Formation of Microcages from a Collagen Mimetic Peptide via Metal-Ligand Interactions.

Here, the hierarchical assembly of a collagen mimetic peptide (CMP) displaying four bipyridine moieties is described. The CMP was capable of forming triple helices followed by self-assembly into disks and domes. Treatment of these disks and domes with metal ions such as Fe(II), Cu(II), Zn(II), Co(II), and Ru(III) triggered the formation of microcages, and micron-sized cup-like structures. Mechanistic studies suggest that the formation of the microcages proceeds from the disks and domes in a metal-dependent fashion. Fluorescently-labeled dextrans were encapsulated within the cages and displayed a time-dependent release using thermal conditions.

Cyclic Peptides for Efficient Detection of Collagen.

We report here a new class of collagen-binding peptides, cyclic collagen-mimetic peptides (cCMPs), that efficiently hybridize with the triple-helix-forming portions of collagen. cCMPs are composed of two parallel collagen-like (Xaa-Yaa-Gly)n strands with both termini tethered by covalent linkages. Enzyme-linked immunosorbent assays and western blotting analysis showed that cCMPs exhibit more potent affinity toward collagen than reported collagen-binding peptides and can specifically detect different collagen polypeptides in a mixture of proteins. Collagen secreted from cultured cells was detected by confocal microscopy with fluorescein-labeled cCMP. The cCMP is also shown to detect sensitively folding intermediates in the endoplasmic reticulum, something that was difficult to visualize with conventional collagen detectors. Molecular-dynamics simulations suggested that a cCMP forms a more stably hybridized product than its single-chain counterpart; this could explain why cCMP has higher affinity toward denatured collagen. These results indicate the usefulness of cCMPs as tools for detecting denatured collagen.

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.

Effects of glycosylated (2S,4R)-hydroxyproline on the stability and assembly of collagen triple helices.

Functionalized collagen-mimetic peptides (CMPs) have been widely used in the preparation of collagen-related biomaterials. Among the reported results, the induced noncovalent interactions between the implanted functional groups or moieties were frequently the key elements to promote the self-assembly of small CMPs. In this work, we designed and synthesized a series of glycosylated CMPs in which 4-O-[ß-D-galactopyranosyl]-(2S,4R)-4-hydroxyproline (Hyp(Gal)) was incorporated to explore the effects of glycosylation on the stability and assembly of collagen triple helices. Circular dichroism measurements showed that glycosylation of hydroxyproline slightly destabilized the collagen triple helices, but did not reduce their refolding rate. Compared to non-glycosylated CMPs, the incorporation of Hyp(Gal) speeded up the assembly of CMPs, indicating that this modification could assist the self-assembly of CMPs into higher-order structures, such as fibrils. O-Galactosylation of hydroxyproline imposes contrary effects on the triple helix stability and the self-assembly rate of collagen triple helices, exhibiting a piece of important and useful information for designing collagen-related biomaterials. Our finding also suggests that instead of stabilizing the triple helical conformation of CMPs, installing additional forces between CMPs could be a crucial factor to promote the assembly of CMPs into large-scale constructs.

Mechanically tough, adhesive, self-healing hydrogel promotes annulus fibrosus repair via autologous cell recruitment and microenvironment regulation.

