Hydrogel foam dressings with angiogenic and immunomodulatory factors from mesenchymal stem cells.

Stem cell therapy and skin substitutes address the stalled healing of chronic wounds in order to promote wound closure; however, the high cost and regulatory hurdles of these treatments limit patient access. A low-cost method to induce bioactive healing has the potential to substantially improve patient care and prevent wound-induced limb loss. A previous study reported that bioactive factors derived from apoptotic-like mesenchymal stem cells (MSCs) demonstrated anti-inflammatory and proangiogenic effects and improved ischemic muscle regeneration. In this work, these MSC-derived bioactive factors were loaded into a hydrogel foam to harness immunomodulatory and angiogenic properties from MSC components to facilitate chronic wound healing without the high cost and translational challenges of cell therapies. After incorporation of bioactive factors, the hydrogel foam retained high absorbency, moisture retention, and target water vapor transmission rate. High loading efficiency was confirmed and release studies indicated that over 90% of loaded factors were released within 24 h. Ethylene oxide sterilization and 4-week storage did not affect the bioactive factor release profile or physical properties of the hydrogel foam dressing. Bioactivity retention of the released factors was also confirmed for as-sterilized, 4°C-stored, and -20°C-stored bioactive hydrogel foams as determined by relevant gene expression levels in treated pro-inflammatory (M1) macrophages. These results support the use of the bioactive dressings as an off-the-shelf product. Overall, this work reports a new method to achieve a first-line wound dressing with the potential to reduce persistent inflammation and promote angiogenesis in chronic wounds.

Injectable hydrogel electrodes as conduction highways to restore native pacing.

There is an urgent clinical need for a treatment regimen that addresses the underlying pathophysiology of ventricular arrhythmias, the leading cause of sudden cardiac death. The current report describes the design of an injectable hydrogel electrode and successful deployment in a pig model with access far more refined than any current pacing modalities allow. In addition to successful cardiac capture and pacing, analysis of surface ECG tracings and three-dimensional electroanatomic mapping revealed a QRS morphology comparable to native sinus rhythm, strongly suggesting the hydrogel electrode captures the deep septal bundle branches and Purkinje fibers. In an ablation model, electroanatomic mapping data demonstrated that the activation wavefront from the hydrogel reaches the mid-myocardium and endocardium much earlier than current single-point pacing modalities. Such uniform activation of broad swaths of tissue enables an opportunity to minimize the delayed myocardial conduction of heterogeneous tissue that underpins re-entry. Collectively, these studies demonstrate the feasibility of a new pacing modality that most closely resembles native conduction with the potential to eliminate lethal re-entrant arrhythmias and provide painless defibrillation.

Hydrogel-polyurethane fiber composites with enhanced microarchitectural control for heart valve replacement.

Polymeric heart valves offer the potential to overcome the limited durability of tissue based bioprosthetic valves and the need for anticoagulant therapy of mechanical valve replacement options. However, developing a single-phase material with requisite biological properties and target mechanical properties remains a challenge. In this study, a composite heart valve material was developed where an electrospun mesh provides tunable mechanical properties and a hydrogel coating confers an antifouling surface for thromboresistance. Key biological responses were evaluated in comparison to glutaraldehyde-fixed pericardium. Platelet and bacterial attachment were reduced by 38% and 98%, respectively, as compared to pericardium that demonstrated the antifouling nature of the hydrogel coating. There was also a notable reduction (59%) in the calcification of the composite material as compared to pericardium. A custom 3D-printed hydrogel coating setup was developed to make valve composites for device-level hemodynamic testing. Regurgitation fraction (9.6 ± 1.8%) and effective orifice area (1.52 ± 0.34 cm2 ) met ISO 5840-2:2021 requirements. Additionally, the mean pressure gradient was comparable to current clinical bioprosthetic heart valves demonstrating preliminary efficacy. Although the hemodynamic properties are promising, it is anticipated that the random microarchitecture will result in suboptimal strain fields and peak stresses that may accelerate leaflet fatigue and degeneration. Previous computational work has demonstrated that bioinspired fiber microarchitectures can improve strain homogeneity of valve materials toward improving durability. To this end, we developed advanced electrospinning methodologies to achieve polyurethane fiber microarchitectures that mimic or exceed the physiological ranges of alignment, tortuosity, and curvilinearity present in the native valve. Control of fiber alignment from a random fiber orientation at a normalized orientation index (NOI) 14.2 ± 6.9% to highly aligned fibers at a NOI of 85.1 ± 1.4%. was achieved through increasing mandrel rotational velocity. Fiber tortuosity and curvilinearity in the range of native valve features were introduced through a post-spinning annealing process and fiber collection on a conical mandrel geometry, respectively. Overall, these studies demonstrate the potential of hydrogel-polyurethane fiber composite as a heart valve material. Future studies will utilize the developed advanced electrospinning methodologies in combination with model-directed fabrication toward optimizing durability as a function of fiber microarchitecture.

