Addi Cyril, Murschel Frédéric, Liberelle Benoît, Riahi Nesrine, De Crescenzo Gregory
Department of Chemical Engineering, Biomedical Science and Technology Research Group, Bio-P(2) Research Unit, École Polytechnique de Montréal, P.O. Box 6079, succ. Centre-Ville, Montréal (QC) H3C 3A7, Canada.
Department of Chemical Engineering, Biomedical Science and Technology Research Group, Bio-P(2) Research Unit, École Polytechnique de Montréal, P.O. Box 6079, succ. Centre-Ville, Montréal (QC) H3C 3A7, Canada.
Acta Biomater. 2017 Mar 1;50:198-206. doi: 10.1016/j.actbio.2017.01.014. Epub 2017 Jan 6.
In the field of tissue engineering, the tethering of growth factors to tissue scaffolds in an oriented manner can enhance their activity and increase their half-life. We chose to investigate the capture of the basic Fibroblast Growth Factor (bFGF) and the Epidermal Growth Factor (EGF) on a gelatin layer, as a model for the functionalization of collagen-based biomaterials. Our strategy relies on the use of two high affinity interactions, that is, the one between two distinct coil peptides as well as the one occurring between a collagen-binding domain (CBD) and gelatin. We expressed a chimeric protein to be used as an adaptor that comprises one of the coil peptides and a CBD derived from the human fibronectin. We proved that it has the ability to bind simultaneously to a gelatin substrate and to form a heterodimeric coiled-coil domain with recombinant growth factors being tagged with the complementary coil peptide. The tethering of the growth factors was characterized by ELISA and surface plasmon resonance-based biosensing. The bioactivity of the immobilized bFGF and EGF was evaluated by a human umbilical vein endothelial cell proliferation assay and a vascular smooth muscle cell survival assay. We found that the tethering of EGF preserved its mitogenic and anti-apoptotic activity. In the case of bFGF, when captured via our adaptor protein, changes in its natural mode of interaction with gelatin were observed.
In an effort to functionalize collagen/gelatin-based biomaterials with growth factors, we have designed an adaptor protein corresponding to a collagen-binding domain fused to a coil peptide. In our strategy, this adaptor protein captures growth factors being tagged with the partner coil peptide in a specific, stable and oriented manner. We have found that the tethering of the Epidermal Growth Factor preserved its mitogenic and anti-apoptotic activity. In the case of the basic Fibroblast Growth Factor, the captured growth factor remained bioactive although its tethering via this adaptor protein modified its natural mode of interaction with gelatin. Altogether this strategy is easily adaptable to the simultaneous tethering of various growth factors.
在组织工程领域，以定向方式将生长因子与组织支架相连可增强其活性并延长其半衰期。我们选择研究碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）在明胶层上的捕获情况，以此作为基于胶原蛋白的生物材料功能化的模型。我们的策略依赖于两种高亲和力相互作用的运用，即两种不同的卷曲肽之间的相互作用以及胶原结合域（CBD）与明胶之间的相互作用。我们表达了一种用作衔接子的嵌合蛋白，其包含一种卷曲肽和源自人纤连蛋白的CBD。我们证明它能够同时结合明胶底物，并与标记有互补卷曲肽的重组生长因子形成异源二聚体卷曲螺旋结构域。通过酶联免疫吸附测定（ELISA）和基于表面等离子体共振的生物传感对生长因子的连接进行了表征。通过人脐静脉内皮细胞增殖测定和血管平滑肌细胞存活测定评估了固定化bFGF和EGF的生物活性。我们发现EGF的连接保留了其促有丝分裂和抗凋亡活性。在bFGF的情况下，当通过我们的衔接子蛋白捕获时，观察到其与明胶的天然相互作用模式发生了变化。
为了用生长因子使基于胶原蛋白/明胶的生物材料功能化，我们设计了一种衔接子蛋白，其对应于与卷曲肽融合的胶原结合域。在我们的策略中，这种衔接子蛋白以特定、稳定和定向的方式捕获标记有伴侣卷曲肽的生长因子。我们发现表皮生长因子的连接保留了其促有丝分裂和抗凋亡活性。就碱性成纤维细胞生长因子而言，捕获的生长因子仍具有生物活性，尽管通过这种衔接子蛋白的连接改变了其与明胶的天然相互作用模式。总之，该策略易于适用于同时连接多种生长因子。
