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Osteoarthritis is characterized by enzymatic breakdown of the articular cartilage via the disruption of chondrocyte homeostasis, ultimately resulting in the destruction of the articular surface. Decades of research have highlighted the importance of inflammation in osteoarthritis progression, with inflammatory cytokines shifting resident chondrocytes into a pro-catabolic state. Inflammation can result in poor outcomes for cells implanted for cartilage regeneration. Therefore, a method to promote the growth of new cartilage and protect the implanted cells from the pro-inflammatory cytokines found in the joint space is required. In this study, we fabricate two gel types: polymer network hydrogels composed of chondroitin sulfate and hyaluronic acid, glycosaminoglycans (GAGs) known for their anti-inflammatory and prochondrogenic activity, and interpenetrating networks of GAGs and collagen I. Compared to a collagen-only hydrogel, which does not provide an anti-inflammatory stimulus, chondrocytes in GAG hydrogels result in reduced production of pro-inflammatory cytokines and enzymes as well as preservation of collagen II and aggrecan expression. Overall, GAG-based hydrogels have the potential to promote cartilage regeneration under pro-inflammatory conditions. Further, the data have implications for the use of GAGs to generally support tissue engineering in pro-inflammatory environments.

Despite advances in breast cancer treatment, there remains a need for local management of noninvasive, low-grade ductal carcinoma in situ (DCIS). These focal lesions are well suited for local intraductal treatment. Intraductal administration supported target site drug retention, improved efficacy, and reduced systemic exposure. Here, we used a poly(N-isopropyl acrylamide, pNIPAM) nanoparticle delivery system loaded with cytotoxic piplartine and an MAPKAP Kinase 2 inhibitor (YARA) for this purpose. For tumor environment targeting, a collagen-binding peptide SILY (RRANAALKAGELYKSILYGSG-hydrazide) was attached to pNIPAM nanoparticles, and the nanoparticle diameter, zeta potential, drug loading, and release were assessed. The system was evaluated for cytotoxicity in a 2D cell culture and 3D spheroids. In vivo efficacy was evaluated using a chemical carcinogenesis model in female Sprague-Dawley rats. Nanoparticle delivery significantly reduced the IC50 of piplartine (4.9 times) compared to the drug in solution. The combination of piplartine and YARA in nanoparticles further reduced the piplartine IC50 (~15 times). Treatment with these nanoparticles decreased the in vivo tumor incidence (5.2 times). Notably, the concentration of piplartine in mammary glands treated with nanoparticles (35.3 ± 22.4 μg/mL) was substantially higher than in plasma (0.7 ± 0.05 μg/mL), demonstrating targeted drug retention. These results indicate that our nanocarrier system effectively reduced tumor development with low systemic exposure.

The popularity of Glycosaminoglycans (GAGs) in drug delivery systems has grown as their innate ability to sequester and release charged molecules makes them adept in the controlled release of therapeutics. However, peptide therapeutics have been relegated to synthetic, polymeric systems, despite their high specificity and efficacy as therapeutics because they are rapidly degraded in vivo when not encapsulated. We present a GAG-based nanoparticle system for the easy encapsulation of cationic peptides, which offers control over particle diameter, peptide release behavior, and swelling behavior, as well as protection from proteolytic degradation, using a singular, organic polymer and no covalent linkages. These nanoparticles can encapsulate cargo with a particle diameter range spanning 130-220 nm and can be tuned to release cargo over a pH range of 4.5 to neutral through the modulation of the degree of sulfation and the molecular weight of the GAG. This particle system also confers better in vitro performance than the unencapsulated peptide via protection from enzymatic degradation. This method provides a facile way to protect therapeutic peptides via the inclusion of the presented binding sequence and can likely be expanded to larger, more diverse cargo as well, abrogating the complexity of previously demonstrated systems while offering broader tunability.

In this study, we developed a novel hybrid collagen-binding nanocarrier for potential intraductal administration and local breast cancer treatment. The particles were formed by the encapsulation of nanostructured lipid carriers (NLCs) containing the cytotoxic drug paclitaxel within a shell of poly(N-isopropylacrylamide) (pNIPAM), and were functionalized with SILY, a peptide that binds to collagen type I (which is overexpressed in the mammary tumor microenvironment) to improve local retention and selectivity. The encapsulation of the NLCs in the pNIPAM shell increased nanoparticle size by approximately 140 nm, and after purification, a homogeneous system of hybrid nanoparticles (∼96%) was obtained. The nanoparticles exhibited high loading efficiency (

Implantable vascular devices are widely used in clinical treatments for various vascular diseases. However, current approved clinical implantable vascular devices generally have high failure rates primarily due to their surface lacking inherent functional endothelium. Here, inspired by the pathological mechanisms of vascular device failure and physiological functions of native endothelium, we developed a new generation of bioactive parylene (poly(p-xylylene))-based conformal coating to address these challenges of the vascular devices. This coating used a polyethylene glycol (PEG) linker to introduce an endothelial progenitor cell (EPC) specific binding ligand LXW7 (cGRGDdvc) onto the vascular devices for preventing platelet adhesion and selectively capturing endogenous EPCs. Also, we confirmed the long-term stability and function of this coating in human serum. Using two vascular disease-related large animal models, a porcine carotid artery interposition model and a porcine carotid artery-jugular vein arteriovenous graft model, we demonstrated that this coating enabled rapid generation of self-renewable "living" endothelium on the blood contacting surface of the expanded polytetrafluoroethylene (ePTFE) grafts after implantation. We expect this easy-to-apply conformal coating will present a promising avenue to engineer surface properties of "off-the-shelf" implantable vascular devices for long-lasting performance in the clinical settings.

A feature of severe COVID-19 is the onset of an acute and intense systemic inflammatory response referred to as the "cytokine storm". The cytokine storm is characterized by high serum levels of inflammatory cytokines and the subsequent transport of inflammatory cells to damaging levels in vital organs (e.g., myocarditis). Immune trafficking and its effect on underlying tissues (e.g., myocardium) are challenging to observe at a high spatial and temporal resolution in mouse models. In this study, we created a vascularized organ-on-a-chip system to mimic cytokine storm-like conditions and tested the effectiveness of a novel multivalent selectin-targeting carbohydrate conjugate (composed of DS - dermatan sulfate and IkL - a selectin-binding peptide, termed DS-IkL) in blocking infiltration of polymorphonuclear leukocytes (PMN). Our data shows that cytokine storm-like conditions induce endothelial cells to produce additional inflammatory cytokines and facilitate infiltration of PMNs into tissue. Treatment of tissues with DS-IkL (60 μM) reduced PMN accumulation in the tissue by >50%. We then created cytokine storm-like conditions in a vascularized cardiac tissue-chip and found that PMN infiltration increases the spontaneous beating rate of the cardiac tissue, and this effect is eliminated by treatment with DS-IkL (60 μM). In summary, we demonstrate the utility of an organ-on-a-chip platform to mimic COVID-19 related cytokine storm and that blocking leukocyte infiltration with DS-IkL could be a viable strategy to mitigate associated cardiac complications.