Annulus fibrosus (AF) defect is an important cause of disc re-herniation after discectomy. The self-regeneration ability of the AF is limited, and AF repair is always hindered by the inflammatory microenvironment after injury. Hydrogels represent one of the most promising materials for AF tissue engineering strategies. However, currently available commercial hydrogels cannot withstand the harsh mechanical load within intervertebral disc. In the present study, an innovative triple cross-linked oxidized hyaluronic acid (OHA)-dopamine (DA)- polyacrylamide (PAM) composite hydrogel, modified with collagen mimetic peptide (CMP) and supplied with transforming growth factor beta 1 (TGF-ß1) (OHA-DA-PAM/CMP/TGF-ß1 hydrogel) was developed for AF regeneration. The hydrogel exhibited robust mechanical strength, strong bioadhesion, and significant self-healing capabilities. Modified with collagen mimetic peptide, the hydrogel exhibited extracellular-matrix-mimicking properties and sustained the AF cell phenotype. The sustained release of TGF-ß1 from the hydrogel was pivotal in recruiting AF cells and promoting extracellular matrix production. Furthermore, the composite hydrogel attenuated LPS-induced inflammatory response and promote ECM synthesis in AF cells via suppressing NFκB/NLRP3 pathway. In vivo, the composite hydrogel successfully sealed AF defects and alleviated intervertebral disk degeneration in a rat tail AF defect model. Histological evaluation showed that the hydrogel integrated well with host tissue and facilitated AF repair. The strategy of recruiting endogenous cells and providing an extracellular-matrix-mimicking and anti-inflammatory microenvironment using the mechanically tough composite OHA-DA-PAM/CMP/TGF-ß1 hydrogel may be applicable for AF defect repair in the clinic. STATEMENT OF SIGNIFICANCE: Annulus fibrosus (AF) repair is challenging due to its limited self-regenerative capacity and post-injury inflammation. In this study, a mechanically tough and highly bioadhesive triple cross-linked composite hydrogel, modified with collagen mimetic peptide (CMP) and supplemented with transforming growth factor beta 1 (TGF-ß1), was developed to facilitate AF regeneration. The sustained release of TGF-ß1 enhanced AF cell recruitment, while both TGF-ß1 and CMP could modulate the microenvironment to promote AF cell proliferation and ECM synthesis. In vivo, this composite hydrogel effectively promoted the AF repair and mitigated the intervertebral disc degeneration. This research indicates the clinical potential of the OHA-DA-PAM/CMP/TGF-ß1 composite hydrogel for repairing AF defects.

A Phase 2 Trial to Test Safety and Efficacy of ST-100, a Unique Collagen Mimetic Peptide Ophthalmic Solution for Dry Eye Disease.

Objective: Dry eye disease (DED) is a worldwide source of ocular discomfort. This first-in-human phase 2 clinical study determined the efficacy of treating signs and symptoms of DED using an ophthalmic solution of synthesized mimetic of human collagen (ST-100). Design: This double-masked, randomized, study compared high (60 µg/mL) and low (22 µg/mL) dose ST-100 to vehicle utilizing the Ora, Inc. Controlled Adverse Environment (CAE) during a 28-day period. Participants: Participants included males and females ≥ 18 years of age with signs and symptoms of DED for ≥ 6 months that worsened during CAE exposure who were not taking any topical prescription therapeutic. Intervention: Participants applied ST-100 or vehicle placebo topically to both corneas (1 drop) twice daily via a blow-fill-sealed preservative-free container. Main Outcome Measures: The prespecified primary efficacy sign end point was mean change from baseline (CFB) in total corneal fluorescein staining, and the primary symptom end point was mean CFB in ocular discomfort. A secondary prespecified efficacy end point was CFB in unanesthetized Schirmer's test for tear film production. Results: Of 160 subjects in the intent-to-treat population (112 female, 48 male, median age 64), 146 completed the study. Total corneal fluorescein staining CFB improved for high-dose ST-100, with superiority over vehicle when both eyes were considered together (2-sample t test: P = 0.0394). High-dose ST-100 was superior to vehicle in Schirmer's CFB for the study eye (least squares mean difference [confidence interval] = 2.3 [0.6, 4.0], P = 0.0094). For study eyes, the proportion of Schirmer's test responders (CFB ≥ 10 mm, Schirmer's responder rate) was 12.2% for high-dose ST-100 versus 0.0% for vehicle (P = 0.0266). The CFB for ocular discomfort score improved in study eyes for high- and low-dose ST-100 (paired t test, P = 0.0133, P = 0.0151, respectively) but without superiority over vehicle (ANCOVA: P = 0.5696, P = 0.8968, respectively). ST-100 Schirmer's responders also demonstrated total elimination of worsening of corneal fluorescein stain during the stress of CAE sessions. Conclusions: ST-100 significantly improved tear production and related outcomes in DED and was well-tolerated in reducing symptoms. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Collagen in the central nervous system: contributions to neurodegeneration and promise as a therapeutic target.

