Attachment of endothelial colony-forming cells onto a surface bearing immobilized anti-CD34 antibodies: Specific CD34 binding versus nonspecific binding

Cardiovascular disease is a leading cause of death worldwide; however, despite substantial advances in medical device surface modifications, no synthetic coatings have so far matched the native endothelium as the optimal hemocompatible surface for blood-contacting implants. A promising strategy for rapid restoration of the endothelium on blood-contacting biomedical devices entails attracting circulating endothelial cells or their progenitors, via immobilized cell-capture molecules; for example, anti-CD34 antibody to attract CD34+ endothelial colony-forming cells (ECFCs). Inherent is the assumption that the cells attracted to the biomaterial surface are bound exclusively via a specific CD34 binding. However, serum proteins might adsorb in-between or on the top of antibody molecules and attract ECFCs via other binding mechanisms. Here, we studied whether a surface with immobilized anti-CD34 antibodies attracts ECFCs via a specific CD34 binding or a nonspecific (non-CD34) binding. To minimize serum protein adsorption, a fouling-resistant layer of hyperbranched polyglycerol (HPG) was used as a “blank slate,” onto which anti-CD34 antibodies were immobilized via aldehyde-amine coupling reaction after oxidation of terminal diols to aldehydes. An isotype antibody, mIgG1, was surface-immobilized analogously and was used as the control for antigen-binding specificity. Cell binding was also measured on the HPG hydrogel layer before and after oxidation. The surface analysis methods, x-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry, were used to verify the intended surface chemistries and revealed that the surface coverage of antibodies was sparse, yet the anti-CD34 antibody grafted surface-bound ECFCs very effectively. Moreover, it still captured the ECFCs after BSA passivation. However, cells also attached to oxidized HPG and immobilized mIgG1, though in much lower amounts. While our results confirm the effectiveness of attracting ECFCs via surface-bound anti-CD34 antibodies, our observation of a nonspecific binding component highlights the importance of considering its consequences in future studies.