The accumulation and growth of Pseudomonas aeruginosa on surfaces is modulated by surface mechanics via cyclic-di-GMP signaling.

Attachment of bacteria onto a surface, consequent signaling, and accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that surface mechanics may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of surface mechanics and modulation of accumulation in response to surface mechanics remain largely unknown. We use thin and thick hydrogels coated on glass to create composite materials with different mechanics (higher elasticity for thin composites; lower elasticity for thick composites) but with the same surface adhesivity and chemistry. The mechanical cue stemming from surface mechanics is elucidated using experiments with the opportunistic human pathogen Pseudomonas aeruginosa combined with finite-element modeling. Adhesion to thin composites results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to thick composites with identical surface chemistry. Using quantitative microscopy, we find that adhesion to thin composites also results in higher cyclic-di-GMP levels, which in turn result in lower motility and less detachment, and thus greater accumulation of bacteria on the surface than does adhesion to thick composites. Mechanics-dependent c-di-GMP production is mediated by the cell-surface-exposed protein PilY1. The biofilm lag phase, which is longer for bacterial populations on thin composites than on thick composites, is also mediated by PilY1. This study shows clear evidence that bacteria actively regulate differential accumulation on surfaces of different stiffnesses via perceiving varied mechanical stress and strain upon surface engagement.

Hydroxyapatite nanoparticle-modified porous bone grafts with improved cell attachment.

Emulsion-templated foams have displayed promise as injectable bone grafts; however, the use of a surfactant as an emulsifier resulted in relatively small pores and impedes cell attachment. Hydroxyapatite nanoparticles were explored as an alternative stabilizer to address these limitations. To this end, hydroxyapatite nanoparticles were first modified with myristic acid to generate the appropriate balance of hydrophobicity to stabilize a water-in-oil emulsion of neopentyl glycol diacrylate and 1,4-butanedithiol. In situ surface modification of the resulting foam with hydroxyapatite was confirmed with elemental mapping and transmission electron microscopy. Nanoparticle-stabilized foams displayed improved human mesenchymal stem cell viability (91 ± 5%) over surfactant-stabilized foams (23 ± 11%). Although the pore size was appropriate for bone grafting applications (115 ± 71 µm), the foams lacked the interconnected architecture necessary for cell infiltration. We hypothesized that a co-stabilization approach with both surfactant and nanoparticles could be used to achieve interconnected pores while maintaining improved cell attachment and larger pore sizes. A range of hydroxyapatite nanoparticle and surfactant concentrations were investigated to determine the effects on microarchitecture and cell behavior. By balancing these interactions, a co-stabilized foam was identified that possessed large, interconnected pores (108 ± 67 µm) and improved cell viability and attachment. The co-stabilized foam was then evaluated as an injectable bone graft including network formation, microscale integration with bone, push out strength, and compressive properties. Overall, this work demonstrated that in situ surface modification with nHA improved cell attachment while retaining desirable bone grafting features and injectability.

A user's guide to degradation testing of polyethylene glycol-based hydrogels: From in vitro to in vivo studies.