Electrically conductive biomaterials direct cell behavior by capitalizing on the effect of bioelectricity in tissue homeostasis and healing. Many studies have leveraged conductive biomaterials to influence cells and improve tissue healing, even in the absence of external stimulation. However, most studies using electroactive materials neglect characterizing how the inclusion of conductive additives affects the material's mechanical properties, and the interplay between substrate electrical and mechanical properties on cell behavior is poorly understood. Furthermore, mechanisms dictating how electrically conductive materials affect cell behavior in the absence of external stimulation are not explicit. In this study, we developed a mechanically and electrically tunable conductive hydrogel using agarose and the conductive polymer PEDOT:PSS. Under certain conditions, we observed that the hydrogel physical and electrical properties were decoupled. We then seeded human mesenchymal stromal cells (MSCs) onto the hydrogels and observed enhanced adhesion and spreading of MSCs on conductive substrates, regardless of the hydrogel mechanical properties, and despite the gels having no cell-binding sites. To explain this observation, we measured protein interaction with the gels and found that charged proteins adsorbed significantly more to conductive hydrogels. These data demonstrate that conductivity promotes cell adhesion, likely by facilitating increased adsorption of proteins associated with cell binding, providing a better understanding of the mechanism of action of electrically conductive materials.

Many endothelial complications, whether from surgical or pathological origins, can result in the denudation of the endothelial layer and the exposure of collagen. Exposure of collagen results in the activation of platelets, leading to thrombotic and inflammatory cascades that ultimately result in vessel stenosis. We have previously reported the use of peptide-GAG compounds to target exposed collagen following endothelial injury. In this paper we optimize the spacer sequence of our collagen binding peptide to increase its conjugation to GAG backbones and increase the peptide-GAG collagen binding affinity by increasing peptide C-terminal cationic charge. Furthermore, we demonstrate the use of these molecules to inhibit platelet activation through collagen blocking, as well as their localization to exposed vascular collagen following systemic delivery. Altogether, optimization of peptide sequence and linkage chemistry can allow for increased conjugation and function, having implications for glycoconjugate use in other clinical applications.

Significant progress has been made in designing bone materials capable of directing endogenous cells to promote vascularized bone regeneration. However, current strategies lack regulation of the specific endogenous cell populations for vascularized bone regeneration, thus leading to adverse tissue formation and decreased regenerative efficiency. Here, we engineered a biomaterial to regulate endogenous cell adhesion and promote vascularized bone regeneration. The biomaterial works by presenting two synthetic ligands, LLP2A and LXW7, explicitly targeting integrins α4β1 and αvβ3, respectively, expressed on the surfaces of the cells related to bone formation and vascularization, such as mesenchymal stem cells (MSCs), osteoblasts, endothelial progenitor cells (EPCs), and endothelial cells (ECs). In vitro, the LLP2A/LXW7 modified biomaterial improved the adhesion of MSCs, osteoblasts, EPCs, and ECs via integrin α4β1 and αvβ3, respectively. In an adult rat calvarial bone defect model, the LLP2A/LXW7 modified biomaterial enhanced bone formation and vascularization by synergistically regulating endogenous cells with osteogenic and angiogenic potentials, such as DLX5+ cells, osteocalcin+ cells, CD34+/CD45- cells and CD31+ cells. In a fetal sheep spinal bone defect model, the LLP2A/LXW7 modified biomaterial augmented bone formation and vascularization without any adverse effects. This innovative biomaterial offers an off-the-shelf, easy-to-use, and biologically safe product suitable for vascularized bone regeneration in both fetal and adult disease environments.

The lack of vascularization associated with deep burns delays the construction of wound beds, increases the risks of infection, and leads to the formation of hypertrophic scars or disfigurement. To address this challenge, we have fabricated a multi-functional pro-angiogenic molecule by grafting integrin αvβ3 ligand LXW7 and collagen-binding peptide (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY (LDS), and further employed this to functionalize collagen-based Integra scaffolds. Using a large deep burn wound model in C57/BLK6 mice (8-10 weeks old, 26-32g, n = 39), we demonstrated that LDS-modified collagen-based Integra scaffolds loaded with endothelial cells (ECs) accelerate wound healing rate, re-epithelialization, vascularization, and collagen deposition. Specifically, a 2 cm × 3 cm full-thickness skin burn wound was created 48 h after the burn, and then wounds were treated with four groups of different dressing scaffolds, including Integra + ECs, Integra + LDS, and Integra + LDS + ECs with Integra-only as the control. Digital photos were taken for wound healing measurement on post-treatment days 1, 7, 14, 21, 28, and 35. Post-treatment photos revealed that treatment with the Intgera + LDS + ECs scaffold exhibited a higher wound healing rate in the proliferation phase. Histology results showed significantly increased re-epithelialization, increased collagen deposition, increased thin and mixed collagen fiber content, increased angiogenesis, and shorter wound length within the Integra + LDS + ECs group at Day 35. On Day 14, the Integra + LDS + ECs group showed the same trend. The relative proportions of collagen changed from Day 14 to Day 35 in the Integra + LDS + ECs and Integra + ECs groups demonstrated decreased thick collagen fiber deposition and greater thin and mixed collagen fiber deposition. LDS-modified Integra scaffolds represent a promising novel treatment to accelerate deep burn wound healing, thereby potentially reducing the morbidity associated with open burn wounds. These scaffolds can also potentially reduce the need for autografting and morbidity in patients with already limited areas of harvestable skin.

Diabetic ischemic wound treatment remains a critical clinical challenge. Neovascularization plays a significant role in wound healing during all stages of the tissue repair process. Strategies that enhance angiogenesis and neovascularization and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic conditions. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvβ3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates vascular endothelial growth factor (VEGF) receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a multi-functional pro-angiogenic molecule by grafting LXW7 and collagen-binding peptides (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY, and further employed this multi-functional molecule to functionalize collagen-based extracellular matrix (ECM) scaffolds. We confirmed that LXW7-DS-SILY modification significantly promoted EPC attachment and growth on the ECM scaffolds in vitro and supported EPC survival in vivo in the ischemic environment. When applied in an established Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model, LXW7-DS-SILY-functionalized ECM scaffolds loaded with EPCs significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.

