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首页> 外文会议>World biomaterials congress >Di-functionalized hyaluronic acid with reactive thiol and azide groups for the neural differentiation of mouse embryonic stem cells
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Di-functionalized hyaluronic acid with reactive thiol and azide groups for the neural differentiation of mouse embryonic stem cells

机译：具有反应性巯基和叠氮基的双功能透明质酸用于小鼠胚胎干细胞的神经分化

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Introduction: The native extracellular matrix (ECM) has a complex composition. Tissue engineering matrices need to possess multiple bioactive signaling molecules in order to adequately mimic the ECM. In the central nervous system, hyaluronic acid (HA) is a regulator of cellular migration, proliferation, differentiation, and angiogenesis. In these experiments, a di-functional HA (diHA), which possesses thiol and azide groups for tethering of bioactive molecules at independent concentrations, is characterized to and initial biological testing is conducted on the unfunctionalized materials using mouse embryonic stem cells (mES). Material and Methods: 60,000 kDa HA was functionalized to create diHA and methacrylated HA (mHA). The thiol group on di-HA was characterized using 1H-NMR, and 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay. The azide group was characterized using 13C-NMR, Fourier transform infrared spectroscopy (FT-IR) spectrum, and radioTLC of 68Ga chelating agent binding. Photopolymerized hydrogels of diHA and mHA of varying compositions ratios were fabricated using Irgacure 2959 and 2.3 mJ cm-2 UVA light for 5 min. The swelling ratio, water contents, and mechanical properties (young's and shear modulus) of 1:2 (diHA: mHA) and 1:5 (diHA: mHA) hydrogels was determined. D3 mES were encapsulated in the HA hydrogel at a density of 1 × 106 cells/mL mES in 3D HA hydrogels were examined proliferation and differentiation potential by MTT assay and b-tubulin Ⅲ (TUJ1) staining, respectively. Results Discussion: On 1 H-NMR, the thiol peak was observed at 2.9 ppm for thiol functionalized samples with and without azide functionalization. A positive staining for thiol was also observed by TNBS assay for these sample groups. An azide peak was observed at 2096 cm-1 on the FT-IR spectrum (Figure 1), and at 50.1 ppm on the 13C-NMR spectrum for only HA samples functionalized with both thiol and azide groups. The average number of chelating moieties capping the azide group in HA was 3.9 ± 0.09 on radio TLC for diHA samples. A higher ratio of mHA (1:5) increased the young's modulus, shear modulus, swelling ratio, and water content, significantly. Encapsulated mES were proliferated on 1:2 and 1:5 ratio of diHA:mHA hydrogel. After 3 and 6 days of differentiation, 44%±7% and55%±12% of mES in 1:2 (diHA: mHA) hydrogels expressed TUJ1, respectively. While mES in 1:5 (diHA: mHA) hydrogels expressed 39%±20% and 40%±14% after 3 and 6 days of differentiation, respectively (Figure 2). mES cultured in 1:2 (diHA: mHA) showed mature neural extension on day 6 of differentiation. Conclusion: These results indicate that thiol and azide groups were present on the modified HA backbone. Encapsulated mES were able to proliferate and differentiate in base matrix without additional bioactive signaling. diHA is a valuable tool to enhance bioactivity and control of bioactive signal concentrations in HA matrices developed to treat central nervous system injuries.
机译：简介：天然细胞外基质（ECM）具有复杂的成分。组织工程基质需要拥有多种生物活性信号分子，才能充分模拟ECM。在中枢神经系统中，透明质酸（HA）是细胞迁移，增殖，分化和血管生成的调节剂。在这些实验中，对具有巯基和叠氮基团的双功能HA（diHA）（具有独立浓度的生物活性分子进行束缚）进行了表征，并使用小鼠胚胎干细胞（mES）对未功能化的材料进行了初步的生物学测试。材料和方法：将60,000 kDa HA官能化以生成diHA和甲基丙烯酸HA（mHA）。使用1 H-NMR和2,4,6-三硝基苯磺酸（TNBS）分析对di-HA上的巯基进行了表征。使用13 C-NMR，傅立叶变换红外光谱（FT-IR）光谱和68Ga螯合剂结合的radioTLC对叠氮化物基团进行表征。使用Irgacure 2959和2.3 mJ cm-2 UVA光5分钟，制备了不同组成比的diHA和mHA的光聚合水凝胶。测定了1：2（diHA：mHA）和1：5（diHA：mHA）水凝胶的溶胀率，水含量和机械性能（杨氏模量和剪切模量）。将D3 mES封装在HA水凝胶中，密度为1×106细胞/ mL mES在3D HA水凝胶中，分别通过MTT分析和b-微管蛋白Ⅲ（TUJ1）染色检查其增殖和分化潜能。结果讨论：在1 H-NMR上，对于有和没有叠氮化物官能化的硫醇官能化样品，在2.9 ppm处都观察到了硫醇峰。通过这些样品组的TNBS测定，还观察到了硫醇的阳性染色。仅用硫醇和叠氮基团官能化的HA样品在FT-IR谱图上在2096 cm-1处观察到叠氮化物峰（图1），在13C-NMR谱上在50.1 ppm处观察到。在diHA样品的放射TLC上，HA中叠氮化物基团的螯合部分的平均数为3.9±0.09。较高的mHA比（1：5）显着提高了杨氏模量，剪切模量，溶胀率和水含量。封装的mES在diHA：mHA水凝胶的1：2和1：5比例下增殖。分化3天和6天后，在1：2（diHA：mHA）水凝胶中，分别有44％±7％和55％±12％的mES表达TUJ1。分化3天和6天后，mES在1：5（diHA：mHA）中的水凝胶分别表达39％±20％和40％±14％（图2）。在分化的第6天，以1：2（diHA：mHA）培养的mES显示出成熟的神经延伸。结论：这些结果表明在修饰的HA主链上存在巯基和叠氮基。封装的mES能够在基础基质中增殖和分化，而无需其他生物活性信号传导。 diHA是在开发用于治疗中枢神经系统损伤的HA基质中增强生物活性和控制生物活性信号浓度的有价值的工具。

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