Poly(ethylene glycol) (PEG)-based hydrogels have gained significant attention in the field of biomedical applications due to their versatility and antifouling properties. Acrylate-derivatized PEG hydrogels (PEGDA) are some of the most widely studied hydrogels; however, there has been debate around the degradation mechanism and predicting resorption rates. Several factors influence the degradation rate of PEG hydrogels, including backbone and endgroup chemistry, macromer molecular weight, and polymer concentration. In addition to hydrogel parameters, it is necessary to understand the influence of biological and environmental conditions (e.g., pH and temperature) on hydrogel degradation. Rigorous methods for monitoring degradation in both in vitro and in vivo settings are also critical to hydrogel design and development. Herein, we provide guidance on tailoring PEG hydrogel chemistry to achieve target hydrolytic degradation kinetics for both resorbable and biostable applications. A detailed overview of accelerated testing methods and hydrogel degradation characterization is provided to aid researchers in experimental design and interpreting in vitro-in vivo correlations necessary for predicting hydrogel device performance.

PEG-Based Hydrogel Coatings: Design Tools for Biomedical Applications.

Device failure due to undesired biological responses remains a substantial roadblock in the development and translation of new devices into clinical care. Polyethylene glycol (PEG)-based hydrogel coatings can be used to confer antifouling properties to medical devices-enabling minimization of biological responses such as bacterial infection, thrombosis, and foreign body reactions. Application of hydrogel coatings to diverse substrates requires careful consideration of multiple material factors. Herein, we report a systematic investigation of two coating methods: (1) traditional photoinitiated hydrogel coatings; (2) diffusion-mediated, redox-initiated hydrogel coatings. The effects of method, substrate, and compositional variables on the resulting hydrogel coating thickness are presented. To expand the redox-based method to include high molecular weight macromers, a mechanistic investigation of the role of cure rate and macromer viscosity was necessary to balance solution infiltration and gelation. Overall, these structure-property relationships provide users with a toolbox for hydrogel coating design for a broad range of medical devices.

Interpenetrating network design of bioactive hydrogel coatings with enhanced damage resistance.

Bioactive hydrogel coatings offer a promising route to introduce sustained thromboresistance to cardiovascular devices without compromising bulk mechanical properties. Poly(ethylene glycol)-based hydrogels provide antifouling properties to limit acute thromobosis and incorporation of adhesive ligands can be used to promote endothelialization. However, conventional PEG-based hydrogels at stiffnesses that promote cell attachment can be brittle and prone to damage in a surgical setting, limiting their utility in clinical applications. In this work, we developed a durable hydrogel coating using interpenetrating networks of polyether urethane diacrylamide (PEUDAm) and poly(N-acryloyl glycinamide) (pNAGA). First, diffusion-mediated redox initiation of PEUDAm was used to coat electrospun polyurethane fiber meshes with coating thickness controlled by the immersion time. The second network of pNAGA was then introduced to enhance damage resistance of the hydrogel coating. The durability, thromboresistance, and bioactivity of the resulting multilayer grafts were then assessed. The IPN hydrogel coatings displayed resistance to surgically-associated damage mechanisms and retained the anti-fouling nature of PEG-based hydrogels as indicated by reduced protein adsorption and platelet attachment. Moreover, incorporation of functionalized collagen into the IPN hydrogel coating conferred bioactivity that supported endothelial cell adhesion. Overall, this conformable and durable hydrogel coating provides an improved approach for cardiovascular device fabrication with targeted biological activity.

Bacterial mechanosensing of surface stiffness promotes signaling and growth leading to biofilm formation by Pseudomonas aeruginosa.

The attachment of bacteria onto a surface, consequent signaling, and the accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that the stiffness of a surface may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of and response to surface stiffness are unknown. Furthermore, whether, and how, the surface stiffness impacts biofilm development, after initial accumulation, is not known. We use thin and thick hydrogels to create stiff and soft composite materials, respectively, with the same surface chemistry. Using quantitative microscopy, we find that the accumulation, motility, and growth of the opportunistic human pathogen Pseudomonas aeruginosa respond to surface stiffness, and that these are linked through cyclic-di-GMP signaling that depends on surface stiffness. The mechanical cue stemming from surface stiffness is elucidated using finite-element modeling combined with experiments - adhesion to stiffer surfaces results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to softer surfaces with identical surface chemistry. The cell-surface-exposed protein PilY1 acts as a mechanosensor, that upon surface engagement, results in higher cyclic-di-GMP levels, lower motility, and greater accumulation on stiffer surfaces. PilY1 impacts the biofilm lag phase, which is extended for bacteria attaching to stiffer surfaces. This study shows clear evidence that bacteria actively respond to different stiffness of surfaces where they adhere via perceiving varied mechanical stress and strain upon surface engagement.

