Assessment of Silver Levels in a Closed-Incision Negative Pressure Therapy Dressing: In Vitro and In Vivo Study.

Objective: In recent years, reticulated open-cell foam-based closed-incision negative pressure therapy (ROCF-ciNPT) has shown effectiveness in management of various postoperative incisions. These dressings consist of a skin interface layer that absorbs fluid from the skin surface and reduces the potential for microbial colonization within the dressing by means of ionic silver. This study examines the ability of silver to reduce the bioburden within the dressing as well as the localized effect due to potential silver mobility. Approach: Ability of silver to reduce bioburden within the ROCF-ciNPT dressing was assessed using Staphylococcus aureus, Pseudomonas aeruginosa, and Candida spp. Furthermore, silver mobility was assessed using an in vitro skin model to study the zone of inhibition along with released silver quantification. Using a porcine model, diffusion of silver into blood and tissue was studied using emission spectrometry and histology. Results: Microbial growth in the ROCF-ciNPT dressing was significantly reduced (∼2.7-4.9 log reduction) compared to a silver-free negative control. No zone of inhibition was observed for microbial colonies for up to 7 days with minimal localized silver release (<5.5 ppm release). In vivo studies demonstrated no measurable concentration (<0.2 µg/g) of silver in the blood, urine, feces, kidney, and liver tissue biopsy. Innovation: This study provides an important insight into silver concentration and mobility within the ROCF-ciNPT dressing, given emerging concerns associated with potential silver cytotoxicity. Conclusion: These results indicate the concentration of silver (0.019% silver by weight) in the ROCF-ciNPT dressings has been adequate to reduce bioburden within the skin interface layer, while severely limiting the amount of silver leaching out.

Fast, irreversible modification of cysteines through strain releasing conjugate additions of cyclopropenyl ketones.

A method of cysteine alkylation using cyclopropenyl ketones is described. Due to the significant release of cyclopropene strain energy, reactions of thiols with cyclopropenyl ketones are both fast and irreversible and give rise to stable conjugate addition adducts. The resulting cyclopropenyl ketones have a low molecular weight and allow for simple attachment of amides via N-hydroxysuccinimide (NHS)-esters. While cyclopropenyl ketones do display slow background reactivity toward water, labeling by thiols is much more rapid. The reaction of a cyclopropenyl ketone with glutathione (GSH) proceeds with a rate of 595 M-1 s-1 in PBS at pH 7.4, which is considerably faster than α-halocarbonyl labeling reagents, and competitive with maleimide/thiol couplings. The method has been demonstrated in protein conjugation, and an arylthiolate conjugate was shown to be stable upon prolonged incubation in either GSH or human plasma. Finally, cyclopropenyl ketones were used to create PEG-based hydrogels that are stable to prolonged incubation in a reducing environment.

Fabrication of Functional Biomaterial Microstructures by in Situ Photopolymerization and Photodegradation.

The in situ fabrication of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microstructures within poly(dimethylsiloxane) (PDMS)-based microfluidic networks is a versatile technique that has enabled unique applications in biosensing, medical diagnostics, and the fundamental life sciences. Hydrogel structures have previously been patterned by the lithographic photopolymerization of PEGDA hydrogel forming solutions, a process that is confounded by oxygen-permeable PDMS. Here, we introduce an alternate PEG patterning technique that relies upon the optical sculpting of features by patterned light-induced erosion of photodegradable PEGDA deemed negative projection lithography. We quantitatively compared the hydrogel micropatterning fidelity of negative projection lithography to positive projection lithography, using traditional PEGDA photopolymerization, within PDMS devices. We found that the channel depth, the local oxygen atmosphere, and the UV exposure time dictated the size and resolution of hydrogel features formed using positive projection lithography. In contrast, negative projection lithography was observed to deliver high-resolution functional features with dimensions on the order of single micrometers enabled by its facilely controlled mechanism of feature formation that is insensitive to oxygen. Next, the utility of photodegradable PEGDA was further assessed by encapsulating or conjugating bioactive molecules within photodegradable PEG matrixes to provide a route to the formation of complex and dynamically reconfigurable chemical microenvironments. Finally, we demonstrated that negative projection lithography enabled photopatterning of multilayered microscale objects without the need for precise mask alignment. The described approach for photopatterning high-resolution photolabile hydrogel microstructures directly within PDMS microchannels could enable novel microsystems of increasing complexity and sophistication for a variety of clinical and biological applications.

Controlling the Release of Small, Bioactive Proteins via Dual Mechanisms with Therapeutic Potential.

