Holmes Róisín, Yang Xue-Bin, Dunne Aishling, Florea Larisa, Wood David, Tronci Giuseppe
Biomaterials and Tissue Engineering Research Group, School of Dentistry, University of Leeds, Wellcome Trust Brenner Building, St James' University Hospital, Leeds LS9 7TF, UK.
Insight Centre for Data Analytics, National Centre for Sensor Research, School of Chemical Sciences, Dublin City University, Dublin 9, Glasnevin, Ireland.
Polymers (Basel). 2017 Jun 14;9(6):226. doi: 10.3390/polym9060226.
Thiol-ene photo-click hydrogels were prepared via step-growth polymerisation using thiol-functionalised type-I collagen and 8-arm poly(ethylene glycol) norbornene-terminated (PEG-NB), as a potential injectable regenerative device. Type-I collagen was thiol-functionalised by a ring opening reaction with 2-iminothiolane (2IT), whereby up to 80 Abs.% functionalisation and 90 RPN% triple helical preservation were recorded via 2,4,6-Trinitrobenzenesulfonic acid (TNBS) colorimetric assay and circular dichroism (CD). Type, i.e., either 2-Hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (I2959) or lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and concentration of photoinitiator were varied to ensure minimal photoinitiator-induced cytotoxicity and to enable thiol-ene network formation of collagen-PEG mixtures. The viability of G292 cells following 24 h culture in photoinitiator-supplemented media was largely affected by the photoinitiator concentration, with I2959-supplemented media observed to induce higher toxic response (0.1 → 0.5% (/) I2959, cell survival: 62 → 2 Abs.%) compared to LAP-supplemented media (cell survival: 86 → 8 Abs.%). In line with the in vitro study, selected photoinitiator concentrations were used to prepare thiol-ene photo-click hydrogels. Gelation kinetics proved to be largely affected by the specific photoinitiator, with LAP-containing thiol-ene mixtures leading to significantly reduced complete gelation time (: 187 s) with respect to I2959-containing mixtures (: 1683 s). Other than the specific photoinitiator, the photoinitiator concentration was key to adjusting the hydrogel storage modulus ('), whereby 15-fold ' increase (232 → 3360 Pa) was observed in samples prepared with 0.5% (/) compared to 0.1% (/) LAP. Further thiol-ene formulations with 0.5% (/) LAP and varied content of PEG-NB were tested to prepare photo-click hydrogels with porous architecture, as well as tunable storage modulus (': 540⁻4810 Pa), gelation time (: 73⁻300 s) and swelling ratio (: 1530⁻2840 wt %). The photoinitiator-gelation-cytotoxicity relationships established in this study will be instrumental to the design of orthogonal collagen-based niches for regenerative medicine.
通过逐步聚合反应，使用硫醇官能化的I型胶原蛋白和8臂聚乙二醇降冰片烯封端的聚合物（PEG-NB）制备了硫醇-烯光点击水凝胶，作为一种潜在的可注射再生装置。I型胶原蛋白通过与2-亚氨基硫杂环戊烷（2IT）的开环反应进行硫醇官能化，通过2,4,6-三硝基苯磺酸（TNBS）比色法和圆二色性（CD）记录，官能化率高达80 Abs.%，三螺旋保留率达90 RPN%。改变光引发剂的类型，即2-羟基-1-[4-(2-羟基乙氧基)苯基]-2-甲基-1-丙酮（I2959）或苯基-2,4,6-三甲基苯甲酰基膦酸锂（LAP）以及光引发剂的浓度，以确保光引发剂诱导的细胞毒性最小，并使胶原蛋白-PEG混合物形成硫醇-烯网络。在补充了光引发剂的培养基中培养24小时后，G292细胞的活力在很大程度上受到光引发剂浓度的影响，与补充LAP的培养基（细胞存活率：86 → 8 Abs.%）相比，补充I2959的培养基观察到诱导更高的毒性反应（0.1 → 0.5%（/）I2959，细胞存活率：62 → 2 Abs.%）。与体外研究一致，使用选定的光引发剂浓度制备硫醇-烯光点击水凝胶。凝胶化动力学在很大程度上受到特定光引发剂的影响，含LAP的硫醇-烯混合物与含I2959的混合物相比，完全凝胶化时间显著缩短（分别为：187秒和1683秒）。除了特定的光引发剂外，光引发剂浓度是调节水凝胶储能模量（'）的关键，与0.1%（/）LAP制备的样品相比，0.5%（/）LAP制备的样品观察到'增加了15倍（232 → 3360 Pa）。进一步测试了含0.5%（/）LAP和不同PEG-NB含量的硫醇-烯配方，以制备具有多孔结构以及可调储能模量（'：540⁻4810 Pa）、凝胶化时间（：73⁻300秒）和溶胀率（：1530⁻2840 wt %）的光点击水凝胶。本研究中建立的光引发剂-凝胶化-细胞毒性关系将有助于设计用于再生医学的基于胶原蛋白的正交微环境。
