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.

Type-I collagen assembles in a stepwise, hierarchic fashion from the folding of the triple helix to the assembly of fibrils into fibers. The mature assembled fibers are crucial for tissue structure and mechanics, cell interactions, and other functions in vivo. Although triple helix folding can be followed with the use of optical methods such as circular dichroism (CD) spectroscopy, fibrillogenesis is typically measured by alternative methods such as turbidity, rheology, and microscopy. Together, these approaches allow for investigation of the mechanical properties and architectures of collagen-based scaffolds and excised tissues. Herein, we demonstrate that CD spectroscopy, a technique that is used primarily to evaluate the secondary structure of proteins, can also be employed to monitor collagen fibrillogenesis. Type-I collagen suspensions demonstrated a strong, negative ellipticity band between 204 and 210 nm under conditions consistent with fibrillogenesis. Deconvolution of CD spectra before, during, and after fibrillogenesis identified a unique fibril spectrum distinct from triple helix and random coil conformations. The ability to monitor multiple states of collagen simultaneously in one experiment using one modality provides a powerful platform for studying this complex assembly process and the effects of other factors, such as collagenases, on fibrillogenesis and degradation.

As a biomaterial, collagen has been used throughout tissue engineering and regenerative medicine. Collagen is native to the body, is highly biocompatible, and naturally promotes cell adhesion and regeneration. However, collagen fibers and the inherent weak mechanical properties of collagen hydrogels interfere with further development of collagen as a bio-ink. Herein, we demonstrate the use of a modified type-I collagen, collagen methacrylamide (CMA), as a fibril-forming bio-ink for free-form fabrication of scaffolds. Like collagen, CMA can self-assemble into a fibrillar hydrogel at physiological conditions. In contrast, CMA is photocrosslinkable and thermoreversible, and photocrosslinking eliminates thermoreversibility. Free-form fabrication of CMA was performed through self-assembly of the CMA hydrogel, photocrosslinking the structure of interest using a photomask, and cooling the entire hydrogel, which results in cold-melting of unphotocrosslinked regions. Printed hydrogels had a resolution on the order of ~350 μm, and can be fabricated with or without cells and maintain viability or be further processed into freeze-dried sponges, all while retaining pattern fidelity. A subcutaneous implant study confirmed the biocompatibility of CMA in comparison to collagen. Free-form fabrication of CMA allows for printing of macroscale, customized scaffolds with good pattern fidelity and can be implemented with relative ease for continued research and development of collagen-based scaffolds in tissue engineering.

We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix.

Plant responses to natural stresses have been the focus of numerous studies; however less is known about plant responses to artificial (i.e., man-made) stress. Chlortetracycline (CTC) is widely used in agriculture and becomes an environmental contaminant when introduced into soil from manure used as fertilizer. We show here that in the model plant Arabidopsis (Arabidopsis thaliana), root uptake of CTC leads to toxicity, with growth reductions and other effects. Analysis of protein accumulation and in vivo synthesis revealed numerous changes in soluble and membrane-associated proteins in leaves and roots. Many representative proteins associated with different cellular processes and compartments showed little or no change in response to CTC. However, differences in accumulation and synthesis of NAD-malic enzyme in leaves versus roots suggest potential CTC-associated effects on metabolic respiration may vary in different tissues. Fluorescence resonance energy transfer (FRET) analysis indicated reduced levels of intracellular calcium are associated with CTC uptake and toxicity. These findings support a model in which CTC uptake through roots leads to reductions in levels of intracellular calcium due to chelation. In turn, changes in overall patterns and levels of protein synthesis and accumulation due to reduced calcium ultimately lead to growth reductions and other toxicity effects.