The extracellular matrix is a richly bioactive composition of substrates that provides biophysical stability, facilitates intercellular signaling, and both reflects and governs the physiological status of the local microenvironment. The matrix in the central nervous system (CNS) is far from simply an inert scaffold for mechanical support, instead conducting an active role in homeostasis and providing broad capacity for adaptation and remodeling in response to stress that otherwise would challenge equilibrium between neuronal, glial, and vascular elements. A major constituent is collagen, whose characteristic triple helical structure renders mechanical and biochemical stability to enable bidirectional crosstalk between matrix and resident cells. Multiple members of the collagen superfamily are critical to neuronal maturation and circuit formation, axon guidance, and synaptogenesis in the brain. In mature tissue, collagen interacts with other fibrous proteins and glycoproteins to sustain a three-dimensional medium through which complex networks of cells can communicate. While critical for matrix scaffolding, collagen in the CNS is also highly dynamic, with multiple binding sites for partnering matrix proteins, cell-surface receptors, and other ligands. These interactions are emerging as critical mediators of CNS disease and injury, particularly regarding changes in matrix stiffness, astrocyte recruitment and reactivity, and pro-inflammatory signaling in local microenvironments. Changes in the structure and/or deposition of collagen impact cellular signaling and tissue biomechanics in the brain, which in turn can alter cellular responses including antigenicity, angiogenesis, gliosis, and recruitment of immune-related cells. These factors, each involving matrix collagen, contribute to the limited capacity for regeneration of CNS tissue. Emerging therapeutics that attempt to rebuild the matrix using peptide fragments, including collagen-enriched scaffolds and mimetics, hold great potential to promote neural repair and regeneration. Recent evidence from our group and others indicates that repairing protease-degraded collagen helices with mimetic peptides helps restore CNS tissue and promote neuronal survival in a broad spectrum of degenerative conditions. Restoration likely involves bolstering matrix stiffness to reduce the potential for astrocyte reactivity and local inflammation as well as repairing inhibitory binding sites for immune-signaling ligands. Facilitating repair rather than endogenous replacement of collagen degraded by disease or injury may represent the next frontier in developing therapies based on protection, repair, and regeneration of neurons in the central nervous system.

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.

Consequences of incorporating thiaproline and its oxidized derivatives into collagen triple helices.

(2R)-4-thiaproline (Thp) is an analog of proline, replacing Cγ in the pyrrolidine ring with sulfur. Its thiazolidine ring easily interconverts between endo and exo puckers due to a small energy barrier, which leads to destabilize polyproline helices. Collagen, composed of three polyproline II helices, mainly consists of X-Y-Gly triplets, where X is often proline and Y is frequently (2S,4R)-hydroxyproline. In this study, we incorporated Thp into either position-X or position-Y to investigate the consequences of such a replacement on the triple helix. Circular dichroism and differential scanning calorimetry analyses showed that the Thp-containing collagen-mimetic peptides (CMPs) can fold into stable triple helices, in which the substitution at position-Y exhibits a larger destabilization effect. Additionally, we also prepared the derivative peptides by oxidizing Thp in the peptide to N-formyl-cysteine or S,S-dioxide Thp. The results showed that the oxidized derivatives at position-X only slightly affect collagen stability, but those at position-Y induce a large destabilization effect. The consequences of incorporating Thp and its oxidized derivatives into CMPs are position dependent. Computational results suggested that the ease of interconversion between exo and endo puckers for Thp and the twist conformation of S,S-dioxide Thp may cause the destabilization effect at position-Y. We have revealed new insights into the impacts of Thp and its oxidized derivatives on collagen and demonstrated that Thp can be used to design collagen-related biomaterials.

VEGF-Encoding, Gene-Activated Collagen-Based Matrices Promote Blood Vessel Formation and Improved Wound Repair.

