Although fibrous collagens are major structural components of extracellular matrix in mammals, collagen overproduction is associated with many human diseases including cancers and fibrosis. Collagen is typically identified in biomedical research by western blot and immunohistochemistry; however anti-collagen antibodies employed in these analyses are difficult to prepare and their affinities to collagen can diminish if collagen becomes denatured during analyses. Previously, we discovered that single-stranded collagen mimetic peptides [CMPs, sequence: (GlyProHyp)9] can bind to denatured collagen chains by triple helix hybridization. Here we present collagen-specific staining methods using simple CMPs conjugated to common fluorophores (e.g. carboxyfluorescein), which allow direct detection of collagens and collagen-like proteins in SDS-PAGE and in various mammalian tissue sections. By directly staining SDS-PAGE gels with fluorescently labeled CMPs, both intact (type I, II, and IV) and MMP-1 cleaved collagen (type I) chains as well as complement factor C1q were detected. Collagen bands containing as little as 5 ng were optically visualized while no staining was observed for fibronectin, laminin, and a collection of proteins from mammalian cell lysate. The CMP was unable to stain collagen-like bacterial protein which contains numerous charged amino acids that are believed to stabilize triple helix in place of Hyp. We also show that fluorescently labeled CMPs can specifically visualize collagens in fixed tissue sections (e.g., skin, cornea, and bone) more effectively than anti-collagen I antibody, and allow facile identification of pathologic conditions in fibrotic liver tissues.

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.

Survival of tissue engineered constructs after implantation depends on proper vascularization. The differentiation of endothelial cells into mature microvasculature requires dynamic interactions between cells, scaffold, and growth factors, which are difficult to recapitulate in artificial systems. Previously, photocrosslinked poly(ethylene glycol) diacrylate (PEGDA) hydrogels displaying collagen mimetic peptides (CMPs), dubbed PEGDA‐CMP, that can be further conjugated with bioactive molecules via CMP‐CMP triple helix hybridization were reported. Here, it is shown that a bifunctional peptide featuring pro‐angiogenic domain mimicking vascular endothelial growth factor (VEGF) and a collagen mimetic domain that can fold into a triple helix conformation can hybridize with CMP side chains of the PEGDA‐CMP hydrogel, which results in presentation of insoluble VEGF‐like signals to endothelial cells. Presentation of VEGF‐like signals on the surface of micropatterned scaffolds in this way transforms cells from a quiescent state to elongated and aligned phenotype suggesting that this system could be used to engineer organized microvasculature. It is also shown that the pro‐angiogenic peptide, when applied topically in combination with modified dextran/PEGDA hydrogels, can enhance neovascularization of burn wounds in mice demonstrating the potential clinical use of CMP‐mediated matrix‐bound bioactive molecules for dermal injuries.

In humans, high level of collagen remodeling is seen during normal physiological events such as bone renewal, as well as in pathological conditions, such as arthritis, tumor growth and other chronic wounds. Our lab recently discovered that collagen mimetic peptide (CMP) is able to hybridize with denatured collagens at these collagen remodeling sites with high affinity. Here, we show that the CMP's high binding affinity to denatured collagens can be utilized to deliver angiogenic signals to scaffolds composed of heat-denatured collagens (gelatins). We first demonstrate hybridization between denatured collagens and QKCMP, a CMP with pro-angiogenic QK domain. We show that high levels of QKCMP can be immobilized to a new artificial matrix containing both fibrous type I collagen and heat denatured collagen through triple helix hybridization, and that the QKCMP is able to stimulate early angiogenic response of endothelial cells (ECs). We also show that the QKCMP can bind to excised tissues from burn injuries in cutaneous mouse model, suggesting its potential for promoting neovascularization of burn wounds.