From Collagen Mimetics to Collagen Hybridization and Back.

ConspectusFacilitated by the unique triple-helical protein structure, fibrous collagens, the principal proteins in animals, demonstrate a dual function of serving as building blocks for tissue scaffolds and as a bioactive material capable of swift renewal in response to environmental changes. While studies of triple-helical collagen mimetic peptides (CMPs) have been instrumental in understanding the molecular forces responsible for the folding and assembly of triple helices, as well as identifying bioactive regions of fibrous collagen molecules, single-strand CMPs that can specifically target and hybridize to denatured collagens (i.e., collagen hybridizing peptides, CHPs) have proven useful in identifying the remodeling activity of collagen-rich tissues related to development, homeostasis, and pathology. Efforts to improve the utility of CHPs have resulted in the development of new skeletal structures, such as dimeric and cyclic CHPs, as well as the incorporation of artificial amino acids, including fluorinated proline and N-substituted glycines (peptoid residues). In particular, dimeric CHPs were used to capture collagen fragments from biological fluid for biomarker study, and the introduction of peptoid-based collagen mimetics has sparked renewed interest in peptidomimetic research because peptoids enable a stable triple-helical structure and the presentation of an extensive array of side chain structures offering a versatile platform for the development of new collagen mimetics.This Account will cover the evolution of our research from CMPs as biomaterials to ongoing efforts in developing triple-helical peptides with practical theranostic potential in targeting denatured and damaged collagens. Our early efforts in functionalizing natural collagen scaffolds via noncovalent modifications led to the discovery of an entirely new use of CMPs. This discovery resulted in the development of CHPs that are now used by many different laboratories for the investigation of pathologies associated with changes in the structures of extracellular matrices including fibrosis, cancer, and mechanical damage to collagen-rich, load-bearing tissues. Here, we delve into the essential design features of CHPs contributing to their collagen binding properties and practical usage and explore the necessity for further mechanistic understanding of not only the binding processes (e.g., binding domain and stoichiometry of the hybridized complex) but also the biology of collagen degradation, from proteolytic digestion of fibrils to cellular processing of collagen fragments. We also discuss the strengths and weaknesses of peptoid-based triple-helical peptides as applied to collagen hybridization touching on thermodynamic and kinetic aspects of triple-helical folding. Finally, we highlight current limitations and future directions in the use of peptoid building blocks to develop bioactive collagen mimetics as new functional biomaterials.

The Chemistry and Biology of Collagen Hybridization.

Collagen provides mechanical and biological support for virtually all human tissues in the extracellular matrix (ECM). Its defining molecular structure, the triple-helix, could be damaged and denatured in disease and injuries. To probe collagen damage, the concept of collagen hybridization has been proposed, revised, and validated through a series of investigations reported as early as 1973: a collagen-mimicking peptide strand may form a hybrid triple-helix with the denatured chains of natural collagen but not the intact triple-helical collagen proteins, enabling assessment of proteolytic degradation or mechanical disruption to collagen within a tissue-of-interest. Here we describe the concept and development of collagen hybridization, summarize the decades of chemical investigations on rules underlying the collagen triple-helix folding, and discuss the growing biomedical evidence on collagen denaturation as a previously overlooked ECM signature for an array of conditions involving pathological tissue remodeling and mechanical injuries. Finally, we propose a series of emerging questions regarding the chemical and biological nature of collagen denaturation and highlight the diagnostic and therapeutic opportunities from its targeting.

Targeting Intratibial Osteosarcoma Using Water-Soluble Copolymers Conjugated to Collagen Hybridizing Peptides.

Osteosarcoma (OS) is the most common form of primary malignant bone cancer in adolescents. Over the years, OS prognosis has greatly improved due to adjuvant and neoadjuvant (preoperative) chemotherapeutic treatment, increasing the chances of successful surgery and reducing the need for limb amputation. However, chemotherapeutic treatment to treat OS is limited by off-target toxicities and requires improved localization at the tumor site. Collagen, the main constituent of bone tissue, is extensively degraded and remodeled in OS, leading to an increased availability of denatured (monomeric) collagen. Collagen hybridizing peptides (CHPs) comprise a class of peptides rationally designed to selectively bind to denatured collagen. In this work, we have conjugated CHPs as targeting moieties to water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers to target OS tumors. We demonstrated increased accumulation of collagen-targeted HPMA copolymer-CHP conjugates compared to nontargeted HPMA copolymers, as well as increased retention compared to both nontargeted copolymers and CHPs, in a murine intratibial OS tumor model. Furthermore, we used microcomputed tomography analysis to evaluate the bone microarchitecture and correlated bone morphometric parameters (porosity, bone volume, and surface area) with maximum accumulation (Smax) and accumulation at 168 h postinjection (S168) of the copolymers at the tumor. Our results provide the foundation for the use of HPMA copolymer-CHP conjugates as targeted drug delivery systems in OS tumors.