Disruption in vascularization during wound repair can severely impair healing. Proangiogenic growth factor therapies have shown great healing potential; however, controlling growth factor activity and cellular behavior over desired healing time scales remains challenging. In this study, we evaluated collagen-mimetic peptide (CMP) tethers for their capacity to control growth factor gene transfer and growth factor activity using our recently developed gene-activated hyaluronic acid-collagen matrix (GAHCM). GAHCM was comprised of DNA/polyethyleneimine (PEI) polyplexes that were retained on hyaluronic acid (HA)-collagen hydrogels using CMPs. We hypothesized that using CMP-collagen tethers to control vascular endothelial growth factor-A (VEGF-A) gene delivery in fibroblasts would provide a powerful strategy to modulate the proangiogenic behaviors of endothelial cells (ECs) for blood vessel formation, resulting in enhanced wound repair. In co-culture experiments, we observed that CMP-modified GAHCM induced tunable gene delivery in fibroblasts as predicted, and correspondingly, VEGF-A produced by the fibroblasts led to increased growth and persistent migration of ECs for at least 7 days, as compared to non-CMP-modified GAHCM. Moreover, when ECs were exposed to fibroblast-containing VEGF-GAHCM with higher levels of CMP modification (50% CMP-PEI, or 50 CP), high CD31 expression was stimulated, resulting in the formation of an interconnected EC network with a significantly higher network volume and a larger diameter network structure than controls. Application of VEGF-GAHCM with 50 CP in murine splinted excisional wounds facilitated prolonged prohealing and proangiogenic responses resulting in increased blood vessel formation, improved granulation tissue formation, faster re-epithelialization, and overall enhanced repair. These findings suggest the benefits of CMP-collagen tethers as useful tools to control gene transfer and growth factor activity for improved treatment of wounds.

Modified hyaluronic acid-collagen matrices trigger efficient gene transfer and prohealing behavior in fibroblasts for improved wound repair.

Growth factor therapy has demonstrated great promise for chronic wound repair, but controlling growth factor activity and cell phenotype over desired time frames remains a critical challenge. In this study, we developed a gene-activated hyaluronic acid-collagen matrix (GAHCM) comprising DNA/polyethylenimine (PEI) polyplexes retained on hyaluronic acid (HA)-collagen hydrogels using collagen mimetic peptides (CMPs). We hypothesized that manipulating both the number of CMP-collagen tethers and the ECM composition would provide a powerful strategy to control growth factor gene transfer kinetics while regulating cell behavior, resulting in enhanced growth factor activity for wound repair. We observed that polyplexes with 50% CMP-modified PEI (50 CP) showed enhanced retention of polyplexes in HCM hydrogels by 2.7-fold as compared to non-CMP modified polyplexes. Moreover, the incorporation of HA in the hydrogel promoted a significant increase in gene transfection efficiency based upon analysis of Gaussia luciferase (GLuc) reporter gene expression, and gene expression could be attenuated by blocking HA-CD44 signaling. Furthermore, when fibroblasts were exposed to vascular endothelial growth factor-A (VEGF-A)-GAHCM, the 50 CP matrix facilitated sustained VEGF-A production for up to 7 days, with maximal expression at day 5. Application of these VEGF-A-50 CP samples stimulated prolonged pro-healing responses, including the TGF-ß1-induced myofibroblast-like phenotypes and enhanced closure of murine splinted wounds. Overall, these findings demonstrate the use of ECM-based materials to stimulate efficient gene transfer and regulate cellular phenotype, resulting in improved control of growth factor activity for wound repair. GAHCM has significant potential to overcome key challenges in growth factor therapy for regenerative medicine. STATEMENT OF SIGNIFICANCE: Despite great promise for growth factor therapies in wound treatment, controlling growth factor activity and providing a microenvironment for cells that maximizes growth factor signaling have continued to limit the success of existing formulations. Our GAHCM strategy, combining CMP gene delivery and a hyaluronic acid-collagen matrix, enabled enhanced wound healing efficacy via the combination of controlled and localized growth factor expression and matrix-mediated regulation of cell behavior. Incorporation of CMPs and HA in the same matrix synergistically enhanced VEGF activity as compared with simpler matrices. Accordingly, GAHCM will advance our ability to leverage growth factor signaling for wound healing, resulting in new long-term treatments for recalcitrant wounds.

Biomaterial functionalization with triple-helical peptides for tissue engineering.