High porosity PEG-based hydrogel foams with self-tuning moisture balance as chronic wound dressings.

A major challenge in chronic wound treatment is maintaining an appropriate wound moisture balance throughout the healing process. Wound dehydration hinders wound healing due to impeded molecule transport and cell migration with associated tissue necrosis. In contrast, wounds that produce excess fluid contain high levels of reactive oxygen species and matrix metalloproteases that impede cell recruitment, extracellular matrix reconstruction, and angiogenesis. Dressings are currently selected based on the relative amount of wound exudate with no universal dressing available that can maintain appropriate wound moisture balance to enhance healing. This work aimed to develop a high porosity poly(ethylene glycol) diacrylate hydrogel foam that can both rapidly remove exudate and provide self-tuning moisture control to prevent wound dehydration. A custom foaming device was used to vary hydrogel foam porosity from 25% to 75% by adjusting the initial air-to-solution volume ratio. Hydrogel foams demonstrated substantial improvements in water uptake volume and rate as compared to bulk hydrogels while maintaining similar hydration benefits with slow dehydration rates. The hydrogel foam with the highest porosity (~75%) demonstrated the greatest water uptake and rate, which outperformed commercial dressing products, Curafoam® and Silvercel®, in water absorption, moisture retention, and exudate management. Investigation of the water vapor transmission rates of each dressing at varied hydration levels was characterized and demonstrated the dynamic moisture-controlling capability of the hydrogel foam dressing. Overall, the self-tuning moisture control of this hydrogel foam dressing holds great promise to improve healing outcomes for both dry and exudative chronic wounds.

Wound swabs versus biopsies to detect methicillin resistant Staphylococcus aureus in experimental equine wounds.

OBJECTIVE: To compare: (1) the load and diversity of cultivatable bacterial species isolated from tissue biopsies with cultures from surface swabs, and (2) the ability of each technique to detect methicillin-resistant Staphylococcus aureus (MRSA) in a model of MRSA-infected equine wounds. STUDY DESIGN: Experimental in vivo study. ANIMALS: Three light-breed adult horses. METHODS: Four 2.5 × 2.5 cm full-thickness skin wounds were created on the dorsolateral aspect of each forelimb. Five days later, each wound was inoculated with a pure culture of MRSA (ATCC 43300). One hundred microlitres of 0, 5 × 108 , 5 × 109 or 5 × 1010 colony forming units (CFU)/ml was used to inoculate each wound. Surface swabs (Levine technique) and tissue biopsy samples (3 mm punch biopsy) were obtained at 2, 7, 14, and 21 days after inoculation. Quantitative aerobic culture was performed using routine clinical techniques. RESULTS: A similar bacterial profile was identified from the culture of each wound-sampling technique and there was moderate correlation (R = 0.49, P < .001) between the bacterial bioburdens. Agreement was fair (κ = 0.31; 95% CI, 0.129-0.505) between the sampling techniques in identification of MRSA. Methicillin-resistant Staphylococcus aureus was isolated more frequently (P = .016) from cultures of tissue biopsies (79%; 76/96) than from surface swabs (62%; 60/96). CONCLUSION: Bacterial load and diversity did not differ between sampling techniques but MRSA was detected more often from the cultures of tissue biopsies. CLINICAL SIGNIFICANCE: Tissue biopsy should be preferred to culture swab in wounds where MRSA is suspected.

Prokaryotic Collagen-Like Proteins as Novel Biomaterials.