Peptoid Residues Make Diverse, Hyperstable Collagen Triple-Helices.

As the only ribosomally encoded N-substituted amino acid, proline promotes distinct secondary protein structures. The high proline content in collagen, the most abundant protein in the human body, is crucial to forming its hallmark structure: the triple-helix. For over five decades, proline has been considered compulsory for synthetic designs aimed at recapitulating collagen's structure and properties. Here we describe that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, enabling synthesis of stable triple-helical collagen mimetic peptides (CMPs) with unprecedented side chain diversity. Supported by atomic-resolution crystal structures as well as circular dichroism and computational characterizations spanning over 30 N-gly-containing CMPs, we discovered that N-glys stabilize the triple-helix primarily by sterically preorganizing individual chains into the polyproline-II helix. We demonstrated that N-glys with exotic side chains including a "click"-able alkyne and a photosensitive side chain enable CMPs for functional applications including the spatiotemporal control of cell adhesion and migration. The structural principles uncovered in this study open up opportunities for a new generation of collagen-mimetic therapeutics and materials.

Enrichment of Collagen Fragments Using Dimeric Collagen Hybridizing Peptide for Urinary Collagenomics.

Collagen remodeling in normal and pathologic conditions releases numerous collagen fragments into biological fluids. Although a few collagen fragments have been tested as biomarkers for disease indication, most occur at trace levels, making them nearly impossible to detect even with modern analytical tools. Here we report a new way to enrich collagen fragments that allows complete peptidomic analysis of collagen fragments in urine. Enrichment is made possible by dimeric collagen hybridizing peptides (CHPs) that bind collagen fragments originating from the triple helical regions of all collagen types with minimal sequence bias. LC-MS/MS analysis of enriched mouse urine revealed an average of 383 collagenous peptide fragments per sample (compared to 34 for unenriched sample), which could be mapped to all types of mouse collagens in the SwissProt database including FACITs and MACITs. Hierarchical clustering of a selected panel of the detected fragments separated osteopenic mice from healthy mice. The results demonstrate dimeric CHP's ability to enrich collagen fragments from biological fluid and its potential to aid peptidomics-based disease detection and biomarker discovery.

Localization of Therapeutic Fab-CHP Conjugates to Sites of Denatured Collagen for the Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in synovial joints and protease-induced cartilage degradation. Current biologic treatments for RA can effectively reduce symptoms, primarily by neutralizing the proinflammatory cytokine TNFα; however, continued, indiscriminate overinhibition of inflammatory factors can significantly weaken the host immune system, leading to opportunistic infections and interrupting treatment. We hypothesize that localizing anti-TNFα therapeutics to denatured collagen (dCol) present at arthritic joints, via conjugation with collagen-hybridizing peptides (CHPs), will reduce off-site antigen binding and maintain local immunosuppression. We isolated the antigen-binding fragment of the clinically approved anti-TNFα therapeutic infliximab (iFab) and prepared iFab-CHP conjugates via lysine-based conjugation with an SMCC linker. After successful conjugation, confirmed by LC-MS, the binding affinity of iFab-CHP was characterized by ELISA-like assays, which showed comparable antigen binding relative to infliximab, comparable dCol binding relative to CHP, and the hybrid ability to bind both dCol and TNFα simultaneously. We further demonstrated localization of Fab-CHP to areas of high dCol in vivo and promising therapeutic efficacy, assessed by histological staining (Safranin-O and H&E), in a pilot mouse study.

Self-Assembled Water-Soluble Nanofibers Displaying Collagen Hybridizing Peptides.

