A Reconfigurable Proangiogenic Hydrogel Patch Enabling Minimally Invasive Drug Delivery.

Hydrogel is widely used for the sustained delivery of bioactive molecules that can treat various injuries, diseases, and tissue defects. However, inserting hydrogel implants without disrupting their functionality and microstructure often requires a large incision, leading to potential complications, such as infection, scarring, and pain. The gel implant is often manually rolled and inserted through a catheter for a minimally invasive delivery. However, success heavily depends on the user's skills, which can inadvertently damage the implant. To address this issue, we developed a reconfigurable hydrogel patch that can self-fold into a small tube and unfold spontaneously after implantation through a catheter. The hydrogel path was assembled by layering a drug-releasing poly(ethylene glycol) diacrylate (PEGDA) hydrogel sheet onto a PEGDA and polyethylenimine (PEI) hydrogel sheet, which rapidly swells and degrades homogeneously at controlled rates. The dynamics of the self-folding and unfolding process could be controlled by differences in the expansion ratio and elastic modulus between the two gel layers according to a mathematical model that closely matched experimental results. The unfolding process triggered a sustained release of the protein cargo. Specifically, the reconfigurable gel loaded with angiopoietin 1 significantly enhanced neovascularization, nearly doubling the vascular density compared to the control group following implantation through a tube with 15% smaller diameter than the original shape of the gel patch. This gel biopatch will be broadly useful for the minimally invasive delivery of a wide array of therapeutic molecules, potentially enhancing therapeutic outcomes.

Wet tissue adhesive polymeric powder hydrogels for skeletal muscle regeneration.

Volumetric muscle loss (VML) frequently results from traumatic incidents and can lead to severe functional disabilities. Hydrogels have been widely employed for VML tissue regeneration, which are unfortunately ineffective because of the lack of intimate contact with injured tissue for structural and mechanical support. Adhesive hydrogels allow for strong tissue connections for wound closure. Nevertheless, conventional adhesive hydrogels exhibit poor tissue adhesion in moist, bleeding wounds due to the hydration layer at the tissue-hydrogel interfaces, resulting in insufficient performance. In this study, we developed a novel, biocompatible, wet tissue adhesive powder hydrogel consisting of dextran-aldehyde (dex-ald) and gelatin for the regeneration of VML. This powder absorbs the interfacial tissue fluid and buffer solution on the tissue, spontaneously forms a hydrogel, and strongly adheres to the tissue via various molecular interactions, including the Schiff base reaction. In particular, the powder composition with a 1:4 ratio of dex-ald to gelatin exhibited optimal characteristics with an appropriate gelation time (258 s), strong tissue adhesion (14.5 kPa), and stability. Dex-ald/gelatin powder hydrogels presented strong adhesion to various organs and excellent hemostasis compared to other wet hydrogels and fibrin glue. A mouse VML injury model revealed that the dex-ald/gelatin powder hydrogel significantly improved muscle regeneration, reduced fibrosis, enhanced vascularization, and decreased inflammation. Consequently, our wet-adhesive powder hydrogel can serve as an effective platform for repairing various tissues, including the heart, muscle, and nerve tissues.

A Gelatin/Alginate Double Network Hydrogel Nerve Guidance Conduit Fabricated by a Chemical-Free Gamma Radiation for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) are widely developed using various materials for the functional repair of injured or diseased peripheral nerves. Especially, hydrogels are considered highly suitable for the fabrication of NGCs due to their beneficial tissue-mimicking characteristics (e.g., high water content, softness, and porosity). However, the practical applications of hydrogel-based NGCs are hindered due to their poor mechanical properties and complicated fabrication processes. To bridge this gap, a novel double-network (DN) hydrogel using alginate and gelatin by a two-step crosslinking process involving chemical-free gamma irradiation and ionic crosslinking, is developed. DN hydrogels (1% alginate and 15% gelatin), crosslinked with 30 kGy gamma irradiation and barium ions, exhibit substantially improved mechanical properties, including tensile strength, elastic modulus, and fracture stain, compared to single network (SN) gelatin hydrogels. Additionally, the DN hydrogel NGC exhibits excellent kink resistance, mechanical stability to successive compression, suture retention, and enzymatic degradability. In vivo studies with a sciatic defect rat model indicate substantially improved nerve function recovery with the DN hydrogel NGC compared to SN gelatin and commercial silicone NGCs, as confirm footprint analysis, electromyography, and muscle weight measurement. Histological examination reveals that, in the DN NGC group, the expression of Schwann cell and neuronal markers, myelin sheath, and exon diameter are superior to the other controls. Furthermore, the DN NGC group demonstrates increased muscle fiber formation and reduced fibrotic scarring. These findings suggest that the mechanically robust, degradable, and biocompatible DN hydrogel NGC can serve as a novel platform for peripheral nerve regeneration and other biomedical applications, such as implantable tissue constructs.

