Rational Design of Genetically Engineered, Gold and Cell-Binding Polypeptides for Fabricating Thermoresponsive Cell Culture Substrates

Chronic wounds do not adequately recover through the natural healing process and have become a significant challenge for healthcare systems worldwide. In the US, chronic wounds affect an estimated 6 million people per year, costing more than $25 billion annually due to complications and over $18.5 billion in associated care. Peripheral arterial disease, neuropathy, and immunodeficiency disorders are conditions that can lead to chronic open wounds and lesions. These conditions are especially prevalent in diabetic patients due to immobility and lack of blood flow to the extremities. Current technologies for chronic wound healing are aimed at dressings which consist of various hydrogels and collagen. Modern tissue engineering methods aim to expand the understanding of cell sheets and cell hydrogels which could form a ‘cellular scaffold’ to improve recovery time. However, these methods often rely on proteolytic enzymes such as trypsin, which damages the extracellular matrix (ECM), cell-cell junctions, and surface cell receptors. Other methods use polymers such as poly(N-isopropyl acrylamide) (pNIPAM), which relies on ECM molecules or chemical conjugations of cells for binding and release with no specific cell binding motifs. For this study, elastin-like-polypeptides (ELPs) were designed with a thermally responsive motif, allowing us to control and target cell binding and detachment in culture without damaging the ECM. These proteins were deposited onto gold surfaces via specific cell-binding cysteine domains. The location of the cell binding motifs within the ELP proved to be an important factor for detachment of the cells. ELP layers with cell binding domains near the surfaces allowed for thermally controlled cell adhesion, similar to those found in pNIPAM. Decreasing temperature below the lower critical solution temperature (LCST) causes the ELPs to adopt a random coil formation, which leads to retraction of the cell binding motifs, thus detaching the adherent cells. The cell lines tested for detachment included bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). This study indicates that varying cell lines can be consistently recovered from the surfaces as cell sheets for tissue engineering purposes.