John E. Paderi

Design of a Synthetic Collagen-Binding Peptidoglycan that Modulates Collagen Fibrillogenesis

The ubiquity of collagen in mammalian tissues, with its host of structural and chemical functions, has motivated its research in many fields, including tissue engineering. The organization of collagen is known to affect cell behavior and the resulting structural integrity of tissues or tissue engineered scaffolds. Of particular interest are proteoglycan (PG) interactions with collagen and their influence on collagen assembly. These natural molecules provide unique chemical and mechanical cues and are known to modulate collagen fibrillogenesis. Research has been limited to PGs extracted and purified from animal sources and has the drawbacks of limited design control and costly purification. Consequently, we have designed a synthetic peptidoglycan based on decorin, a collagen-binding PG. The synthetic peptidoglycan containing a collagen-binding peptide with a single dermatan sulfate side chain specifically binds to collagen, delays fibrillogenesis, and increases collagen gel stiffness as decorin does. This design can be tailored with respect to the peptide sequence and attached glycosaminoglycan chain, offering unique control with relative ease of manufacturing.

Collagen-Binding Peptidoglycans Inhibit MMP Mediated Collagen Degradation and Reduce Dermal Scarring

Scarring of the skin is a large unmet clinical problem that is of high patient concern and impact. Wound healing is complex and involves numerous pathways that are highly orchestrated, leaving the skin sealed, but with abnormal organization and composition of tissue components, namely collagen and proteoglycans, that are then remodeled over time. To improve healing and reduce or eliminate scarring, more rapid restoration of healthy tissue composition and organization offers a unique approach for development of new therapeutics. A synthetic collagen-binding peptidoglycan has been developed that inhibits matrix metalloproteinase-1 and 13 (MMP-1 and MMP-13) mediated collagen degradation. We investigated the synthetic peptidoglycan in a rat incisional model in which a single dose was delivered in a hyaluronic acid (HA) vehicle at the time of surgery prior to wound closure. The peptidoglycan treatment resulted in a significant reduction in scar tissue at 21 days as measured by histology and visual analysis. Improved collagen architecture of the treated wounds was demonstrated by increased tensile strength and transmission electron microscopy (TEM) analysis of collagen fibril diameters compared to untreated and HA controls. The peptidoglycans mechanism of action includes masking existing collagen and inhibiting MMP-mediated collagen degradation while modulating collagen organization. The peptidoglycan can be synthesized at low cost with unique design control, and together with demonstrated preclinical efficacy in reducing scarring, warrants further investigation for dermal wound healing.

Incorporation of a decorin biomimetic enhances the mechanical properties of electrochemically aligned collagen threads.

Orientational anisotropy of collagen molecules is integral to the mechanical strength of collagen-rich tissues. We have previously reported a novel methodology to synthesize highly oriented electrochemically aligned collagen (ELAC) threads with mechanical properties approaching those of native tendon. Decorin, a small leucine-rich proteoglycan (SLRP), binds to fibrillar collagen and has been suggested to enhance the mechanical properties of tendon. Based on the structure of natural decorin, we have previously designed and synthesized a peptidoglycan (DS-SILY) that mimics decorin both structurally and functionally. In this study, we investigated the effect of the incorporation of DS-SILY on the mechanical properties and structural organization of ELAC threads. The results indicated that the addition of DS-SILY at a molar ratio of 30:1 (collagen:DS-SILY) significantly enhanced the ultimate stress and ultimate strain of the ELAC threads. Furthermore, differential scanning calorimetry revealed that the addition of DS-SILY at a molar ratio of 30:1 resulted in a more thermally stable collagen structure. However, addition of DS-SILY at a higher concentration (10:1 collagen:DS-SILY) yielded weaker threads with mechanical properties comparable to collagen control threads. Transmission electron microscopy revealed that the addition of DS-SILY at a higher concentration (10:1) resulted in pronounced aggregation of collagen fibrils. More importantly, these aggregates were not aligned along the long axis of the ELAC, thereby compromising the overall tensile properties of the material. We conclude that incorporation of an optimal amount of DS-SILY is a promising approach to synthesize mechanically competent collagen-based biomaterials for tendon tissue engineering applications.

The inhibition of platelet adhesion and activation on collagen during balloon angioplasty by collagen-binding peptidoglycans

Collagen is a potent stimulator for platelet adhesion, activation, and thrombus formation, and provides a means for controlling blood loss due to injury, and recruiting inflammatory cells for fighting infection. Platelet activation is not desirable however, during balloon angioplasty/stent procedures in which balloon expansion inside an artery exposes collagen, initiating thrombosis, and inflammation. We have developed biomimetic polymers, termed peptidoglycans, composed of a dermatan sulfate backbone with covalently attached collagen-binding peptides. The peptidoglycan binds to collagen, effectively masking it from platelet activation. The lead peptidoglycan binds to collagen with high affinity (K(D) = 24 nm) and inhibits platelet binding and activation on collagen in both static studies and under flow, while promoting endothelial regrowth on collagen. Application for angioplasty is demonstrated in the Ossabaw miniature pig by fast delivery to the vessel wall through a therapeutic infusion catheter with a proprietary PTFE porous balloon. The peptidoglycan is an approach for locally preventing platelet deposition and activation on collagen. It can be used during angioplasty to prevent platelet deposition on target vessels and could be used in any vessel, including those not amenable to stent deployment.