Adding chondroitin sulfate (CS) to collagen scaffolds has been shown to improve the outcomes for articular cartilage tissue engineering. Instead of physical entrapment or chemical crosslinking of CS within a scaffold, this study investigated the use of CS with attached collagen-binding peptides (termed CS-SILY). This method better recapitulates the aspects of native cartilage while retaining CS within a collagen type I and II blend (Col I/II) hydrogel. CS retention, average fibril diameter, and mechanical properties were altered by varying the number of SILY peptides attached to the CS backbone. When mesenchymal stromal cells (MSCs) were encapsulated within the scaffolds, the addition of CS-SILY molecules resulted in higher sulfated glycosaminoglycan production, and these results suggest that CS-SILY promotes MSC differentiation into chondrocytes. Taken together, our study shows the promise of adding a CS-SILY molecule to a Col I/II hydrogel with encapsulated MSCs to promote cartilage repair.

AimsOne of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardioprotective effects of DS-IkL after acute myocardial infarction.Methods and resultsIn vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 min of left anterior descending coronary artery ligation-induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischaemia/reperfusion of the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischaemia/reperfusion and 24 h later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls.ConclusionsOur findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.

Although stem cell-derived extracellular vesicles (EVs) have remarkable therapeutic potential for various diseases, the therapeutic efficacy of EVs is limited due to their degradation and rapid diffusion after administration, hindering their translational applications. Here, we developed a new generation of collagen-binding EVs, by chemically conjugating a collagen-binding peptide SILY to EVs (SILY-EVs), which were designed to bind to collagen in the extracellular matrix (ECM) and form an EV-ECM complex to improve EVs' in situ retention and therapeutic efficacy after transplantation. Methods: SILY was conjugated to the surface of mesenchymal stem/stromal cell (MSC)-derived EVs by using click chemistry to construct SILY-EVs. Nanoparticle tracking analysis (NTA), ExoView analysis, cryogenic electron microscopy (cryo-EM) and western-blot analysis were used to characterize the SILY-EVs. Fluorescence imaging (FLI), MTS assay, ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to evaluate the collagen binding and biological functions of SILY-EVs in vitro. In a mouse hind limb ischemia model, the in vivo imaging system (IVIS), laser doppler perfusion imaging (LDPI), micro-CT, FLI and RT-qPCR were used to determine the SILY-EV retention, inflammatory response, blood perfusion, gene expression, and tissue regeneration. Results:In vitro, the SILY conjugation significantly enhanced EV adhesion to the collagen surface and did not alter the EVs' biological functions. In the mouse hind limb ischemia model, SILY-EVs presented longer in situ retention, suppressed inflammatory responses, and significantly augmented muscle regeneration and vascularization, compared to the unmodified EVs. Conclusion: With the broad distribution of collagen in various tissues and organs, SILY-EVs hold promise to improve the therapeutic efficacy of EV-mediated treatment in a wide range of diseases and disorders. Moreover, SILY-EVs possess the potential to functionalize collagen-based biomaterials and deliver therapeutic agents for regenerative medicine applications.

Glycosaminoglycans (GAGs), such as hyaluronic acid (HA) and chondroitin sulfate (CS), have seen widespread adoption as components of tissue engineering scaffolds because of their potent bioactive properties and ease of chemical modification. However, modification of the biopolymers will impair biological recognition of the GAG and reduce the bioactive properties of the material. In this work, we studied how the degree of thiolation of HA and CS, along with other key hydrogel design parameters, affected the physical and bioactive properties of the bulk hydrogel. Although properties, such as the HA molecular weight, did not have a major effect, increasing the degree of thiolation of both HA and CS decreased their biorecognition in experimental analogues for cell/matrix remodeling and binding. Furthermore, combining HA and CS into dual polymer network hydrogels also modulated the physical and bioactive properties, as seen with differences in gel stiffness, degradation rate, and encapsulated cell viability.

Hydrogels have been widely investigated in biomedical fields due to their similar physical and biochemical properties to the extracellular matrix (ECM). Collagen and hyaluronic acid (HA) are the main components of the ECM in many tissues. As a result, hydrogels prepared from collagen and HA hold inherent advantages in mimicking the structure and function of the native ECM. Numerous studies have focused on the development of collagen and HA hydrogels and their biomedical applications. In this extensive review, we provide a summary and analysis of the sources, features, and modifications of collagen and HA. Specifically, we highlight the fabrication, properties, and potential biomedical applications as well as promising commercialization of hydrogels based on these two natural polymers.

Joint trauma results in the production of inflammatory cytokines that stimulate the secretion of catabolic enzymes, which degrade articular cartilage. Molecular fragments of the degraded articular cartilage further stimulate inflammatory cytokine production, with this process eventually resulting in post-traumatic osteoarthritis (PTOA). The loss of matrix component aggrecan occurs early in the progression of PTOA and results in the loss of compressive stiffness in articular cartilage. Aggrecan is highly sulfated, associates with hyaluronic acid (HA), and supports the compressive stiffness in cartilage. Presented here, we conjugated the HA-binding peptide GAHWQFNALTVRGSG (GAH) to anionic nanoparticles (hNPs). Nanoparticles conjugated with roughly 19 GAH peptides, termed 19 GAH-hNP, bound to HA in solution and increased the dynamic viscosity by 94.1% compared to an HA solution treated with unconjugated hNPs. Moreover, treating aggrecan-depleted (AD) cartilage explants with 0.10 mg of 19 GAH-hNP restored the cartilage compressive stiffness to healthy levels six days after a single nanoparticle treatment. Treatment of AD cartilage with 0.10 mg of 19 GAH-hNP inhibited the degradation of articular cartilage. Treated AD cartilage had 409% more collagen type II and 598% more GAG content than untreated-AD explants. The 19 GAH-hNP therapeutic slowed ECM degradation in AD cartilage explants, restored the compressive stiffness of damaged cartilage, and showed promise as a localized treatment for PTOA.

Bioelectricity plays an important role in cell behavior and tissue modulation, but is understudied in tissue engineering research. Endogenous electrical signaling arises from the transmembrane potential inherent to all cells and contributes to many cell behaviors, including migration, adhesion, proliferation, and differentiation. Electrical signals are also involved in tissue development and repair. Synthetic and natural conductive materials are under investigation for leveraging endogenous electrical signaling cues in tissue engineering applications due to their ability to direct cell differentiation, aid in maturing electroactive cell types, and promote tissue functionality. In this review, we provide a brief overview of bioelectricity and its impact on cell behavior, report recent literature using conductive materials for tissue engineering, and discuss opportunities within the field to improve experimental design when using conductive substrates.