Collagen hybridizing peptides (CHP) have been demonstrated as a powerful vehicle for targeting denatured collagen (dColl) produced by disease or injury. Conjugation of ß-sheet peptide motif to the CHP results in self-assembly of nonaggregating ß-sheet nanofibers with precise structure. Due to the molecular architecture of the nanofibers which puts high density of hydrophilic CHPs on the nanofiber surface at fixed distance, the nanofibers exhibit high water solubility, without any signs of intramolecular triple helix formation or fiber-fiber aggregation. Other molecules that are flanked with the triple helical forming GlyProHyp repeats can readily bind to the nanofibers by triple helical folding, allowing facile display of bioactive molecules at high density. In addition, the multivalency of CHPs allows the nanofibers to bind to dColl in vitro and in vivo with extraordinary affinity, particularly without preactivation that unravels the CHP homotrimers. The length of the nanofibers can be tuned from micrometers down to 100 nm by simple heat treatment, and when injected intravenously into mice, the small nanofibers can specifically target dColl in the skeletal tissues with little target-associated signals in the skin and other organs. The CHP nanofibers can be a useful tool for detecting and capturing dColl, understanding how ECM remodelling impacts disease progression, and development of new delivery systems that target such diseases.

High Serum Stability of Collagen Hybridizing Peptides and Their Fluorophore Conjugates.

Collagen hybridizing peptides (CHPs) have a great potential for use in targeted drug delivery, diagnostics, and regenerative medicine due to their ability to specifically bind to denatured collagens associated with many pathologic conditions. Since peptides generally suffer from poor enzymatic stability, resulting in rapid degradation and elimination in vivo, CHP's serum stability is a critical parameter that may dictate its pharmacokinetic behavior. Here, we report the serum stability of a series of monomeric CHP derivatives and establish how peptide length, amino acid composition, terminal modification, and linker chemistry influence their availability in serum. We show that monomeric CHPs comprised of the collagen-like Gly-Pro-Hyp motif are resistant to common serum proteinases and that their stability can be further increased by simple N-terminal labeling which negates CHP's susceptibility to proline-specific exopeptidases. When fluorescent dyes are conjugated to a CHP via maleimide-thiol reaction, the dye can transfer from CHP onto serum proteins (e.g., albumin), resulting in an unexpected drop in signal during serum stability assays and off-target accumulation during in vivo tests. This work is the crucial first step toward understanding the pharmacokinetic behavior of CHPs, which can facilitate the development of CHP-based theranostics.

Targeting collagen strands by photo-triggered triple-helix hybridization.

Collagen remodeling is an integral part of tissue development, maintenance, and regeneration, but excessive remodeling is associated with various pathologic conditions. The ability to target collagens undergoing remodeling could lead to new diagnostics and therapeutics as well as applications in regenerative medicine; however, such collagens are often degraded and denatured, making them difficult to target with conventional approaches. Here, we present caged collagen mimetic peptides (CMPs) that can be photo-triggered to fold into triple helix and bind to collagens denatured by heat or by matrix metalloproteinase (MMP) digestion. Peptide-binding assays indicate that the binding is primarily driven by stereo-selective triple-helical hybridization between monomeric CMPs of high triple-helical propensity and denatured collagen strands. Photo-triggered hybridization allows specific staining of collagen chains in protein gels as well as photo-patterning of collagen and gelatin substrates. In vivo experiments demonstrate that systemically delivered CMPs can bind to collagens in bones, as well as prominently in articular cartilages and tumors characterized by high MMP activity. We further show that CMP-based probes can detect abnormal bone growth activity in a mouse model of Marfan syndrome. This is an entirely new way to target the microenvironment of abnormal tissues and could lead to new opportunities for management of numerous pathologic conditions associated with collagen remodeling and high MMP activity.

Planar microphone based on piezoelectric electrospun poly(γ-benzyl-α,L-glutamate) nanofibers.

Velocity and pressure microphones composed of piezoelectric poly(γ-benzyl-α,L-glutamate) (PBLG) nanofibers were produced by adhering a single layer of PBLG film to a Mylar diaphragm. The device exhibited a sensitivity of -60 dBV/Pa in air, and both pressure and velocity response showed a broad frequency response that was primarily controlled by the stiffness of the supporting diaphragm. The pressure microphone response was ±3 dB between 200 Hz and 4 kHz when measured in a semi-anechoic chamber. Thermal stability, easy fabrication, and simple design make this single element transducer ideal for various applications including those for underwater and high temperature use.

HPMA copolymer-collagen hybridizing peptide conjugates targeted to breast tumor extracellular matrix.