Injectable delivery systems that respond to biologically relevant stimuli present an attractive strategy for tailorable drug release. Here, the design and synthesis of unique polymers are reported for the creation of hydrogels that are formed in situ and degrade in response to clinically relevant endogenous and exogenous stimuli, specifically reducing microenvironments and externally applied light. Hydrogels are formed with polyethylene glycol and heparin-based polymers using a Michael-type addition reaction. The resulting hydrogels are investigated for the local controlled release of low molecular weight proteins (e.g., growth factors and cytokines), which are of interest for regulating various cellular functions and fates in vivo yet remain difficult to deliver. Incorporation of reduction-sensitive linkages and light-degradable linkages affords significant changes in the release profiles of fibroblast growth factor-2 (FGF-2) in the presence of the reducing agent glutathione or light, respectively. The bioactivity of the released FGF-2 is comparable to pristine FGF-2, indicating the ability of these hydrogels to retain the bioactivity of cargo molecules during encapsulation and release. Further, in vivo studies demonstrate degradation-mediated release of FGF-2. Overall, our studies demonstrate the potential of these unique stimuli-responsive chemistries for controlling the local release of low molecular weight proteins in response to clinically relevant stimuli.

Tuning and Predicting Mesh Size and Protein Release from Step Growth Hydrogels.

Hydrogel-based depots are of growing interest for release of biopharmaceuticals; however, a priori selection of hydrogel compositions that will retain proteins of interest and provide desired release profiles remains elusive. Toward addressing this, in this work, we have established a new tool for the facile assessment of protein release from hydrogels and applied it to evaluate the effectiveness of mesh size estimations on predicting protein retention or release. Poly(ethylene glycol) (PEG)-based hydrogel depots were formed by photoinitiated step growth polymerization of four-arm PEG functionalized with norbornene (PEG-norbornene, 4% w/w to 20% w/w, Mn ∼ 5 to 20 kDa) and different dithiol cross-linkers (PEG Mn ∼ 1.5 kDa or enzymatically degradable peptide), creating well-defined, robust materials with a range of mesh sizes estimated with Flory-Rehner or rubber elasticity theory (∼5 to 15 nm). A cocktail of different model proteins was released from compositions of interest, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to facilely and quantitatively analyze temporal release profiles. Mesh size was predictive of retention of relatively large proteins and release of relatively small proteins. Proteins with diameters comparable to the mesh size, which is often the case for growth factors, were released by hindered diffusion and required experimental assessment of retention and release. With this knowledge, hydrogels were designed for the controlled release of a therapeutically relevant growth factor, PDGF-BB.

Aortic adventitial fibroblast sensitivity to mitogen activated protein kinase inhibitors depends on substrate stiffness.

Adventitial fibroblasts (AFs) are key determinants of arterial function and critical mediators of arterial disease progression. The effects of altered stiffness, particularly those observed across individuals during normal vascular function, and the mechanisms by which AFs respond to altered stiffness, are not well understood. To study the effects of matrix stiffness on AF phenotype, cytokine production, and the regulatory pathways utilized to interpret basic cell-matrix interactions, human aortic AFs were grown in 5%, 7.5%, and 10% (w/v%) PEG-based hydrogels with Young's moduli of 1.2, 3.3, and 9.6 kPa, respectively. In 5% gels, AFs had higher proliferation rates, elevated monocyte chemoattractant protein-1 secretion, and enhanced monocyte recruitment. Significantly more AFs were α-smooth muscle actin positive in 7.5% gels, indicating myofibroblast development. AFs in 10% gels had low proliferation rates but produced high levels of interleukin-6 and vascular endothelial growth factor-A. Importantly, these modulus-dependent changes in AF phenotype were accompanied by alterations in the mitogen-activated protein kinase (MAPK) pathways contributing to the production of cytokines. These data indicate that complex cell regulatory changes occur with altered tissue stiffness and suggest that therapeutics affecting MAPK pathways may have altered effects on AFs depending on substrate stiffness.

Thiol-ene click hydrogels for therapeutic delivery.

Hydrogels are of growing interest for the delivery of therapeutics to specific sites in the body. For use as a delivery vehicle, hydrophilic precursors are usually laden with bioactive moieties and then directly injected to the site of interest for in situ gel formation and controlled release dictated by precursor design. Hydrogels formed by thiol-ene click reactions are attractive for local controlled release of therapeutics owing to their rapid reaction rate and efficiency under mild aqueous conditions, enabling in situ formation of gels with tunable properties often responsive to environmental cues. Herein, we will review the wide range of applications for thiol-ene hydrogels, from the prolonged release of anti-inflammatory drugs in the spine to the release of protein-based therapeutics in response to cell-secreted enzymes, with a focus on their clinical relevance. We will also provide a brief overview of thiol-ene click chemistry and discuss the available alkene chemistries pertinent to macromolecule functionalization and hydrogel formation. These chemistries include functional groups susceptible to Michael type reactions relevant for injection and radically-mediated reactions for greater temporal control of formation at sites of interest using light. Additionally, mechanisms for the encapsulation and controlled release of therapeutic cargoes are reviewed, including i) tuning the mesh size of the hydrogel initially and temporally for cargo entrapment and release and ii) covalent tethering of the cargo with degradable linkers or affinity binding sequences to mediate release. Finally, myriad thiol-ene hydrogels and their specific applications also are discussed to give a sampling of the current and future utilization of this chemistry for delivery of therapeutics, such as small molecule drugs, peptides, and biologics.