Glycosaminoglycans are native components of the extracellular matrix that drive cell behavior and control the microenvironment surrounding cells, making them promising therapeutic targets for a myriad of diseases. Recent studies have shown that recapitulation of cell interactions with the extracellular matrix are key in tissue engineering, where the aim is to mimic and regenerate endogenous tissues. Because of this, incorporation of glycosaminoglycans to drive stem cell fate and promote cell proliferation in engineered tissues has gained increasing attention. This review summarizes the role glycosaminoglycans can play in tissue engineering and the recent advances in their use in these constructs. We also evaluate the general trend of research in this niche and provide insight into its future directions.

Glycosaminoglycans are native components of the extracellular matrix that drive cell behavior and control the microenvironment surrounding cells, making them promising therapeutic targets for a myriad of diseases. Recent studies have shown that recapitulation of cell interactions with the extracellular matrix are key in tissue engineering, where the aim is to mimic and regenerate endogenous tissues. Because of this, incorporation of glycosaminoglycans to drive stem cell fate and promote cell proliferation in engineered tissues has gained increasing attention. This review summarizes the role glycosaminoglycans can play in tissue engineering and the recent advances in their use in these constructs. We also evaluate the general trend of research in this niche and provide insight into its future directions.

Collagen, the most abundant protein in mammals, has garnered the interest of scientists for over 50 years. Its ubiquitous presence in all body tissues combined with its excellent biocompatibility has led scientists to study its potential as a biomaterial for a wide variety of biomedical applications with a high degree of success and widespread clinical approval. More recently, in order to increase their tunability and applicability, collagen hydrogels have frequently been co-polymerized with other natural and synthetic polymers. Of special significance is the use of bioactive glycosaminoglycans-the carbohydrate-rich polymers of the ECM responsible for regulating tissue homeostasis and cell signaling. This review covers the recent advances in the development of collagen-based hydrogels and collagen-glycosaminoglycan blend hydrogels for biomedical research. We discuss the formulations and shortcomings of using collagen in isolation, and the advantages of incorporating glycosaminoglycans (GAGs) in the hydrogels. We further elaborate on modifications used on these biopolymers for tunability and discuss tissue specific applications. The information presented herein will demonstrate the versatility and highly translational value of using collagen blended with GAGs as hydrogels for biomedical engineering applications.

Collagen and elastin proteins are major components of the extracellular matrix of many organs. The presence of collagen and elastin networks, and their associated properties, in different tissues have led scientists to study collagen and elastin composites for use in tissue engineering. In this study, we characterized physical, biochemical, and optical properties of gels composed of collagen and elastin blends. We demonstrated that the addition of varying amounts of elastin to the constructs alters collagen fibrillogenesis, D-banding pattern length, and storage modulus. However, the addition of elastin does not affect collagen fibril diameter. We also evaluated the autofluorescence properties of the different collagen and elastin blends with fluorescence lifetime imaging (FLIm). Autofluorescence emission showed a red shift with the addition of elastin to the hydrogels. The fluorescence lifetime values of the gels increased with the addition of elastin and were strongly correlated with the storage moduli measurements. These results suggest that FLIm can be used to monitor the gels' mechanical properties nondestructively. These collagen and elastin constructs, along with the FLIm capabilities, can be used to develop and study collagen and elastin composites for tissue engineering and regenerative medicine.

Osteoarthritis is a debilitating disease that results in pain and joint stiffness. Currently, steroidal and nonsteroidal anti-inflammatory drugs and supplements aimed at restoring lubrication to the affected joint are the most successful with respect to improving patient comfort. Due to the success in lubricating therapies, there exists a keen interest to develop better therapies that mimic how lubrication occurs naturally in the joint. Here we describe the results obtained using a chondroitin sulfate chain to which is conjugated peptides that bind to either hyaluronic acid (found in high concentrations in the synovial fluid) or collagen type II (present on the cartilage surface). Our study investigates the effect of binding to the cartilage surface and interacting with hyaluronic acid on lubrication at the cartilage surface. The results described here suggest that binding to the cartilage surface is critical to supporting lubrication and did not require the addition of hyaluronic acid to reduce friction.

AimsOne of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardio-protective effects of DS-IkL after acute myocardial infarction.Methods and resultsIn vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 minutes of left anterior descending coronary artery ligation induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischemia/reperfusion at the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischemia/reperfusion and 24 hours later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls.ConclusionsOur findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.Translational perspectiveCardiovascular disease remains the leading cause of mortality worldwide. Acute inflammation from myocardial infarction (MI) results in damaged endothelial cells, leading to the loss of glycocalyx. Here, we designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. DS-IkL limits neutrophil binding and platelet activation on inflamed endothelial cells. Treatment with DS-IkL in a preclinical model of MI reduces infarct size by preventing neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis. Our findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.

Severe fibrotic and thrombotic events permeate the healthcare system, causing suffering for millions of patients with inflammatory disorders. As late-state consequences of chronic inflammation, fibrosis and thrombosis are the culmination of pathological interactions of activated endothelium, neutrophils and platelets after vessel injury. Coupling of these three cell types ensures a pro-coagulant, cytokine-rich environment that promotes the capture, activation and proliferation of circulating immune cells and recruitment of key pro-fibrotic cell types such as myofibroblasts. As the first responders to sterile inflammatory injury, it is important to understand how endothelial cells, neutrophils and platelets help create this environment. There has been a growing interest in this intersection over the past decade that has helped shape the development of therapeutics to target these processes. Here, we review recent insights into how neutrophils, platelets and endothelial cells guide the development of pathological vessel repair that can also result in underlying tissue fibrosis. We further discuss recent efforts that have been made to translate this knowledge into therapeutics and provide perspective as to how a compound or combination therapeutics may be most efficacious when tackling fibrosis and thrombosis that is brought upon by chronic inflammation.

The native extracellular matrix (ECM) contains a host of matricellular proteins and bioactive factors that regulate cell behavior, and many ECM components have been leveraged to guide cell fate. However, the large size and chemical characteristics of these constituents complicate their incorporation into biomaterials without interfering with material properties, motivating the need for alternative approaches to regulate cellular responses. Mesenchymal stromal cells (MSCs) can promote osseous regeneration in vivo directly or indirectly through multiple means including (1) secretion of proangiogenic and mitogenic factors to initiate formation of a vascular template and recruit host cells into the tissue site or (2) direct differentiation into osteoblasts. As MSC behavior is influenced by the properties of engineered hydrogels, we hypothesized that the biochemical and biophysical properties of alginate could be manipulated to promote the dual contributions of encapsulated MSCs toward bone formation. We functionalized alginate with QK peptide to enhance proangiogenic factor secretion and RGD to promote adhesion, while biomechanical-mediated osteogenic cues were controlled by modulating viscoelastic properties of the alginate substrate. A 1:1 ratio of QK:RGD resulted in the highest levels of both proangiogenic factor secretion and mineralization in vitro. Viscoelastic alginate outperformed purely elastic gels in both categories, and this effect was enhanced by stiffness up to 20 kPa. Furthermore, viscoelastic constructs promoted vessel infiltration and bone regeneration in a rat calvarial defect over 12 weeks. These data suggest that modulating viscoelastic properties of biomaterials, in conjunction with dual peptide functionalization, can simultaneously enhance multiple aspects of MSC regenerative potential and improve neovascularization of engineered tissues.

