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首页> 外文会议>World biomaterials congress >Hyaluronan-based cryogels are effective scaffolds for human dermal fibroblasts supporting long term growth and matrix deposition in vitro
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Hyaluronan-based cryogels are effective scaffolds for human dermal fibroblasts supporting long term growth and matrix deposition in vitro

机译：基于透明质酸的冷冻凝胶是用于人类皮肤成纤维细胞的有效支架，可在体外支持长期生长和基质沉积

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Introduction: Hyaluronan (HA), a native extracellular matrix component, is an attractive starting material to generate cell scaffolds in tissue regeneration. However, the formation of dimensionally and mechanically stable scaffolds with defined inter-connective porosity is hampered by the high solution viscosity of HA even at low concentrations, its high swellability and susceptibility against degradation. A promising approach to stable and biocompatible scaffolds avoiding cytotoxic reagents is the electron-beam initiated cryogel formation using (meth)acrylated polysaccharides. Here we present data on the fabrication, characterization and biological compatibility of HA-cryogels for the culture of primary human dermal fibroblasts with the perspective to develop skin compatible scaffolds. Materials and Methods: Two HA acrylates (DS = 0.8 and 1.0, resp., MW = 70 -90 kDa) were synthesized by phase-transfer catalyzed acylation of HA with acryloyl chloride. Cryogels were prepared as described by Reichelt et al. Briefly, 2-3 wt.-% aqueous solutions of HA acrylates were degassed and frozen in centrifuge tubes at -20 °C for 2 h. The irradiation of 12 kGy total was applied in 3 kGy steps using a 10 MeV LINAC at -17 °C. The cryogels were characterized with respect to mechanical and swelling properties, gel content and thermal stability. For biological testing the cryogels were seeded with primary foreskin fibroblasts and cultured with continuous shaking. The cells were investigated concerning cell proliferation, gene expression and matrix deposition within the cryogels by XTT assay, quantitative qPCR and (immuno)histochemical staining techniques, respectively. Results and Discussion: Thin cryogel films (2 mm thick) were successfully synthesized by electron-beam initiated cross-linking. The degree of swelling and the gel content were determined to be 43 and 80 %, resp. The pore size was in the typical range (50-70 μm). We established an effective seeding protocol enabling the growth of huFb for at least 28 days in vitro. The cell proliferation was as effective as on collagen-coated reference surfaces during the first 48h and could be quantified for further four days. Gene expression analysis demonstrated that huFb were regulated by TGFβ and PDGF-BB with induction of known target genes like collagen Ⅰ(αⅠ) or HA synthase 2 suggesting an adequate physiological behavior of the cells grown within the cryogels. Histochemical analysis demonstrates that Fb settled the cryogels completely and deposited their own matrix of collagens and glycosaminoglycans. Human Fb were viable after 4 weeks of culture and the cryogel structure remained stable. Conclusions: Cryogels were prepared by an electron-beam initiated crosslinking reaction of HA acrylates omitting the use of potentially toxic reagents. The morphology and mechanical properties of the obtained cryogels may be tailored by variation of the composition of the HA derivatives (e. g. degree of acrylation) and the reaction parameters (e. g. polymer concentration). The cryogels are very effective growth substrates for huFb which proliferate and deposit their own matrix within the pores. Taken together, HA-based cryogels are promising biomaterials for the development of pre-seeded tissue equivalents.
机译：简介：透明质酸（HA）是一种天然的细胞外基质成分，是在组织再生中产生细胞支架的一种有吸引力的起始材料。然而，即使在低浓度下，HA的高溶液粘度，其高溶胀性和抗降解敏感性也阻碍了具有确定的互连孔隙率的尺寸和机械稳定性支架的形成。避免细胞毒性试剂的稳定且生物相容性支架的一种有前途的方法是使用（甲基）丙烯酸酯化多糖的电子束引发的冷冻凝胶形成。在这里，我们从发展皮肤相容性支架的角度介绍了用于原始人类皮肤成纤维细胞培养的HA冷冻凝胶的制备，表征和生物相容性的数据。材料与方法：通过相转移催化HA与丙烯酰氯的酰化反应，合成了两种HA丙烯酸酯（DS = 0.8和1.0，分别为MW = 70 -90 kDa）。如Reichelt等人所述制备冰冻蛋白。简而言之，将2-3重量％的HA丙烯酸酯的水溶液脱气并在-20℃的离心管中冷冻2小时。在-17°C下使用10 MeV LINAC分3 kGy进行总辐射12 kGy的辐射。相对于机械和溶胀性能，凝胶含量和热稳定性，对冷凝胶进行了表征。为了进行生物学测试，将冷冻凝胶接种原皮包皮成纤维细胞，并连续摇动培养。分别通过XTT测定，定量qPCR和（免疫）组织化学染色技术研究了细胞在冷冻凝胶中的细胞增殖，基因表达和基质沉积。结果与讨论：通过电子束引发的交联成功地合成了2mm厚的冷冻凝胶薄膜。溶胀度和凝胶含量被确定为分别为43％和80％。孔径在典型范围内（50-70μm）。我们建立了有效的接种方案，可使huFb在体外至少生长28天。在最初的48小时内，细胞增殖与在胶原蛋白包被的参考表面上一样有效，并且可以在接下来的四天内进行定量。基因表达分析表明，huFb受TGFβ和PDGF-BB的调控，并诱导了已知的靶基因如胶原Ⅰ（αⅠ）或HA合酶2，提示冰晶体内生长的细胞具有适当的生理行为。组织化学分析表明，Fb完全沉淀了冷冻凝胶，并沉积了自己的胶原蛋白和糖胺聚糖基质。培养4周后，人Fb仍能存活，并且冷冻凝胶结构保持稳定。结论：冰冷凝胶是通过电子束引发的HA丙烯酸酯的交联反应制备的，而无需使用潜在的有毒试剂。可以通过改变HA衍生物的组成（例如丙烯酸化度）和反应参数（例如聚合物浓度）来调整获得的冰凝胶的形态和机械性能。冷冻凝胶是huFb的非常有效的生长底物，可在孔中增殖并沉积其自身的基质。总而言之，基于HA的冷冻凝胶是用于开发预接种组织等效物的有前途的生物材料。

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