The extracellular matrix (ECM) is dynamically involved in many aspects of cell growth and survival, and it plays an active role in cancer etiology. In comparison to healthy ECM, tumor associated ECM shows high collagen deposition and remodeling activity, which results in an increased amount of denatured collagen strands in tumor tissues. Capitalizing on this distinguishing feature, we developed tumor-localizing polymeric carriers that selectively bind to denatured collagen in the tumor ECM. We synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with their side chains conjugated to collagen hybridizing peptides (CHPs). HPMA copolymer-CHP conjugates exhibited selective affinity to denatured collagen and localized to tumors in an orthotopic MDA-MB-231 murine breast cancer model. The conjugates had increased tumor localization compared to copolymers with scrambled peptides in the side chains, as well as increased retention compared to free CHPs. Such conjugates show promise as carriers for ECM-acting drugs and imaging agents in the management of diseases characterized by high ECM remodeling activity.

Strain-induced collagen denaturation is rate dependent in failure of cerebral arteries.

While soft tissues are commonly damaged by mechanical loading, the manifestation of this damage at the microstructural level is not fully understood. Specifically, while rate-induced stiffening has been previously observed in cerebral arteries, associated changes in microstructural damage patterns following high-rate loading are largely undefined. In this study, we stretched porcine middle cerebral arteries to failure at 0.01 and >150 s-1, both axially and circumferentially, followed by probing for denatured tropocollagen using collagen hybridizing peptide (CHP). We found that collagen fibrils aligned with the loading direction experienced less denaturation following failure tests at high than low rates. Others have demonstrated similar rate dependence in tropocollagen denaturation during soft tissue failure, but this is the first study to quantify this behavior using CHP and to report it for cerebral arteries. These findings may have significant implications for traumatic brain injury and intracranial balloon angioplasty. We additionally observed possible tropocollagen denaturation in vessel layers primarily composed of fibrils transversely aligned to the loading axis. To our knowledge, this is the first observation of collagen denaturation due to transverse loading, but further research is needed to confirm this finding. STATEMENT OF SIGNIFICANCE: Previous work shows that collagen hybridizing peptide (CHP) can be used to identify collagen molecule unfolding and denaturation in mechanically overloaded soft tissues, including the cerebral arteries. But experiments have not explored collagen damage at rates relevant to traumatic brain injury. In this work, we quantified collagen damage in cerebral arteries stretched to failure at both high and low rates. We found that the collagen molecule is less damaged at high than at low rates, suggesting that damage mechanisms of either the collagen molecule or other elements of the collagen superstructure are rate dependent. This work implies that arteries failed at high rates, such as in traumatic brain injury, will have different molecular-level damage patterns than arteries failed at low rates. Consequently, improved understanding of damage characteristics may be expanded in the future to better inform clinically relevant cases of collagen damage such as angioplasty and injury healing.

Collagen fibrils from both positional and energy-storing tendons exhibit increased amounts of denatured collagen when stretched beyond the yield point.

Collagen molecules are the base structural unit of tendons, which become denatured during mechanical overload. We recently demonstrated that during tendon stretch, collagen denaturation occurs at the yield point of the stress-strain curve in both positional and energy-storing tendons. We were interested in investigating how this load is transferred throughout the collagen hierarchy, and sought to determine the onset of collagen denaturation when collagen fibrils are stretched. Fibrils are one level above the collagen molecule in the collagen hierarchy, allowing more direct probing of the effect of strain on collagen molecules. We isolated collagen fibrils from both positional and energy-storing tendon types and stretched them using a microelectromechanical system device to various levels of strain. We stained the fibrils with fluorescently labeled collagen hybridizing peptides that specifically bind to denatured collagen, and examined whether samples stretched beyond the yield point of the stress-strain curve exhibited increased amounts of denatured collagen. We found that collagen denaturation in collagen fibrils from both tendon types occurs at the yield point. Greater amounts of denatured collagen were found in post-yield positional fibrils than in energy-storing fibrils. This is despite a greater yield strain and yield stress in fibrils from energy-storing tendons compared to positional tendons. Interestingly, the peak modulus of collagen fibrils from both tendon types was the same. These results are likely explained by the greater crosslink density found in energy-storing tendons compared to positional tendons. The insights gained from this study could help management of tendon and other musculoskeletal injuries by targeting collagen molecular damage at the fibril level. STATEMENT OF SIGNIFICANCE: When tendons are stretched or torn, this can lead to collagen denaturation (damage). Depending on their biomechanical function, tendons are considered positional or energy-storing with different crosslink profiles. By stretching collagen fibrils instead of fascicles from both tendon types, we can more directly examine the effect of tensile stretch on the collagen molecule in tendons. We found that regardless of tendon type, collagen denaturation in fibrils occurs when they are stretched beyond the yield point of the stress-strain curve. This provides insight into how load affects different tendon sub-structures during tendon injuries and failure, which will help clinicians and researchers understand mechanisms of injuries and potentially target collagen molecular damage as a treatment strategy, leading to improved clinical outcomes following injury.

