Collagen is the primary protein component of the extracellular matrix (ECM) and plays a major role in the attachment, migration, and growth of cells in most tissues. The key building block of collagen’s supramolecular structure is the triple helix assembled from three individual collagen strands.1
This unique helical conformation is made possible by the prevailing (Glycine-X-Y) repeating amino acid sequence in which X and Y positions are typically occupied by proline and hydroxyproline, respectively. Collagen’s triple helical structure and subsequent assembly into larger fibrous structures lend structural integrity to the ECM of many mammalian tissues. In addition to providing structural stability, collagen presents sites for cell binding and proteolytic degradation, securing its role as a bioactive player in tissue development.
Collagen-based biomaterials, especially bovine collagen, have been used extensively in biomedical applications.2
The biocompatibility of collagen and its bio-resorbability make it a well-suited biomaterial for tissue regeneration, and collagen is one of the most cost-effective biomaterials due to its abundance in nature. Collagen can be prepared with controlled porosity to enhance cellular migration or diffusion of bioactive factors. Unfortunately, animal-derived collagen can pose inherent problems such as immunogenicity and pathogen transmission.3
In addition, regenerated collagen gels exhibit low mechanical strength that limits their use in tissue scaffolds that are subject to high stress. These shortcomings highlight the need to develop artificial scaffolds that mimic the structural and biofunctional capacity of natural collagens in the ECM.
The design of artificial scaffolds poses many challenges including properly mimicking tissue stiffness and incorporating insoluble bioactive molecules that induce cells to differentiate, proliferate, and migrate into the scaffold. Therefore, creating functional and effective synthetic collagen tissue scaffolds requires a solid understanding of the interplay between cells and the ECM. Cell surface receptors link ECM proteins to the interior cytoskeleton, enabling cells to sense the mechanical nature of their environment and to respond by converting those mechanical cues into chemical signals.4
As a result, the viscoelastic properties of the ECM control many cellular properties including shape, viability, adhesion, and migration.5-7
The ECM is comprised of a highly hydrated network of various protein fibrils and glycosaminoglycan chains.8
This suggests that crosslinked, hydrophilic polymers exhibiting viscoelastic properties similar to the natural ECM could be effective substitutes for natural ECM for tissue engineering applications. Various hydrogels have been synthesized and successfully used as tissue substitutes after implantation via
minimally invasive surgery.9
PEG-based hydrogels are the most popular among these synthetic scaffolds because their use in humans has been approved by the FDA. The extreme hydrophilicity of PEG generates a water-swollen gel that mimics the high water content of the ECM and simultaneously reduces non-specific protein adsorption and cell adhesion.10
Furthermore, PEG conjugation reactions are well understood, allowing biofunctionalities to be easily incorporated into PEG-based hydrogels to spur cell activity.
Collagen mimetic peptides (CMPs) are extensively studied as a model system for understanding the triple helix formation and stability of natural collagens in the ECM.11, 12
CMPs are typically 15-40 amino acid long peptides that mimic collagen in their ability to form the triple helix. CMPs feature the characteristic Gly-X-Y repeat sequence found in natural collagen and exhibit thermally reversible triple helix melting behavior.13
When heated above their melting temperature (Tm
), the CMPs exist as single strands that, unlike natural collagen, completely reform the triple helix when cooled back below the melting temperature.
More recently, the biomimetic properties of CMPs have been employed in hydrogels for tissue engineering applications. CMPs have been shown to bind to natural collagen, presumably through a triple helical strand invasion process.14
This behavior has been exploited in the studies of photo-polymerized poly(ethylene oxide)-diacrylate (PEODA) hydrogels displaying CMPs; the CMPs allowed the gel to better retain the cell-secreted collagens within the hydrogel and enhanced ECM buildup.15
Other research efforts employed temperature-sensitive CMP folding as a way to create hydrogels with temperature dependent macroscopic stability. Koide and coworkers created hydrogels consisting solely of staggered CMPs connected by cysteine disulfide bonds. At high concentrations and below the peptide’s Tm
, the triple helix formation between different staggered CMP molecules led to the assembly of supramolecular peptide structures that created a self-supporting gel.16
De Wolf and coworkers employed recombinant protein methods to create triblock copolymers featuring collagen mimetic sequences.17
These triblock copolymers also exhibited temperature-sensitive gelling behavior based on the melting of the triple helical crosslinks that hold the gel together.
In this study, we present multi-armed PEG-CMP hydrogels containing triple helical crosslinks that provide a convenient means to change both the mechanical and biochemical properties of 3D scaffolds. The temperature-sensitive folding of the CMPs enables the creation and subsequent modulation of CMP-mediated triple helical physical crosslinks within the gel. Furthermore, the CMP triple helices provide an avenue to attach insoluble factors by the addition of unbound CMP complexes to a preformed PEG-CMP hydrogel.
We used particle tracking microrheology to investigate the viscoelastic nature of PEG-CMP hydrogels and to assess the variations in local stiffness of the PEG-CMP gels. Mason et al
and Gittes et al
developed single-particle tracking techniques to study local viscoelastic properties of solutions and gels, and such microrheology approaches have become valuable tools to investigate the viscoelastic properties of small volume samples such as cells. Video-based particle tracking techniques can monitor the Brownian motion of multiple nanoparticles suspended in the investigated medium.20
The stiffness or compliance of the suspending medium is determined by tracking particle positions and calculating the mean squared displacement (MSD) as a function of time lag.18
Particle tracking has been extensively used to study intracellular viscoelastic properties using ballistic intracellular nanorheology.21-24
It has also been used to study the local and global heterogeneity of reconstituted lamin in biological gels and to measure their micromechanical properties.25
Unlike bulk rheology (e.g. cone and plate rheology), particle tracking techniques can investigate localized variations in stiffness within a small sample, which can prove valuable when analyzing stiffness gradients in novel tissue engineering scaffolds.