Decorin mimic inhibits vascular smooth muscle proliferation and migration.

Over the past 10 years, the number of percutaneous coronary intervention procedures performed in the United States increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, complicates this procedure. A wide range of anti-restenotic therapeutics have been developed, although many elicit non-specific effects that compromise vessel healing. Drawing inspiration from biologically-relevant molecules, our lab developed a mimic of the natural proteoglycan decorin, termed DS-SILY, which can mask exposed collagen and thereby effectively decrease platelet activation, thus contributing to suppression of vascular intimal hyperplasia. Here, we characterize the effects of DS-SILY on both proliferative and quiescent human SMCs to evaluate the potential impact of DS-SILY-SMC interaction on restenosis, and further characterize in vivo platelet interactions. DS-SILY decreased proliferative SMC proliferation and pro-inflammatory cytokine secretion in vitro in a concentration dependent manner as compared to untreated controls. The addition of DS-SILY to in vitro SMC cultures decreased SMC migration and protein synthesis by 95% and 37%, respectively. Furthermore, DS-SILY decreased platelet activation, as well as reduced neointimal hyperplasia by 60%, in vivo using Ossabaw swine. These results indicate that DS-SILY demonstrates multiple biological activities that may all synergistically contribute to an improved treatment paradigm for balloon angioplasty.

Modification of native collagen with cell‐adhesive peptide to promote RPE cell attachment on Bruch's membrane

Current efforts to reverse loss of visual function due to Age-related Macular Degeneration point to the restoration of the Retinal Pigment Epithelial (RPE) layer. Restoration of the RPE layer involves replacing lost RPE cells as well as addressing the degeneration of the underlying Bruchs membrane (BM). To advance the potential of using donor BM, we present a strategy to achieve specific and controllable modification of the inner collagenous layer (ICL) of the Bruchs membrane. In particular, interaction between a collagen binding peptide (CBP) sequence with exposed collagen fibers on the ICL surface is utilized to anchor bioactive molecules. Here, a cell-adhesion sequence is added to the collagen binding sequence to promote attachment and survival of ARPE-19. First, the binding specificity of the CBP sequence is verified with a fluorescent binding assay. Subsequently, the effect of modification using the peptide is studied qualitatively using confocal fluorescent imaging and quantitatively through a cell proliferation assay. Results of these experiments indicate that the peptide sequence binds specifically to collagen fibers. Additionally, modification using the peptide enhanced cell adhesion, allowing large uniform cell networks to be formed on the surface. Furthermore, modification with the peptide also delayed the onset of apoptosis on adherent cells.

Glycan Therapeutics: Resurrecting an Almost Pharma-Forgotten Drug Class

Despite their enormous potential, glycans as therapeutics yet remain a widely untapped drug class. This overview shares the viewpoint that glycans have been aptly termed the “dark matter” of biology and have thus been largely ignored for decades. Provided herein is a background on the multiple structures and functions of glycan therapeutics, and focuses on examples and case studies of the glycan therapeutics in clinical use or in a clinical development. Perspectives on various hurdles are also provided, such as regulatory or scientific messaging and how these can influence the clinical development of this drug category. Finally some of the necessary changes in perception, education, and research infrastructure for continued support and advancement of this promising category of therapeutics are described.

Incorporation of a decorin biomimetic enhances the mechanical properties of electrochemically aligned collagen threads

Damaged tendons often do not heal completely and lack full functionality. Tissue engineering employing collagen based biomaterials is a viable option to repair damaged tendons. However, most existing constructs lack the desired mechanical strength needed to reconstruct such load bearing tissues. We have previously reported a novel methodology to synthesize highly ordered electrochemically aligned collagen (ELAC) threads that are mechanically stronger and more amenable to cell migration compared to randomly oriented collagen constructs. While the ELAC mimics the orientational anisotropy of tendon it can be further improved by the incorporation of small leucine rich proteoglycans like decorin. Decorin consists of a protein core that binds to collagen and a glycosaminoglycan (GAG) chain. The GAG chains of adjacent collagen fibrils associate with one another to form crosslinks and are suggested to enhance the mechanical properties of tendon by allowing fibrillar slippage. Based on the structure of natural decorin, we have previously synthesized a novel peptidoglycan (DS-SILY) containing a collagen binding peptide (SILY) and a dermatan sulfate (DS) GAG chain. DS-SILY mimics decorin both structurally and functionally. In this study, we investigated the effects of the incorporation of DS-SILY on the mechanical properties and structural organization of ELAC threads by monotonic mechanical testing, swelling ratio and differential scanning calorimetry.Copyright