Collagen Molecular Damage is a Hallmark of Early Atherosclerosis Development.

Remodeling of extracellular matrix proteins underlies the development of cardiovascular disease. Herein, we utilized a novel molecular probe, collagen hybridizing peptide (CHP), to target collagen molecular damage during atherogenesis. The thoracic aorta was dissected from ApoE-/- mice that had been on a high-fat diet for 0-18 weeks. Using an optimized protocol, tissues were stained with Cy3-CHP and digested to quantify CHP with a microplate assay. Results demonstrated collagen molecular damage, inferred from Cy3-CHP fluorescence, was a function of location and time on the high-fat diet. Tissue from the aortic arch showed a significant increase in collagen molecular damage after 18 weeks, while no change was observed in tissue from the descending aorta. No spatial differences in fluorescence were observed between the superior and inferior arch tissue. Our results provide insight into the early changes in collagen during atherogenesis and present a new opportunity in the subclinical diagnosis of atherosclerosis.

Encoding Cell-Instructive Cues to PEG-Based Hydrogels via Triple Helical Peptide Assembly.

Effective synthetic tissue engineering scaffolds mimic the structure and composition of natural extracellular matrix (ECM) to promote optimal cellular adhesion, proliferation, and differentiation. Among many proteins of the ECM, collagen and fibronectin are known to play a key role in the scaffold's structural integrity as well as its ability to support cell adhesion. Here, we present photocrosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels displaying collagen mimetic peptides (CMPs) that can be further conjugated to bioactive molecules via CMP-CMP triple helix association. Pre-formed PEGDA-CMP hydrogels can be encoded with varying concentration of cell-signaling CMP-RGD peptides similar to cell adhesive fibronectin decorating the collagen fibrous network by non-covalent binding. Furthermore, the triple helix mediated encoding allows facile generation of spatial gradients and patterns of cell-instructive cues across the cell scaffold that simulate distribution of insoluble factors in the natural ECM.

Terminal repeats impact collagen triple-helix stability through hydrogen bonding.

Nearly 30% of human proteins have tandem repeating sequences. Structural understanding of the terminal repeats is well-established for many repeat proteins with the common α-helix and ß-sheet foldings. By contrast, the sequence-structure interplay of the terminal repeats of the collagen triple-helix remains to be fully explored. As the most abundant human repeat protein and the most prevalent structural component of the extracellular matrix, collagen features a hallmark triple-helix formed by three supercoiled polypeptide chains of long repeating sequences of the Gly-X-Y triplets. Here, with CD characterization of 28 collagen-mimetic peptides (CMPs) featuring various terminal motifs, as well as DSC measurements, crystal structure analysis, and computational simulations, we show that CMPs only differing in terminal repeat may have distinct end structures and stabilities. We reveal that the cross-chain hydrogen bonding mediated by the terminal repeat is key to maintaining the triple-helix's end structure, and that disruption of it with a single amide to carboxylate substitution can lead to destabilization as drastic as 19 °C. We further demonstrate that the terminal repeat also impacts how strong the CMP strands form hybrid triple-helices with unfolded natural collagen chains in tissue. Our findings provide a spatial profile of hydrogen bonding within the CMP triple-helix, marking a critical guideline for future crystallographic or NMR studies of collagen, and algorithms for predicting triple-helix stability, as well as peptide-based collagen assemblies and materials. This study will also inspire new understanding of the sequence-structure relationship of many other complex structural proteins with repeating sequences.

Matrix-Bound VEGF Mimetic Peptides: Design and Endothelial Cell Activation in Collagen Scaffolds.