In the growing field of tissue engineering, providing cells in biomaterials with the adequate biological cues represents an increasingly important challenge. Yet, biomaterials with excellent mechanical properties are often biologically inert to many cell types. To address this issue, researchers resort to functionalization, i.e. the surface modification of a biomaterial with active molecules or substances. Functionalization notably aims to replicate the native cellular microenvironment provided by the extracellular matrix, and in particular by collagen, its major component. As our understanding of biological processes regulating cell behavior increases, functionalization with biomolecules binding cell surface receptors constitutes a promising strategy. Among these, triple-helical peptides (THPs) that reproduce the architectural and biological properties of collagen are especially attractive. Indeed, THPs containing binding sites from the native collagen sequence have successfully been used to guide cell response by establishing cell-biomaterial interactions. Notably, the GFOGER motif recognizing the collagen-binding integrins is extensively employed as a cell adhesive peptide. In biomaterials, THPs efficiently improved cell adhesion, differentiation and function on biomaterials designed for tissue repair (especially for bone, cartilage and heart), vascular graft fabrication, wound dressing, drug delivery or immunomodulation. This review describes the key characteristics of THPs, their effect on cells when combined to biomaterials and their strong potential as biomimetic tools for regenerative medicine. STATEMENT OF SIGNIFICANCE: This review article describes how triple-helical peptides constitute efficient tools to improve cell-biomaterial interactions in tissue engineering. Triple helical peptides are bioactive molecules that mimic the architectural and biological properties of collagen. They have been successfully used to specifically recognize cell-surface receptors and provide cells seeded on biomaterials with controlled biological cues. Functionalization with triple-helical peptides has enabled researchers to improve cell function for regenerative medicine applications, such as tissue repair. However, despite encouraging results, this approach remains limited and under-exploited, and most functionalization strategies reported in the literature rely on biomolecules that are unable to address collagen-binding receptors. This review will assist researchers in selecting the correct tools to functionalize biomaterials, in efforts to guide cellular response.

A Collagen-Mimetic Organic-Inorganic Hydrogel for Cartilage Engineering.

Promising strategies for cartilage regeneration rely on the encapsulation of mesenchymal stromal cells (MSCs) in a hydrogel followed by an injection into the injured joint. Preclinical and clinical data using MSCs embedded in a collagen gel have demonstrated improvements in patients with focal lesions and osteoarthritis. However, an improvement is often observed in the short or medium term due to the loss of the chondrocyte capacity to produce the correct extracellular matrix and to respond to mechanical stimulation. Developing novel biomimetic materials with better chondroconductive and mechanical properties is still a challenge for cartilage engineering. Herein, we have designed a biomimetic chemical hydrogel based on silylated collagen-mimetic synthetic peptides having the ability to encapsulate MSCs using a biorthogonal sol-gel cross-linking reaction. By tuning the hydrogel composition using both mono- and bi-functional peptides, we succeeded in improving its mechanical properties, yielding a more elastic scaffold and achieving the survival of embedded MSCs for 21 days as well as the up-regulation of chondrocyte markers. This biomimetic long-standing hybrid hydrogel is of interest as a synthetic and modular scaffold for cartilage tissue engineering.

Application of "Magnetic Anchors" to Align Collagen Fibres for Axonal Guidance.

The use of neural scaffolds with a highly defined microarchitecture, fabricated with standard techniques such as electrospinning and microfluidic spinning, requires surgery for their application to the site of injury. To circumvent the risk associated with aciurgy, new strategies for treatment are sought. This has led to an increase in the quantity of research into injectable hydrogels in recent years. However, little research has been conducted into controlling the building blocks within these injectable hydrogels to produce similar scaffolds with a highly defined microarchitecture. "Magnetic particle string" and biomimetic amphiphile self-assembly are some of the methods currently available to achieve this purpose. Here, we developed a "magnetic anchor" method to improve the orientation of collagen fibres within injectable 3D scaffolds. This procedure uses GMNP (gold magnetic nanoparticle) "anchors" capped with CMPs (collagen mimetic peptides) that "chain" them to collagen fibres. Through the application of a magnetic field during the gelling process, these collagen fibres are aligned accordingly. It was shown in this study that the application of CMP functionalised GMNPs in a magnetic field significantly improves the alignment of the collagen fibres, which, in turn, improves the orientation of PC12 neurites. The growth of these neurite extensions, which were shown to be significantly longer, was also improved. The PC12 cells grown in collagen scaffolds fabricated using the "magnetic anchor" method shows comparable cellular viability to that of the untreated collagen scaffolds. This capability of remote control of the alignment of fibres within injectable collagen scaffolds opens up new strategic avenues in the research for treating debilitating neural tissue pathologies.

