Mulyasasmita Widya, Cai Lei, Dewi Ruby E, Jha Arshi, Ullmann Sabrina D, Luong Richard H, Huang Ngan F, Heilshorn Sarah C
Department of Bioengineering, Stanford University, Stanford, CA, USA.
Department of Materials Science and Engineering, Stanford University, Stanford, CA, USA; Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA.
J Control Release. 2014 Oct 10;191:71-81. doi: 10.1016/j.jconrel.2014.05.015. Epub 2014 May 18.
To translate recent advances in induced pluripotent stem cell biology to clinical regenerative medicine therapies, new strategies to control the co-delivery of cells and growth factors are needed. Building on our previous work designing Mixing-Induced Two-Component Hydrogels (MITCHs) from engineered proteins, here we develop protein-polyethylene glycol (PEG) hybrid hydrogels, MITCH-PEG, which form physical gels upon mixing for cell and growth factor co-delivery. MITCH-PEG is a mixture of C7, which is a linear, engineered protein containing seven repeats of the CC43 WW peptide domain (C), and 8-arm star-shaped PEG conjugated with either one or two repeats of a proline-rich peptide to each arm (P1 or P2, respectively). Both 20kDa and 40kDa star-shaped PEG variants were investigated, and all four PEG-peptide variants were able to undergo a sol-gel phase transition when mixed with the linear C7 protein at constant physiological conditions due to noncovalent hetero-dimerization between the C and P domains. Due to the dynamic nature of the C-P physical crosslinks, all four gels were observed to be reversibly shear-thinning and self-healing. The P2 variants exhibited higher storage moduli than the P1 variants, demonstrating the ability to tune the hydrogel bulk properties through a biomimetic peptide-avidity strategy. The 20kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40kDa variants. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) adopted a well-spread morphology within three-dimensional MITCH-PEG cultures, and MITCH-PEG provided significant protection from cell damage during ejection through a fine-gauge syringe needle. In a mouse hindlimb ischemia model of peripheral arterial disease, MITCH-PEG co-delivery of hiPSC-ECs and VEGF was found to reduce inflammation and promote muscle tissue regeneration compared to a saline control.
为了将诱导多能干细胞生物学的最新进展转化为临床再生医学疗法，需要新的策略来控制细胞和生长因子的共同递送。基于我们之前利用工程蛋白设计混合诱导双组分水凝胶（MITCHs）的工作，在此我们开发了蛋白 - 聚乙二醇（PEG）杂化水凝胶MITCH - PEG，其在混合时形成物理凝胶以实现细胞和生长因子的共同递送。MITCH - PEG是C7的混合物，C7是一种线性工程蛋白，包含CC43 WW肽结构域（C）的七个重复序列，以及与每个臂上一个或两个富含脯氨酸肽重复序列（分别为P1或P2）共轭的8臂星形PEG。研究了20kDa和40kDa星形PEG变体，并且由于C和P结构域之间的非共价异二聚化，所有四种PEG - 肽变体在恒定生理条件下与线性C7蛋白混合时都能够经历溶胶 - 凝胶相转变。由于C - P物理交联的动态性质，观察到所有四种凝胶都具有可逆的剪切变稀和自愈特性。P2变体表现出比P1变体更高的储能模量，证明了通过仿生肽亲和力策略调节水凝胶整体性质的能力。20kDa PEG变体表现出封装的血管内皮生长因子（VEGF）释放较慢，这是由于相对于40kDa变体水凝胶网孔尺寸减小。人诱导多能干细胞衍生的内皮细胞（hiPSC - ECs）在三维MITCH - PEG培养物中呈现良好的铺展形态，并且MITCH - PEG在通过细针注射器注射时为细胞损伤提供了显著的保护。在小鼠外周动脉疾病后肢缺血模型中，与盐水对照相比，发现MITCH - PEG共同递送hiPSC - ECs和VEGF可减轻炎症并促进肌肉组织再生。