Biomaterials for 4D stem cell culture.

Stem cells reside in complex three-dimensional (3D) environments within the body that change with time, promoting various cellular functions and processes such as migration and differentiation. These complex changes in the surrounding environment dictate cell fate yet, until recently, have been challenging to mimic within cell culture systems. Hydrogel-based biomaterials are well suited to mimic aspects of these in vivo environments, owing to their high water content, soft tissue-like elasticity, and often-tunable biochemical content. Further, hydrogels can be engineered to achieve changes in matrix properties over time to better mimic dynamic native microenvironments for probing and directing stem cell function and fate. This review will focus on techniques to form hydrogel-based biomaterials and modify their properties in time during cell culture using select addition reactions, cleavage reactions, or non-covalent interactions. Recent applications of these techniques for the culture of stem cells in four dimensions (i.e., in three dimensions with changes over time) also will be discussed for studying essential stem cell processes.

Design of Thiol- and Light-sensitive Degradable Hydrogels using Michael-type Addition Reactions.

Injectable depots that respond to exogenous and endogenous stimuli present an attractive strategy for tunable, patient-specific drug delivery. Here, the design of injectable and multimodal degradable hydrogels that respond to externally applied light and physiological stimuli, specifically aqueous and reducing microenvironments, is reported. Rapid hydrogel formation was achieved using a thiol-maleimide click reaction between multifunctional poly(ethylene glycol) macromers. Hydrogel degradation kinetics in response to externally applied cytocompatible light, reducing conditions, and hydrolysis were characterized, and degradation of the gel was controlled over multiple time scales from seconds to days. Further, tailored release of an encapsulated model cargo, fluorescent nanobeads, was demonstrated.

Decreasing matrix modulus of PEG hydrogels induces a vascular phenotype in human cord blood stem cells.

Adult and congenital cardiovascular diseases are significant health problems that are often managed using surgery. Bypass grafting is a principal therapy, but grafts fail at high rates due to hyperplasia, fibrosis, and atherosclerosis. Biocompatible, cellularized materials that attenuate these complications and encourage healthy microvascularization could reduce graft failure, but an improved understanding of biomaterial effects on human stem cells is needed to reach clinical utility. Our group investigates stem-cell-loaded biomaterials for placement along the adventitia of at-risk vessels and grafts. Here, the effects of substrate modulus on human CD34+ stem cells from umbilical cord blood were evaluated. Cells were isolated by immunomagnetic separation and encapsulated in 3, 4, and 6 weight% PEG hydrogels containing 0.032% gelatin and 0.0044% fibronectin. Gels reached moduli of 0.34, 4.5, and 9.1 kPa. Cell viability approached 100%. Cell morphologies appeared similar across gels, but proliferation was significantly lower in 6 wt% gels. Expression profiling using stem cell signaling arrays indicated enhanced self-renewal and differentiation into vascular endothelium among cells in the lower weight percent gels. Thus, modulus was associated with cell proliferation and function. Gels with moduli in the low kilopascal range may be useful in stimulating cell engraftment and microvascularization of graft adventitia.

Designing degradable hydrogels for orthogonal control of cell microenvironments.

Degradable and cell-compatible hydrogels can be designed to mimic the physical and biochemical characteristics of native extracellular matrices and provide tunability of degradation rates and related properties under physiological conditions. Hence, such hydrogels are finding widespread application in many bioengineering fields, including controlled bioactive molecule delivery, cell encapsulation for controlled three-dimensional culture, and tissue engineering. Cellular processes, such as adhesion, proliferation, spreading, migration, and differentiation, can be controlled within degradable, cell-compatible hydrogels with temporal tuning of biochemical or biophysical cues, such as growth factor presentation or hydrogel stiffness. However, thoughtful selection of hydrogel base materials, formation chemistries, and degradable moieties is necessary to achieve the appropriate level of property control and desired cellular response. In this review, hydrogel design considerations and materials for hydrogel preparation, ranging from natural polymers to synthetic polymers, are overviewed. Recent advances in chemical and physical methods to crosslink hydrogels are highlighted, as well as recent developments in controlling hydrogel degradation rates and modes of degradation. Special attention is given to spatial or temporal presentation of various biochemical and biophysical cues to modulate cell response in static (i.e., non-degradable) or dynamic (i.e., degradable) microenvironments. This review provides insight into the design of new cell-compatible, degradable hydrogels to understand and modulate cellular processes for various biomedical applications.