Inflammation following joint trauma contributes to cartilage degradation and progression of post traumatic osteoarthritis (PTOA). Therefore, drug delivery vehicles that deliver effective anti-inflammatory treatments have the potential to prevent PTOA. We have developed solid and hollow, thermoresponsive nanoparticles for the controlled release of our anti-inflammatory MK2-inhibiting (MK2i) peptide for intra-articular injection to halt inflammation that contributes to the advancement of PTOA. This system exploits the thermosensitive characteristic of N-isopropyl acrylamide (NIPAm) to transition phases when passing through its lower critical solution temperature (LCST). The nanoparticles (NPs) swell below the LCST and constrict above it. Non-crosslinked poly(NIPAm) (pNIPAm), held above its LCST, formed hydrophobic cores around which shells composed of NIPAm, degradable crosslinker N, N'-bis (acryloyl) cystamine (BAC), sulfated 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), and acrylic acid (AAc) were polymerized. Removal of the non-crosslinked pNIPAm cores via diffusion produced thermosensitive, degradable nanoparticles with low density, or hollow, cores. The data presented here revealed low-density, termed hollow, nanoparticles (hNPs) load and release significantly more MK2i than solid nanoparticles (sNPs). Furthermore, drug loading below the LCST of NIPAm results in roughly 2.5 times more therapeutic encapsulation compared to loading particles in their constricted state. Hollow nanoparticles increase drug loading compared to solid nanoparticles, are taken up into chondrocytes within 24 h, cleared from the cells within 6 days, significantly decrease the secretion of the proinflammatory cytokine IL-6, and, via intra-articular injection, are successfully delivered into the joint space of rats. The peptide loaded nanoparticles provide a reproducible platform for intra-articular delivery of therapeutics.

Establishing and maintaining a healthy endothelium on vascular and intravascular devices is crucial for the prevention of thrombosis and stenosis. Generating a biofunctional surface on vascular devices to recruit endothelial progenitor cells (EPCs) and endothelial cells (ECs) has proven efficient in promoting in situ endothelialization. However, molecules conventionally used for EPC/EC capturing generally lack structural stability, capturing specificity, and biological functionalities, which have limited their applications. Discovery of effective, specific, and structurally stable EPC/EC capturing ligands is desperately needed. Using the high-throughput One-Bead One-Compound combinatorial library screening technology, we recently identified a disulfide cyclic octa-peptide LXW7 (cGRGDdvc), which possesses strong binding affinity and functionality to EPCs/ECs, weak binding to platelets, and no binding to inflammatory cells. Because LXW7 is cyclic and 4 out of the 8 amino acids are unnatural D-amino acids, LXW7 is highly proteolytically stable. In this study, we applied LXW7 to modify small diameter vascular grafts using a Click chemistry approach. In vitro studies demonstrated that LXW7-modified grafts significantly improved EPC attachment, proliferation and endothelial differentiation and suppressed platelet attachment. In a rat carotid artery bypass model, LXW7 modification of the small diameter vascular grafts significantly promoted EPC/EC recruitment and rapidly achieved endothelialization. At 6 weeks after implantation, LXW7-modified grafts retained a high patency of 83%, while the untreated grafts had a low patency of 17%. Our results demonstrate that LXW7 is a potent EPC/EC capturing and platelet suppressing ligand and LXW7-modified vascular grafts rapidly generate a healthy and stable endothelial interface between the graft surface and the circulation to reduce thrombosis and improve patency. STATEMENT OF SIGNIFICANCE: In this study, One-Bead One-Compound (OBOC) technology has been applied for the first time in discovering bioactive ligands for tissue regeneration applications. Current molecules used to modify artificial vascular grafts generally lack EPC/EC capturing specificity, biological functionalities and structural stability. Using OBOC technology, we identified LXW7, a constitutionally stable disulfide cyclic octa-peptide with strong binding affinity and biological functionality to EPCs/ECs, very weak binding to platelets and no binding to inflammatory cells. These characteristics are crucial for promoting rapid endothelialization to prevent thrombosis and improve patency of vascular grafts. LXW7 coating technology could be applied to a wide range of vascular and intravascular devices, including grafts, stents, cardiac valves, and catheters, where a "living" endothelium and healthy blood interface are needed.

Proteoglycans have emerged as biomacromolecules with important roles in matrix remodeling, homeostasis, and signaling in the past two decades. Due to their negatively charged glycosaminoglycan chains as well as distinct core protein structures, they interact with a variety of molecules, including matrix proteins, growth factors, cytokines and chemokines, pathogens, and enzymes. This led to the dawn of glycan therapies in the 20th century, but this research was quickly overshadowed by readily available DNA and protein-based therapies. The recent development of recombinant technology and advances in our understanding of proteoglycan function have led to a resurgence of these molecules as potential therapeutics. This review focuses on the recent preclinical efforts that are bringing proteoglycan research and therapies back to the forefront. Examples of studies using proteoglycan cores and mimetics have also been included to give the readers a perspective on the wide-ranging and extensive applications of these versatile molecules. Collectively, these advances are opening new avenues for targeting diseases at a molecular level, and providing avenues for the development of new and exciting treatments in regenerative medicine.

Endothelial cell (EC) transplantation via injectable collagen hydrogel has received much attention as a potential treatment for various vascular diseases. However, the therapeutic effect of transplanted ECs is limited by their poor viability, which partially occurs as a result of cellular apoptosis triggered by the insufficient cell-extracellular matrix (ECM) engagement. Integrin binding to the ECM is crucial for cell anchorage to the surrounding matrix, cell spreading and migration, and further activation of intracellular signaling pathways. Although collagen contains several different types of integrin binding sites, it still lacks sufficient specific binding sites for ECs. Previously, using one-bead one-compound (OBOC) combinatorial technology, we identified LXW7, an integrin αvβ3 ligand, which possessed a strong binding affinity to and enhanced functionality of ECs. In this study, to improve the EC-matrix interaction, we developed an approach to molecularly conjugate LXW7 to the collagen backbone, via a collagen binding peptide SILY, in order to increase EC specific integrin binding sites on the collagen hydrogel. Results showed that in the in vitro 2-dimensional (2D) culture model, the LXW7-treated collagen surface significantly improved EC attachment and survival and decreased caspase 3 activity in an ischemic-mimicking environment. In the in vitro 3-dimensional (3D) culture model, LXW7-modified collagen hydrogel significantly improved EC spreading, proliferation, and survival. In a mouse subcutaneous implantation model, LXW7-modified collagen hydrogel improved the engraftment of transplanted ECs and supported ECs to form vascular network structures. Therefore, LXW7-functionalized collagen hydrogel has shown promising potential to improve vascularization in tissue regeneration and may be used as a novel tool for EC delivery and the treatment of vascular diseases.