Long term survival and success of artificial tissue constructs depend greatly on vascularization. Endothelial cell (EC) differentiation and vasculature formation are dependent on spatio-temporal cues in the extracellular matrix that dynamically interact with cells, a process difficult to reproduce in artificial systems. Here we present a novel bifunctional peptide that mimics matrix-bound vascular endothelial growth factor (VEGF) and can be used to encode spatially controlled angiogenic signals in collagen scaffolds. The peptide is comprised of a collagen mimetic domain that was previously reported to bind to type I collagen by a unique hybridization mechanism, and a VEGF mimetic domain with pro-angiogenic activity. Circular dichroism and collagen binding studies confirm the triple helical structure and the collagen binding affinity of the collagen mimetic domain, and EC culture studies demonstrate the peptide's ability to induce endothelial cell morphogenesis and network formation as a matrix-bound factor in 2D and 3D collagen scaffolds. We also show spatial modification of collagen substrates with this peptide that allows localized EC activation and network formation. These results demonstrate that the peptide can be used to present spatially directed angiogenic cues in collagen scaffolds, which may be useful for engineering organized microvasculature.

A cellular model for the investigation of Fuchs' endothelial corneal dystrophy.

Fuchs' endothelial corneal dystrophy is the most common corneal endotheliopathy, and a leading indication for corneal transplantation in the US. Relatively little is known about its underlying pathology. We created a cellular model of the disease focusing on collagen VIII alpha 2 (COL8A2), a collagen which is normally present in the cornea, but which is found in abnormal amounts and distribution in both early and late-onset forms of the disease. We performed cellular transfections using COL8A2 cDNAs including both wild-type and mutant alleles which are known to result in early-onset FECD. We used this cell model to explore the cellular production of wild-type and mutant monomeric and trimeric collagen VIII and measured production levels and patterns using Western blotting and immunofluorescence. We studied the thermal stability of the mutated collagen VIII helices using computer modeling, and further investigated these differences using collagen mimetic peptides. The Western blots demonstrated that similar amounts of wild-type and mutant collagen VIII monomers were produced in the cells. However, the levels of trimeric collagen peptide in the mutant-transfected cells were elevated. Intracellular accumulation of trimeric collagen VIII was confirmed on immunofluorescence studies. Both the computer model and the collagen mimetic peptides demonstrated that the L450W mutant was less thermally stable than either the Q455K or wild-type collagen VIII. Thus, although both mutant collagen VIII peptides were retained intracellularly, the biochemical reasons for the retention varied between genotypes. Collagen VIII mutations, which clinically result in Fuchs' dystrophy, are associated with abnormal cellular accumulation of collagen VIII. Different collagen VIII mutations may act via distinct biochemical mechanisms to produce the FECD phenotype.

Collagen Mimetic Peptides: Progress Towards Functional Applications.

Traditionally, collagen mimetic peptides (CMPs) have been used for elucidating the structure of the collagen triple helix and the factors responsible for its stabilization. The wealth of fundamental knowledge on collagen structure and cell-extracellular matrix (ECM) interactions accumulated over the past decades has led to a recent burst of research exploring the potential of CMPs to recreate the higher order assembly and biological function of natural collagens for biomedical applications. Although a large portion of such research is still at an early stage, the collagen triple helix has become a promising structural motif for engineering self-assembled, hierarchical constructs similar to natural tissue scaffolds which are expected to exhibit unique or enhanced biological activities. This paper reviews recent progress in the field of collagen mimetic peptides that bears both direct and indirect implications to engineering collagen-like materials for potential biomedical use. Various CMPs and collagen-like proteins that mimic either structural or functional characteristics of natural collagens are discussed with particular emphasis on providing helpful information to bioengineers and biomaterials scientists interested in collagen engineering.

PEG-based hydrogels with collagen mimetic peptide-mediated and tunable physical cross-links.

Mechanical properties of tissue scaffolds have major effects on the morphology and differentiation of cells. In contrast to two-dimensional substrates, local biochemical and mechanical properties of three-dimensional hydrogels are difficult to control due to the geometrical confinement. We designed synthetic 3D hydrogels featuring complexes of four-arm poly(ethylene glycol) (PEG) and collagen mimetic peptides (CMPs) that form hydrogels via physical cross-links mediated by thermally reversible triple helical assembly of CMPs. Here we present the fabrication of various PEG-CMP 3D hydrogels and their local mechanical properties determined by particle tracking microrheology. Results show that CMP mediated physical cross-links can be disrupted by altering the temperature of the gel or by adding free CMPs that compete for triple helix formation. This allowed modulation of both bulk and local stiffness as well as the creation of stiffness gradients within the PEG-CMP hydrogel, which demonstrates its potential as a novel scaffold for encoding physicochemical signals for tissue formation.