Collagens are the major structural component in animal extracellular matrices and are critical signaling molecules in various cell-matrix interactions. Its unique triple helical structure is enabled by tripeptide Gly-X-Y repeats. Understanding of sequence requirements for animal-derived collagen led to the discovery of prokaryotic collagen-like protein in the early 2000s. These prokaryotic collagen-like proteins are structurally similar to mammalian collagens in many ways. However, unlike the challenges associated with recombinant expression of mammalian collagens, these prokaryotic collagen-like proteins can be readily expressed in E. coli and are amenable to genetic modification. In this review article, we will first discuss the properties of mammalian collagen and provide a comparative analysis of mammalian collagen and prokaryotic collagen-like proteins. We will then review the use of prokaryotic collagen-like proteins to both study the biology of conventional collagen and develop a new biomaterial platform. Finally, we will describe the application of Scl2 protein, a streptococcal collagen-like protein, in thromboresistant coating for cardiovascular devices, scaffolds for bone regeneration, chronic wound dressing and matrices for cartilage regeneration.

Model-Directed Design of Tissue Engineering Scaffolds.

Scaffold-based tissue engineering requires a resorbable scaffold that can restore function and guide regeneration. Recent advances in material fabrication have expanded our control of compositional and architectural features to approach the complexity of native tissue. However, iterative scaffold design to balance multiple design targets toward optimizing regenerative performance remains both challenging and time-consuming. The number of design parameter combinations for scaffold manufacturing to achieve target properties is nearly limitless. Although a trial-and-error experimental approach may lead to a favorable scaffold design, the time and costs associated with such an approach are enormous. Computational optimization approaches are well suited to sample such a multidimensional design space and streamline the identification of target scaffold parameters for experimental evaluation. In this computational biomechanics approach, target fabrication parameters are identified by using a computational model to iterate across design parameter combinations (input) and predict the resulting graft properties (output). Herein, we describe the three key stages of this model-directed scaffold design: (1) model development, verification, and validation; (2) in silico optimization; and (3) model-directed scaffold fabrication and testing. Although there are several notable examples that have demonstrated the potential of this approach, additional accurate and physiologically appropriate computational simulations are needed to expand its utility across the field. In addition, continued advances in material fabrication are needed to provide the requisite control and resolution to produce the model-directed design. Finally, and most importantly, active collaboration between experts in materials science and computational modeling are critical to realize the full potential of this approach to accelerate scaffold development.

PoreScript: Semi-automated pore size algorithm for scaffold characterization.

The scaffold pore size influences many critical physical aspects of tissue engineering, including tissue infiltration, biodegradation rate, and mechanical properties. Manual measurements of pore sizes from scanning electron micrographs using ImageJ/FIJI are commonly used to characterize scaffolds, but these methods are both time-consuming and subject to user bias. Current semi-automated analysis tools are limited by a lack of accessibility or limited sample size in their verification process. The work here describes the development of a new MATLAB algorithm, PoreScript, to address these limitations. The algorithm was verified using three common scaffold fabrication methods (e.g., salt leaching, gas foaming, emulsion templating) with varying pore sizes and shapes to demonstrate the versatility of this new tool. Our results demonstrate that the pore size characterization using PoreScript is comparable to manual pore size measurements. The PoreScript algorithm was further evaluated to determine the effect of user-input and image parameters (relative image magnification, pixel intensity threshold, and pore structure). Overall, this work validates the accuracy of the PoreScript algorithm across several fabrication methods and provides user-guidance for semi-automated image analysis and increased throughput of scaffold characterization.

Anisotropic elastic behavior of a hydrogel-coated electrospun polyurethane: Suitability for heart valve leaflets.