BackgroundGlaucoma is characterized by the progressive dysfunction and loss of retinal ganglion cells. Recent work in animal models suggests that a critical neuroinflammatory event damages retinal ganglion cell axons in the optic nerve head during ocular hypertensive injury. We previously demonstrated that monocyte-like cells enter the optic nerve head in an ocular hypertensive mouse model of glaucoma (DBA/2 J), but their roles, if any, in mediating axon damage remain unclear.MethodsTo understand the function of these infiltrating monocyte-like cells, we used RNA-sequencing to profile their transcriptomes. Based on their pro-inflammatory molecular signatures, we hypothesized and confirmed that monocyte-platelet interactions occur in glaucomatous tissue. Furthermore, to test monocyte function we used two approaches to inhibit their entry into the optic nerve head: (1) treatment with DS-SILY, a peptidoglycan that acts as a barrier to platelet adhesion to the vessel wall and to monocytes, and (2) genetic targeting of Itgam (CD11b, an immune cell receptor that enables immune cell extravasation).ResultsMonocyte specific RNA-sequencing identified novel neuroinflammatory pathways early in glaucoma pathogenesis. Targeting these processes pharmacologically (DS-SILY) or genetically (Itgam / CD11b knockout) reduced monocyte entry and provided neuroprotection in DBA/2 J eyes.ConclusionsThese data demonstrate a key role of monocyte-like cell extravasation in glaucoma and demonstrate that modulating neuroinflammatory processes can significantly lessen optic nerve injury.

Vocal fold scarring is the fibrotic manifestation of a variety of voice disorders, and is difficult to treat. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. However, major challenges remain in capturing the complexity of the native tissue and sustaining regeneration. We hypothesized that hydrogels with tunable viscoelastic properties that present relevant biological cues to cells might be better suited as therapeutics. Herein, we characterized the response of human vocal fold fibroblasts to four different biomimetic hydrogels: thiolated hyaluronan (HA) crosslinked with poly(ethylene glycol) diacrylate (PEGDA), HA-PEGDA with type I collagen (HA-Col I), HA-PEGDA with type III collagen (HA-Col III) and HA-PEGDA with type I and III collagen (HA-Col I-Col III). Collagen incorporation allowed for interpenetrating fibrils of collagen within the non-fibrillar HA network, which increased the mechanical properties of the hydrogels. The addition of collagen fibrils also reduced hyaluronidase degradation of HA and hydrogel swelling ratio. Fibroblasts encapsulated in the HA-Col gels adopted a spindle shaped fibroblastic morphology by day 7 and exhibited extensive cytoskeletal networks by day 21, suggesting that the incorporation of collagen was essential for cell adhesion and spreading. Cells remained viable and synthesized new DNA throughout 21 days of culture. Gene expression levels significantly differed between the cells encapsulated in the different hydrogels. Relative fold changes in gene expression of MMP1, COL1A1, fibronectin and decorin suggest higher degrees of remodeling in HA-Col I-Col III gels in comparison to HA-Col I or HA-Col III hydrogels, suggesting that the former may better serve as a natural biomimetic hydrogel for tissue engineering applications. STATEMENT OF SIGNIFICANCE: Voice disorders affect about 1/3rd of the US population and significantly reduce quality of life. Patients with vocal fold fibrosis have few treatment options. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. Various studies have used collagen or thiolated hyaluronan (HA) with gelatin as potential tissue engineering therapies. However, there is room for improvement in providing cells with more relevant biological cues that mimic the native tissue microenvironment and sustain regeneration. The present study introduces the use of type I collagen and type III collagen along with thiolated HA as a natural biomimetic hydrogel for vocal fold tissue engineering applications.

IntroductionThe glycocalyx is a layer of glycoproteins, proteoglycans and glycosaminoglycans that coats the luminal surface of most blood vessels. It effectively regulates adhesive interactions between leukocytes in flowing blood and the endothelium, where during inflammation, binding to E- and P-selectins and intercellular adhesion molecule-1 (ICAM-1) promotes cell tethering and arrest under shear flow.MethodsIn this study, we examine the targeting of E-selectin by an engineered peptide moiety bound to a dermatan sulfate backbone. We further investigate this conjugate, denoted as EC-SEAL, by observing its binding to inflamed endothelium, and quantifying its ability to modulate neutrophil-endothelium interactions.ResultsBinding data reveal that EC-SEAL recognizes domains on E-selectin, and to a lesser degree on P- and L-selectin, and ICAM-1. Further, EC-SEAL increases neutrophil rolling velocity, and decreases neutrophil arrest and migration on inflamed human microvascular endothelial cells under physiologically relevant flow conditions.ConclusionsWe conclude that simple targeting strategies can mimic glycocalyx function under inflammatory conditions, effectively reducing neutrophil recruitment.

The growing demand for tissue engineered vascular grafts (TEVG) motivates the development of optimized fabrication and monitoring procedures. Bioreactors which provide physiologically-relevant conditions are important for improving holistic TEVG properties and performance. Herein we describe a fiber-based intraluminal imaging system that allows for in situ assessment of vascular materials and re-cellularization processes inside a bioreactor by simultaneous and co-registered measurements of endogenous fluorescence lifetime and exogenous marker fluorescence intensity. The lumen of 6 vascular grafts (∼4 mm diameter) were scanned by reciprocally rotating a 41° angle polished multimode optical fiber inside a protective glass tube with outer diameter of 3 mm. Tubular bovine pericardium constructs were recellularized using enhanced Green Fluorescent Protein (eGFP) transfected cells in a custom bioreactor. The imaging system has resolved consistently the cellular autofluorescence from that of tissue matrix in situ based on the lifetime fluorescence properties of endogenous molecular species. The location of the re-cellularized area was validated by the eGFP emission. Current results demonstrate the potential of this system as a valuable tool in tissue engineering for in situ studies of cell-tissue interactions in cylindrical or other 3-dimensional structures.

