Methacrylation Induces Rapid, Temperature-Dependent, Reversible Self-Assembly of Type-I Collagen

Abstract

Type-I collagen self-assembles into a fibrillar gel at physiological temperature and pH to provide a cell-adhesive, supportive, structural network. As such, it is an attractive, popular scaffold for in vitro evaluations of cellular behavior and for tissue engineering applications. In this study, type-I collagen is modified to introduce methacrylate groups on the free amines of the lysine residues to create collagen methacrylamide (CMA). CMA retains the properties of collagen such as self-assembly, biodegradability, and natural bioactivity but is also photoactive and can be rapidly cross-linked or functionalized with acrylated molecules when irradiated with ultraviolet light in the presence of a photoinitiator. CMA also demonstrates unique temperature-dependent behavior. For natural type-I collagen, the overall structure of the fiber network remains largely static over time scales of a few hours upon heating and cooling at temperatures below its denaturation point. CMA, however, is rapidly thermoreversible and will oscillate between a liquid macromer suspension and a semisolid fibrillar hydrogel when the temperature is modulated between 10 and 37 °C. Using a series of mechanical, scattering, and spectroscopic methods, we demonstrate that structural reversibility is manifest across multiple scales from the protein topology of the triple helix up through the rheological properties of the CMA hydrogel. Electron microscopy imaging of CMA after various stages of heating and cooling shows that the canonical collagen-like D-periodic banding ultrastructure of the fibers is preserved. A rapidly thermoreversible collagen-based hydrogel is expected to have wide utility in tissue engineering and drug delivery applications as a biofunctional, biocompatible material. Thermal reversibility also makes CMA a powerful model for studying the complex process of hierarchical collagen self-assembly.

Circular Dichroism Spectroscopy of Collagen Fibrillogenesis: A New Use for an Old Technique

“…Herein, we describe the observation of fibrillogenesis using CD spectroscopy under conditions in which both triple helix and fibril states can be observed simultaneously. Previously, a unique CD spectrum was observed during temperature-induced self-assembly of a chemically modified collagen derivative that was coincident with fibrillogenesis and distinct from that of a triple helix (24). Here, we establish the spectral shape of this component and demonstrate its utility for studying hierarchic assembly.…”

“…2). The concentration of type-III collagen necessary for rheological comparison was prohibitive, so self-assembly measurements were performed via light scattering (24). The derived count rate of FIGURE 1 (A) MRE at 222 nm (dashed lines) and the storage modulus (G 0 ) (solid lines) were measured from 4 C to 60 C for Telo-EPC in AcA versus neutral PBS buffers.…”

“…However, many of the structural and functional features of assembled fibrils can be recreated in vitro under appropriate conditions using collagen extracted from a variety of source animal tissues into neutral salt or buffers, or, more frequently, into dilute acidic solutions (1). Fibrillogenesis can be controlled by varying the pH (17)(18)(19)(20), temperature (18,19,21,22), and buffer conditions (17,18,23,24). Under acidic conditions, collagen exists primarily as a soluble triple helix (17) below its melting transition around 42 C (24).…”

“…Fibrillogenesis can be controlled by varying the pH (17)(18)(19)(20), temperature (18,19,21,22), and buffer conditions (17,18,23,24). Under acidic conditions, collagen exists primarily as a soluble triple helix (17) below its melting transition around 42 C (24). At neutral pH, collagen readily forms fibrils above 20 C (17).…”

A thermoreversible, photocrosslinkable collagen bio-ink for free-form fabrication of scaffolds for regenerative medicine

“…We have developed a methacrylate-derivatized type-I collagen — collagen methacrylamide (CMA) — that retains the ability to form D-banded fibrillar hydrogels 35,36 . CMA has two unique properties that allow for free-form fabrication: 1) following fibrillogenesis, CMA can be photocrosslinked using a photoinitiator and UV light to stiffen the hydrogel where it is exposed to light 35 , and 2) fibrillogenesis of CMA is thermoreversible — it can reversibly cycle between liquid and fibrillar hydrogel states when cycled between 4 °C and 37 °C 36 .…”