A collagen mimetic peptide-modified hyaluronic acid hydrogel system with enzymatically mediated degradation for mesenchymal stem cell differentiation.

We have successfully designed and synthesized a biomimetic hydrogel system with maleimide-modified hyaluronic acid (HA) as the backbone and conjugated it to the collagen mimetic peptide (GPO)8-CG-RGDS. The matrix metalloproteinase (MMP)-sensitive peptide GCRDGPQGI↓WGQDRCG was the cross-linker. HA has high biocompatibility, low immunogenicity, and the capacity to interact with extracellular molecules. Recent studies have found that matrix metalloproteinases (MMPs) are involved in regulating the differentiation of bone mesenchymal stem cells and play a pivotal role in cartilage formation. (GPO)8-CG-RGDS has a natural collagen partial structure that follows the (Gly-Xaa-Yaa)n sequence, which is controllable in quality and can mimic the structure and biological activity of natural collagen. We found that combining this CMP with a MMP-sensitive peptide may have the potential to induce the differentiation of BMSCs into cartilage and inhibit the hypertrophic phenotype during differentiation. This design allows HA hydrogels to not only bind RGD sequences but also graft other functional peptide sequences to achieve a highly flexible platform with potential for multiple biomedical applications.

Encapsulation of collagen mimetic peptide-tethered vancomycin liposomes in collagen-based scaffolds for infection control in wounds.

Wound infections are a significant clinical problem affecting millions of people worldwide. Topically applied antibacterial formulations with longer residence time and controlled antimicrobial release would offer significant benefits for improved prevention and treatment of infected wounds. In this study, we developed collagen mimetic peptide (CMP) tethered vancomycin (Van)-containing liposomes (Lipo) (CMP-Van-Lipo) hybridized to collagen-based hydrogels ('co-gels,' e.g., collagen/fibrin combination hydrogels) for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections in vitro and in vivo. Tethering CMP-Van-Lipo nanostructures to co-gels enabled sustained Van release and enhanced in vitro antibacterial effects against MRSA as compared to Van loaded co-gels or Van-Lipo loaded co-gels following multiple fresh bacterial inoculations over a period of 48 h. These results were successfully translated in vivo wherein MRSA infected wounds were effectively treated with CMP-Van-Lipo loaded co-gels for up to 9 days, whereas the activity of Van loaded co-gels and Van-Lipo loaded co-gels were limited to <2 days. Moreover, CMP-Van-Lipo retained in vivo antibacterial activity even after re-inoculation with bacteria; however, Van loaded co-gels and Van-Lipo loaded co-gels allowed significant bacterial growth demonstrating their limited efficacy. Altogether, these results provide proof-of-concept that CMP-Van-Lipo loaded co-gels can be effective topical formulations for preventive treatment of MRSA wound infections. STATEMENT OF SIGNIFICANCE: Current topical antimicrobial formulations (e.g., creams, gels, and ointments) do not control release, leaving antimicrobial concentrations either too high or too low at different time points, and provoking the development of antibacterial resistance and recurrence of wound infections. Here, collagen mimetic peptides (CMPs) were used to stably hybridize vancomycin-containing liposomal nanocarriers (CMP-Van-Lipo) within collagen-fibrin co-gels via triple-helical integration with collagen, enabling control over Van release for prolonged time periods and minimizing the adverse effects of the Lipo formulations on fibroblast cell viability in the wound bed. The CMP-Van-Lipo loaded co-gel's higher antibacterial effects in vitro were successfully translated in vivo for treatment of MRSA-infected mouse wounds, and thus the co-gels can be a potentially translatable treatment for improved clinical wound management.