Volumetric muscle loss (VML) occurs when skeletal muscle injury is too large for the body to fully self-repair. Typically, fibrotic tissue fills the void, which reduces muscle functionality and limb movement. Although a wide variety of natural and synthetic scaffolds have been studied with the purpose of providing the appropriate structural support, to date no scaffold has significantly restored muscle functionality after VML. Satellite cells, adult stem cells within the muscle capable of restoring smaller injuries, are sensitive to the stiffness and composition of the surrounding environment. Scaffolds that only address structural support are not sufficient to restore functionality and instead need to be designed to both promote satellite cell activation and prevent excessive fibroblast recruitment. The objective of this study was to design a scaffold that mimicked the regenerative environment and determine how the biomechanical properties differentially influence myogenic precursor and connective tissue cells. One of the main extracellular matrix (ECM) molecules upregulated during regeneration is hyaluronic acid (HA). Therefore, thiol-modified HA and poly(ethylene glycol) diacrylate hydrogels were generated and functionalized with peptides based on ECM known to influence regeneration, including fibronectin, laminin and tenascin-C. Scaffolds with different stiffness were created by varying HA content. The influence of HA stiffness and peptide functionalization on myogenic precursor and connective tissue cell proliferation, migration and gene expression was quantified. Our results indicated that HA hydrogels functionalized with the laminin peptide, IKVAV, show potential due to the enhanced promotion of myogenic cell behaviors including migration, proliferation and an increase in relevant transcription factors. STATEMENT OF SIGNIFICANCE: The goal of this study was to identify hyaluronic acid (HA) hydrogels with peptide and stiffness combinations that will direct muscle-derived cells towards regenerating phenotypes. While the interaction of skeletal muscle with RGD-functionalized HA hydrogels has been investigated, none of the other peptides described in this study had been used in the context of HA-based scaffolds and skeletal muscle-derived cells. Notably, the response of cells to variations in mechanics was dependent on ECM coating and lineage. The 3% HA functionalized with the laminin peptide, IKVAV, showed the most promise for future in vivo studies, as these hydrogels best promoted myoblast cell proliferation, attachment and spreading, enhanced migration over connective tissue cells and upregulated transcription factors associated with activated satellite cells.

Xenogeneic biomaterials contain biologically relevant extracellular matrix (ECM) composition and organization, making them potentially ideal surgical grafts and tissue engineering scaffolds. Defining the effect of ECM niche (e.g., basement membrane vs. non-basement membrane) on repopulating cell phenotype and function has important implications for use of xenogeneic biomaterials, particularly in vascular applications. We aim to understand how serous (i.e., basement membrane) versus fibrous (i.e., non-basement membrane) ECM niche of antigen-removed bovine pericardium (AR-BP) scaffolds influence human aortic endothelial cell (hAEC) adhesion, growth, phenotype, inflammatory response and laminin production. At low and moderate seeding densities hAEC proliferation was significantly increased on the serous side. Similarly, ECM niche modulated cellular morphology, with serous side seeding resulting in a more rounded aspect ratio and intact endothelial layer formation. At moderate seeding densities, hAEC production of human laminin was enhanced following serous seeding. Finally, inflammatory marker and pro-inflammatory cytokine expression decreased following long-term cell growth regardless of seeding side. This work demonstrates that at low and moderate seeding densities AR-BP sidedness significantly impacts endothelial cell growth, morphology, human laminin production, and inflammatory state. These findings suggest that ECM niche has a role in modulating response of repopulating recipient cells toward AR-BP scaffolds for vascular applications.

New techniques able to monitor the maturation of tissue engineered constructs over time are needed for a more efficient control of developmental parameters. Here, a label-free fluorescence lifetime imaging (FLIm) approach implemented through a single fiber-optic interface is reported for nondestructive in situ assessment of vascular biomaterials. Recellularization processes of antigen removed bovine pericardium scaffolds with endothelial cells and mesenchymal stem cells were evaluated on the serous and the fibrous sides of the scaffolds, 2 distinct extracellular matrix niches, over the course of a 7 day culture period. Results indicated that fluorescence lifetime successfully report cell presence resolved from extracellular matrix fluorescence. The recellularization process was more rapid on the serous side than on the fibrous side for both cell types, and endothelial cells expanded faster than mesenchymal stem cells on antigen-removed bovine pericardium. Fiber-based FLIm has the potential to become a nondestructive tool for the assessment of tissue maturation by allowing in situ imaging of intraluminal vascular biomaterials.

Vocal fold scarring is a common cause of dysphonia. Current treatments involving vocal fold augmentation do not yield satisfactory outcomes in the long term. Tissue engineering and regenerative medicine offer an attractive treatment option for vocal fold scarring, with the aim to restore the native extracellular matrix microenvironment and biomechanical properties of the vocal folds by inhibiting progression of scarring and thus leading to restoration of normal vocal function. Hyaluronic acid is a bioactive glycosaminoglycan responsible for maintaining optimum viscoelastic properties of the vocal folds and hence is widely targeted in tissue engineering applications. This review covers advances in hyaluronic acid-based vocal fold tissue engineering and regeneration strategies.

Targeted delivery of anti-inflammatory osteoarthritis treatments have the potential to significantly decrease undesirable systemic side effects and reduce required therapeutic dosage. Here we present a targeted, non-invasive drug delivery system to decrease inflammation in an osteoarthritis model. Hollow thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) nanoparticles have been synthesized via degradation of a N,N'-bis(acryloyl)cystamine (BAC) cross-linked core out of a non-degradable pNIPAM shell. Sulfated 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) was copolymerized in the shell to increase passive loading of an anti-inflammatory mitogen-activated protein kinase-activated protein kinase 2 (MK2)-inhibiting cell-penetrating peptide (KAFAK). The drug-loaded hollow nanoparticles were effective at delivering a therapeutically active dose of KAFAK to bovine cartilage explants, suppressing pro-inflammatory interleukin-6 (IL-6) expression after interleukin-1 beta (IL-1β) stimulation. This thermosensitive hollow nanoparticle system provides an excellent platform for the delivery of peptide therapeutics into highly proteolytic environments such as osteoarthritis.

Objectives/hypothesisPhysiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1.Study designIn vitro.MethodsCell metabolic activity of immortalized human vocal fold fibroblasts incubated in different medium combinations was confirmed with an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay. Fibroblasts were grown to confluence, and primary bronchial/tracheal epithelial cells suspended in coculture medium were seeded directly over the base layer of the fibroblasts. Cells were treated with transforming growth factor β1 (TGFβ1) to induce myofibroblast formation. Cell shape and position were confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (αSMA), and phenotype was confirmed by immunostaining for vimentin and E-cadherin.ResultsFibroblasts retain metabolic activity in coculture epithelial medium. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. αSMA expression was enhanced in TGFβ1-treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture.ConclusionsThese data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.Level of evidenceNA Laryngoscope, 127:E185-E192, 2017.