Although xenograft biomaterials have been used for decades in replacement heart valves, they continue to face multiple limitations, including limited durability, mineralization, and restricted design space due to their biological origins. These issues necessitate the need for novel replacement heart valve biomaterials that are durable, non-thrombogenic, and compatible with transcatheter aortic valve replacement devices. In this study, we explored the suitability of an electrospun poly(carbonate urethane) (ES-PCU) mesh coated with a poly(ethylene glycol) diacrylate (PEGDA) hydrogel as a synthetic biomaterial for replacement heart valve leaflets. In this material design, the mesh provides the mechanical support, while the hydrogel provides the required surface hemocompatibility. We conducted a comprehensive study to characterize the structural and mechanical properties of the uncoated mesh as well as the hydrogel-coated mesh (composite biomaterial) over the estimated operational range. We found that the composite biomaterial was functionally robust with reproducible stress-strain behavior within and beyond the functional ranges for replacement heart valves, and was able to withstand the rigors of mechanical evaluation without any observable damage. In addition, the composite biomaterial displayed a wide range of mechanical anisotropic responses, which were governed by fiber orientation of the mesh, which in turn, was controlled with the fabrication process. Finally, we developed a novel constitutive modeling approach to predict the mechanical behavior of the composite biomaterial under in-plane extension and shear deformation modes. This model identified the existence of fiber-fiber mechanical interactions in the mesh that have not previously been reported. Interestingly, there was no evidence of fiber-hydrogel mechanical interactions. This important finding suggests that the hydrogel coating can be optimized for hemocompatibility independent of the structural mechanical responses required by the leaflet. This initial study indicated that the composite biomaterial has mechanical properties well-suited for replacement heart valve applications and that the electrospun mesh microarchitecture and hydrogel biological properties can be optimized independently. It also reveals that the structural mechanisms contributing to the mechanical response are more complicated than what was previously established and paves the pathway for more detailed future studies.

A Prevascularized Polyurethane-Reinforced Fibrin Patch Improves Regenerative Remodeling in a Rat Right Ventricle Replacement Model.

Congenital heart defects (CHDs) affect 1 in 120 newborns in the United States. Surgical repair of structural heart defects often leads to arrhythmia and increased risk of heart failure. The laboratory has previously developed an acellular fibrin patch reinforced with a biodegradable poly(ether ester urethane) urea mesh that result in improved heart function when tested in a rat right ventricle wall replacement model compared to fixed pericardium. However, this patch does not drive significant neotissue formation. The patch materials are modified here and this patch is prevascularized with human umbilical vein endothelial cells and c-Kit+ human amniotic fluid stem cells. Rudimentary capillary-like networks form in the fibrin after culture of cell-encapsulated patches for 3 d in vitro. Prevascularized patches and noncell loaded patch controls are implanted onto full-thickness heart wall defects created in the right ventricle of athymic nude rats. Two months after surgery, defect repair with prevascularized patches results in improved heart function and the patched heart area exhibited greater vascularization and muscularization, less fibrosis, and increased M2 macrophage infiltration compared to acellular patches.

Reconstituting electrical conduction in soft tissue: the path to replace the ablationist.

Cardiac arrhythmias are a leading cause of morbidity and mortality in the developed world. A common mechanism underlying many of these arrhythmias is re-entry, which may occur when native conduction pathways are disrupted, often by myocardial infarction. Presently, re-entrant arrhythmias are most commonly treated with antiarrhythmic drugs and myocardial ablation, although both treatment methods are associated with adverse side effects and limited efficacy. In recent years, significant advancements in the field of biomaterials science have spurred increased interest in the development of novel therapies that enable restoration of native conduction in damaged or diseased myocardium. In this review, we assess the current landscape of materials-based approaches to eliminating re-entrant arrhythmias. These approaches potentially pave the way for the eventual replacement of myocardial ablation as a preferred therapy for such pathologies.

Engineering Toolbox for Systematic Design of PolyHIPE Architecture.

Polymerization of high internal phase emulsions (polyHIPEs) is a well-established method for the production of high porosity foams. Researchers are often regulated to using a time-intensive trial and error approach to achieve target pore architectures. In this work, we performed a systematic study to identify the relative effects of common emulsion parameters on pore architecture (mixing speed, surfactant concentration, organic phase viscosity, molecular hydrophobicity). Across different macromer chemistries, the largest magnitude of change in pore size was observed across surfactant concentration (~6 fold, 5-20 wt%), whereas changing mixing speeds (~4 fold, 500-2000 RPM) displayed a reduced effect. Furthermore, it was observed that organic phase viscosity had a marked effect on pore size (~4 fold, 6-170 cP) with no clear trend observed with molecular hydrophobicity in this range (logP = 1.9-4.4). The efficacy of 1,4-butanedithiol as a reactive diluent was demonstrated and provides a means to reduce organic phase viscosity and increase pore size without affecting polymer fraction of the resulting foam. Overall, this systematic study of the microarchitectural effects of these macromers and processing variables provides a framework for the rational design of polyHIPE architectures that can be used to accelerate design and meet application needs across many sectors.