Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA, USA.
Department of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Japan.
FASEB J. 2020 May;34(5):6729-6740. doi: 10.1096/fj.201902794R. Epub 2020 Apr 5.
The extracellular matrix (ECM) is comprised of a large network of proteins that are essential for tissue development and repair. A bioactive RGD-containing peptide from laminin α1 chain, A99 (AGTFALRGDNPQG), promotes strong cell attachment and has demonstrated utility in cell culture and tissue engineering. Various materials can be utilized as a scaffold for bioactive peptides; however, it may be advantageous to design materials that use bioconjugation strategies that do not affect bioactivity, generate homogenous products, and can be produced at scale. This report is the first to compare the methods for preparing chemically conjugated and recombinant A99 to elastin-like polypeptides (ELPs) as the scaffold and characterize the biological and cell attachment activity using human dermal fibroblasts (HDFs). ELPs are biocompatible protein-polymers that are also thermo-responsive. Below a lower critical solution temperature (LCST), they are highly soluble. Above the LCST, ELPs phase separate into a polymer-rich liquid, known as a coacervate. Both chemically conjugated and recombinant fusion between A99 and an ELP (A99-ELP-R) show dose-dependent cell attachment. In addition, coating above the LCST provides better cell spreading compared to coating at 4°C. ELPs provide an excellent structural framework for deposition of bioactive peptides of the ECM, and their intrinsic biophysical properties make laminin peptide-ELPs promising biomaterials for cell culture and tissue engineering.
细胞外基质 (ECM) 由大量蛋白质组成，对于组织发育和修复至关重要。层粘连蛋白 α1 链的一种具有生物活性的 RGD 肽 A99 (AGTFALRGDNPQG) 可促进细胞强烈附着，并已在细胞培养和组织工程中得到应用。各种材料可作为生物活性肽的支架；然而，使用不会影响生物活性、生成均质产物且可规模化生产的生物偶联策略设计材料可能具有优势。本报告首次比较了用化学方法制备化学偶联和重组 A99 与弹性蛋白样多肽 (ELP) 作为支架的方法，并使用人真皮成纤维细胞 (HDF) 对其生物学和细胞附着活性进行了表征。ELP 是一种生物相容性的蛋白质聚合物，也具有温度响应性。低于下临界溶液温度 (LCST) 时，它们高度可溶。高于 LCST 时，ELP 相分离成聚合物丰富的液体，称为凝聚体。A99 与 ELP 之间的化学偶联和重组融合 (A99-ELP-R) 均显示出剂量依赖性的细胞附着。此外，在 LCST 以上进行涂层比在 4°C 下进行涂层更有利于细胞展开。ELP 为 ECM 中生物活性肽的沉积提供了极好的结构框架，其固有生物物理特性使层粘连蛋白肽-ELP 成为细胞培养和组织工程有前途的生物材料。
