1323. doi: 10.1021/acsbiomaterials.5b00436. Epub 2015 Oct 29.
Gelatin-based hydrogels are increasingly used to promote cell fate processes in 3D. Here, we report the use of orthogonal thiol-norbornene photochemistry to prepare modularly cross-linked gelatin-based hydrogels for studying the influence of independent matrix properties on hepatocellular carcinoma cell fate . In addition to demonstrating the ability to independently tune the mechanical and biological properties of modular gelatin-norbornene (GelNB) hydrogels, we also determined that network cross-linking density plays a key role in the mechanisms of proteolytic gel degradation. During degradation studies, GelNB hydrogels with lower cross-linking density degraded faster and followed a surface erosion mechanism, whereas dense GelNB hydrogels degraded in a bulk degradation mechanism. Hepatocellular carcinoma cells, Huh7, were encapsulated and grown in GelNB hydrogels with modularly tuned stiffness, bioactive motifs, and heparin content. We systematically evaluated the effect of matrix properties on cell viability and functions , including CYP3A4 activity and urea secretion. We found that encapsulated Huh7 cells exhibited higher cellular metabolic activity when encapsulated in modular GelNB hydrogels composed of higher gelatin contents or gels with lower stiffness. Interestingly, altering gelatin content and matrix stiffness did not significantly affect hepatocyte-specific cellular functions. To improve cellular function, we prepared norbornene and heparin dual-functionalized gelatin through a two-step synthesis protocol. Heparin-functionalized GelNB (i.e., GelNB-Hep) hydrogels were able to sequester and slowly release hepatocyte growth factor (HGF) . Finally, the conjugation of heparin on GelNB led to suppressed Huh7 cell metabolic activity and improved CYP3A4 activity and urea secretion.
基于明胶的水凝胶越来越多地用于促进三维空间中的细胞命运进程。在此，我们报道了使用正交硫醇-降冰片烯光化学来制备模块化交联的基于明胶的水凝胶，以研究独立的基质特性对肝癌细胞命运的影响。除了证明能够独立调节模块化明胶-降冰片烯（GelNB）水凝胶的机械和生物学特性外，我们还确定网络交联密度在蛋白水解凝胶降解机制中起关键作用。在降解研究中，交联密度较低的GelNB水凝胶降解更快，并遵循表面侵蚀机制，而致密的GelNB水凝胶则以整体降解机制降解。将肝癌细胞Huh7封装在具有模块化调节硬度、生物活性基序和肝素含量的GelNB水凝胶中并使其生长。我们系统地评估了基质特性对细胞活力和功能的影响，包括CYP3A4活性和尿素分泌。我们发现，当封装在由较高明胶含量或较低硬度的凝胶组成的模块化GelNB水凝胶中时，封装的Huh7细胞表现出更高的细胞代谢活性。有趣的是，改变明胶含量和基质硬度并没有显著影响肝细胞特异性细胞功能。为了改善细胞功能，我们通过两步合成方案制备了降冰片烯和肝素双功能化明胶。肝素功能化的GelNB（即GelNB-Hep）水凝胶能够螯合并缓慢释放肝细胞生长因子（HGF）。最后，肝素与GelNB的共轭导致Huh7细胞代谢活性受到抑制，同时改善了CYP3A4活性和尿素分泌。