Enhanced wound healing via collagen-turnover-driven transfer of PDGF-BB gene in a murine wound model.

Wound healing is a complex biological process that requires coordinated cell proliferation, migration, and extracellular matrix production/remodeling, all of which are inhibited/delayed in chronic wounds. In this study, a formulation was developed that marries a fibrin-based, provisional-like matrix with collagen mimetic peptide (CMP)/PDGF gene-modified collagens, leading to the formation of robust gels that supported temporally controlled PDGF expression and facile application within the wound bed. Analysis employing in vitro co-gel scaffolds confirmed sustained and temporally controlled gene release based on matrix metalloproteinase (MMP) activity, with ~30% higher PDGF expression in MMP producing fibroblasts as-compared with non-MMP-expressing cells. The integration of fibrin with the gene-modified collagens resulted in co-gels that strongly supported both fibroblast cell recruitment/invasion as well as multiple aspects of the longer-term healing process. The excisional wound healing studies in mice established faster wound closure using CMP-modified PDGF polyplex-loaded co-gels, which exhibited up to 24% more wound closure (achieved with ~2 orders of magnitude lower growth factor dosing) after 9 days as compared to PDGF-loaded co-gels, and 19% more wound closure after 9 days as compared to CMP-free polyplex loaded co-gels. Moreover, minimal scar formation as well as improved collagen production, myofibroblast activity, and collagen orientation was observed following CMP-modified PDGF polyplex-loaded co-gel application on wounds. Taken together, the combined properties of the co-gels, including their stability and capacity to control both cell recruitment and cell phenotype within the murine wound bed, strongly supports the potential of the co-gel scaffolds for improved treatment of chronic non-healing wounds.

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels.

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell-cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

Characterization of Self-Assembled Protein Scaffolds from Collagen-Mimetic Peptides.

Collagen-mimetic peptides have been utilized to form structures of different morphology for various biomedical and nanotechnology applications. This chapter describes the characterization of collagen-mimetic peptide self-assembled structures formed by tuning the interactions between peptides. Inclusion of varying hydrophobicity, electrostatic forces, and stereoselectivity was mainly employed in CMP designs discussed herein. The role of these forces can be assessed using multiple characterization techniques. Light scattering techniques have been employed to study the aggregation kinetics of self-assembled nanostructures and to investigate the net charge distribution of peptides. Spectroscopy techniques like circular dichroism, fluorescence, and absorption spectroscopy have been utilized to decipher the secondary structures of peptide and binding of the peptides with dyes. Imaging techniques helped in resolving the morphology of the self-assembled structures. Confocal fluorescence microscopy and differential scanning calorimetry helped in indirect assessment of hydrophobicity and X-ray studies to determine the inter-helical spacing between the triple helical peptides of the higher order structures.

Improved Cell Adhesion and Osteogenesis of op-HA/PLGA Composite by Poly(dopamine)-Assisted Immobilization of Collagen Mimetic Peptide and Osteogenic Growth Peptide.

A nanocomposite of poly(lactide-co-glycolide) (PLGA) and hydroxyapatite (HA) with a different grafting ratio of l-lactic acid oligomer (op-HA) showed better interface compatibility, mineralization, and osteogenetic abilities. However, surface modification of the composite is crucial to improve the osteointegration for bone regeneration. In this study, a biomimetic process via poly(dopamine) coating was utilized to prepare functional substrate surfaces with immobilized bioactive peptides that efficiently regulate the osteogenic differentiation of preosteoblasts (MC3T3-E1). Our study demonstrated that incorporation of collagen mimetic peptide significantly enhanced cell adhesion and proliferation. The immobilization of osteogenic growth peptide induced the osteodifferentiation of cells, as indicated by the alkaline phosphate activity test, quantitative real-time polymerase chain reaction analysis, and immunofluorescence staining. The mineralization on the peptide-modified substrates was also enhanced greatly. Findings from this study revealed that this biofunctionalized layer on op-HA/PLGA substrate improved mineralization and osteogenic differentiation. In conclusion, the surface modification strategy with bioactive peptides shows potential to enhance the osteointegration of bone implants.