Non-specific cytotoxins, including paclitaxel and sirolimus analogues, currently utilized as anti-restenotic therapeutics, affect not only smooth muscle cells (SMCs) but also neighbouring vascular endothelial cells (ECs). These drugs inhibit the formation of an intact endothelium following vessel injury, thus emphasizing the critical need for new candidate therapeutics. Utilizing our in vitro models, including EC monolayers and both hyperplastic and quiescent EC-SMC co-cultures, we investigated the ability of DS-SILY20 , a decorin mimic, to promote EC health. DS-SILY20 increased EC proliferation and migration by 1.5- and 2-fold, respectively, which corresponded to increased phosphorylation of ERK-1/2. Interestingly, IL-6 secretion and the production of both E-selectin and P-selectin were reduced in the presence of 10 μm DS-SILY20 , even in the presence of the potent pro-inflammatory cytokine platelet-derived growth factor (PDGF). In hyperplastic and quiescent EC-SMC co-cultures, DS-SILY20 treatment reduced the secretion of IFNγ, IL-1β, IL-6 and TNFα, corresponding to a 23% decrease in p38 phosphorylation. E-selectin and P-selectin expression was further reduced following DS-SILY20 treatment in both co-culture models. These results indicate that DS-SILY20 promotes EC health and that this decorin mimic could serve as a potential therapeutic to promote vessel healing following percutaneous coronary intervention (PCI). Copyright © 2015 John Wiley & Sons, Ltd.

Endothelial cell (EC) dysfunction is associated with many disease states including deep vein thrombosis (DVT), chronic kidney disease, sepsis and diabetes. Loss of the glycocalyx, a thin glycosaminoglycan (GAG)-rich layer on the EC surface, is a key feature of endothelial dysfunction and increases exposure of EC adhesion molecules such as selectins, which are involved in platelet binding to ECs. Once bound, platelets cause thrombus formation and an increased inflammatory response. We have developed a GAG derived, selectin targeting anti-adhesive coating (termed EC-SEAL) consisting of a dermatan sulfate backbone and multiple selectin-binding peptides designed to bind to inflamed endothelium and prevent platelet binding to create a more quiescent endothelial state. Multiple EC-SEAL variants were evaluated and the lead variant was found to preferentially bind to selectin-expressing ECs and smooth muscle cells (SMCs) and inhibit platelet binding and activation in a dose-dependent manner. In an in vivo model of DVT, treatment with the lead variant resulted in reduced thrombus formation. These results indicate that EC-SEAL has promise as a potential therapeutic in the treatment of endothelial dysfunction.

Peripheral artery disease is an atherosclerotic stenosis in the peripheral vasculature that is typically treated via percutaneous transluminal angioplasty. Deployment of the angioplasty balloon damages the endothelial layer, exposing the underlying collagen and allowing for the binding and activation of circulating platelets which initiate an inflammatory cascade leading to eventual restenosis. Here, we report on collagen-binding sulfated poly(N-isopropylacrylamide) nanoparticles that are able to target to the denuded endothelium. Once bound, these nanoparticles present a barrier that reduces cellular and platelet adhesion to the collagenous surface by 67% in whole blood and 59% in platelet-rich plasma under biologically relevant shear rates. In vitro studies indicate that the collagen-binding nanoparticles are able to load and release therapeutic quantities of anti-inflammatory peptides, with the particles reducing inflammation in endothelial and smooth muscle cells by 30% and 40% respectively. Once bound to collagen, the nanoparticles increased endothelial migration while avoiding uptake by smooth muscle cells, indicating that they may promote regeneration of the damaged endothelium while remaining anchored to the collagenous matrix and locally releasing anti-inflammatory peptides into the injured area. Combined, these collagen-binding nanoparticles have the potential to reduce inflammation, and the subsequent restenosis, while simultaneously promoting endothelial regeneration following balloon angioplasty.Statement of significanceIn this manuscript, we present our work on the development and characterization of a novel temperature sensitive collagen-binding nanoparticle system. We demonstrate that when bound to a collagenous matrix, the nanoparticles are able to promote endothelial migration while avoiding cellular uptake. We also show that the nanoparticles are able to reduce inflammation via the release of anti-inflammatory peptides which, when combined with its ability to inhibit platelet binding, could lead to reduced intimal hyperplasia following balloon angioplasty. The drug delivery platform presented represents a unique dual therapy biomaterial wherein the nanoparticle itself plays a crucial role in the system's overall therapeutic potential while simultaneously releasing anti-inflammatory peptides.

Biomaterials that provide signals present in the native extracellular matrix have been proposed as scaffolds to support improved cartilage regeneration. This study harnesses the biological activity of collagen type II and the superior mechanical properties of collagen type I by characterizing gels made of collagen type I and II blends. The collagen blend hydrogels were able to incorporate both types of collagen and retained chondroitin sulfate and hyaluronic acid. Cryo-scanning electron microscopy images showed that the 3:1 ratio of collagen type I to type II gels had a lower void space percentage (36.4%) than the 1:1 gels (46.5%). The complex modulus was larger for the 3:1 gels (G* = 5.0 Pa) compared to the 1:1 gels (G* = 1.2 Pa). The 3:1 blend consistently formed gels with superior mechanical properties compared to the other blends and has the potential to be implemented as a scaffold for articular cartilage engineering.

Characterized by pain, cartilage degradation, and inflammation, osteoarthritis is often treated with anti-inflammatory therapies that provide short-term relief but can have adverse side effects; intra-articular drug delivery systems with controlled release of anti-inflammatory peptides using degradable poly(N-isopropylacrylamide) (pNIPAM) nanoparticles could prolong relief and minimize these side effects. Nanoparticles provide a biocompatible drug carrier that can protect encapsulated therapeutics from enzymatic degradation and increase payload delivery upon encountering a degradation stimulus. Here we demonstrate passive targeting of inflamed cartilage ex vivo by uptake of PEGylated pNIPAM nanoparticles with degradable disulfide crosslinks (abbreviated as NGPEGSS) into chondrocytes and subsequent intracellular release of an anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK). The KAFAK-loaded NGPEGSS treatment reduced ex vivo inflammation to a greater extent compared to its non-degradable counterparts. This study highlights a nanoparticle system that delivers therapeutics intracellularly with improved efficacy by triggered degradation and suppresses inflammation in multiple cell types within an inflamed joint.