Strategies to balance covalent and non-covalent biomolecule attachment within collagen-GAG biomaterials

Abstract

Strategies to integrate instructive biomolecular signals into a biomaterial are becoming increasingly complex and bioinspired. While a large majority of reports still use repeated treatments with soluble factors, this approach can be prohibitively costly and difficult to translate in vivo for applications where spatial control over signal presentation is necessary. Recent efforts have explored the use of covalent immobilization of biomolecules to the biomaterial, via both bulk (ubiquitous) as well as spatially-selective light-based crosslinking, as a means to both enhance stability and bioactivity. However, little is known about how processing conditions during immobilization impact the degree of unintended non-covalent interactions, or fouling, that takes place between the biomaterial and the biomolecule of interest. Here we demonstrate the impact of processing conditions for bulk carbodiimide (EDC) and photolithography-based benzophenone (BP) crosslinking on specific attachment vs. fouling of a model protein (Concanavalin A, ConA) within collagen-glycosaminoglycan (CG) scaffolds. Collagen source significantly impacts the selectivity of biomolecule immobilization. EDC crosslinking intensity and ligand concentration significantly impacted selective immobilization. For benzophenone photoimmobilization we observed that increased UV exposure time leads to increased ConA immobilization. Immobilization efficiency for both EDC and BP strategies was maximal at physiological pH. Increasing ligand concentration during immobilization process led to enhanced immobilization for EDC chemistry, no impact on BP immobilization, but significant increases in non-specific fouling. Given recent efforts to covalently immobilize biomolecules to a biomaterial surface to enhance bioactivity, improved understanding of the impact of crosslinking conditions on selective attachment versus non-specific fouling will inform the design of instructive biomaterials for applications across tissue engineering.

Nanotopography-Induced Structural Anisotropy and Sarcomere Development in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells

“…In such cases, 1-ethyl-3-(3 dimethylaminopropyl)-carbodiimide (EDC) chemistry is often used to covalently link polymeric and biological molecules together. 28 Although previous groups have demonstrated some success in binding biologically relevant peptides or proteins to synthetic polymers, 29–31 the process is limited by chemical cross-linking reagents and suboptimal reaction environments. In addition, conjugation chemistry results in poor yields, and does not provide strict control over surface distribution of the biological molecule.…”

The induction of pro‐angiogenic processes within a collagen scaffold via exogenous estradiol and endometrial epithelial cells

“…Scaffolds were further crosslinked (room temperature, 1.5 h) in a solution of 1‐ethyl‐3‐[3‐dimethylaminopropyl] carbodiimide hydrochloride (EDC) (Sigma–Aldrich) and N ‐hydroxysulfosuccinimide (NHS) (Sigma–Aldrich) at a molar ratio of 5:2:1 EDC:NHS:COOH. For immobilized estradiol experiments, BSA‐E2 (0–1,000 nM E2; Sigma–Aldrich) was immobilized into the scaffolds during crosslinking at a molar ratio of 5:12.5:1 EDC:NHS:COOH using a previously described stepwise reaction (Pence et al, ; Shen et al, ). Equivalent dosages of soluble E2 versus covalently‐bound BSA‐E2 were determined as equivalent molar ratios.…”

“…Scaffolds support significant cell invasion and metabolite biotransport (O'Brien et al, 2007). Recently, methods have been described to incorporate exogenous biomolecular signals in soluble (Caliari and Harley, 2014), covalently-bound (Alsop et al, 2014), or transiently sequestered (Hortensius and Harley, 2013;Pence et al, 2014) forms to instruct cell behaviors. Here, we examined the impact of exogenous E2 on separate and combined cultures of endometrial epithelial cells from an adenocarcinoma (Ishikawa 3-H-12 cells) and endothelial cells (human umbilical vein endothelial cells, HUVECs) commonly used in many investigations of angiogenesis.…”

Bioinspired Collagen Scaffolds in Cranial Bone Regeneration: From Bedside to Bench

“…[100] When evaluated in the laboratory, the efficiency of EDC crosslinking of Concanavalin A (ConA) to collagen glycosaminoglycan (CG) scaffolds was significantly increased in the presence of greater concentrations of ConA, a 5:12.5:1 EDC-NHS-COOH ratio, and with the step crosslinking approach versus bulk crosslinking. [8] Pence and Harley demonstrated that the source of collagen can significantly change the immobilization of proteins during crosslinking. [8] Murphy and O’Brien evaluated a range of pore sizes in collagen glycosaminoglycan scaffolds of identical composition, crosslinking, and synthesis methods.…”

“…[8] Pence and Harley demonstrated that the source of collagen can significantly change the immobilization of proteins during crosslinking. [8] Murphy and O’Brien evaluated a range of pore sizes in collagen glycosaminoglycan scaffolds of identical composition, crosslinking, and synthesis methods. [13,101] They demonstrated that wide differences in adhesion, migration, and differentiation in a range of 85–325 μm in pore sizes, with the larger pore size demonstrating the best results.…”