Implantable polypyrrole bioelectrodes inducing anti-inflammatory macrophage polarization for long-term in vivo signal recording.

Bioelectrodes are critical components of implantable electronic devices that enable precise electrical signal transmission in close contact with living tissues. However, their in vivo performance is often compromised by inflammatory tissue reactions mainly induced by macrophages. Hence, we aimed to develop implantable bioelectrodes with high performance and high biocompatibility by actively modulating the inflammatory response of macrophages. Consequently, we fabricated heparin-doped polypyrrole electrodes (PPy/Hep) and immobilized anti-inflammatory cytokines (interleukin-4 [IL-4]) via non-covalent interactions. IL-4 immobilization did not alter the electrochemical performance of the original PPy/Hep electrodes. In vitro primary macrophage culture revealed that IL-4-immobilized PPy/Hep electrodes induced anti-inflammatory polarization of macrophages, similar to the soluble IL-4 control. In vivo subcutaneous implantation indicated that IL-4 immobilization on PPy/Hep promoted the anti-inflammatory polarization of host macrophages and significantly mitigated scarring around the implanted electrodes. In addition, high-sensitivity electrocardiogram signals were recorded from the implanted IL-4-immobilized PPy/Hep electrodes and compared to bare gold and PPy/Hep electrodes, which were maintained for up to 15 days post-implantation. This simple and effective surface modification strategy for developing immune-compatible bioelectrodes will facilitate the development of various electronic medical devices that require high sensitivities and long-term stabilities. STATEMENT OF SIGNIFICANCE: To fabricate highly immunocompatible conductive polymer-based implantable electrodes with high performance and stability in vivo, we introduced the anti-inflammatory activity to PPy/Hep electrodes by immobilizing IL-4 via non-covalent surface modification. IL-4-immobilized PPy/Hep could significantly mitigate inflammatory responses and scarring around implants by skewing macrophages to an anti-inflammatory phenotype. The IL-4-immobilized PPy/Hep electrodes could successfully record in vivo electrocardiogram signals for up to 15 days with no substantial sensitivity loss, retaining their superior sensitivity compared to bare gold and pristine PPy/Hep electrodes. Our simple and effective surface modification strategy for developing immune-compatible bioelectrodes will facilitate the development of various electronic medical devices that require high sensitivities and long-term stabilities, such as neural electrode arrays, biosensors, and cochlear electrodes.

A Conductive and Adhesive Hydrogel Composed of MXene Nanoflakes as a Paintable Cardiac Patch for Infarcted Heart Repair.

Myocardial infarction (MI) is a major cause of death worldwide. After the occurrence of MI, the heart frequently undergoes serious pathological remodeling, leading to excessive dilation, electrical disconnection between cardiac cells, and fatal functional damage. Hence, extensive efforts have been made to suppress pathological remodeling and promote the repair of the infarcted heart. In this study, we developed a hydrogel cardiac patch that can provide mechanical support, electrical conduction, and tissue adhesiveness to aid in the recovery of an infarcted heart function. Specifically, we developed a conductive and adhesive hydrogel (CAH) by combining the two-dimensional titanium carbide (Ti3C2Tx) MXene with natural biocompatible polymers [i.e., gelatin and dextran aldehyde (dex-ald)]. The CAH was formed within 250 s of mixing the precursor solution and could be painted. The hydrogel containing 3.0 mg/mL MXene, 10% gelatin, and 5% dex-ald exhibited appropriate material characteristics for cardiac patch applications, including a uniform distribution of MXene, a high electrical conductivity (18.3 mS/cm), cardiac tissue-like elasticity (30.4 kPa), strong tissue adhesion (6.8 kPa), and resistance to various mechanical deformations. The CAH was cytocompatible and induced cardiomyocyte (CM) maturation in vitro, as indicated by the upregulation of connexin 43 expression and a faster beating rate. Furthermore, CAH could be painted onto the heart tissue and remained stably adhered to the beating epicardium. In vivo animal studies revealed that CAH cardiac patch treatment significantly improved cardiac function and alleviated the pathological remodeling of an infarcted heart. Thus, we believe that our MXene-based CAH can potentially serve as a promising platform for the effective repair of various electroactive tissues including the heart, muscle, and nerve tissues.

Injectable Conductive Hydrogels with Tunable Degradability as Novel Implantable Bioelectrodes.

Bioelectrodes have been developed to efficiently mediate electrical signals of biological systems as stimulators and recording devices. Recently, conductive hydrogels have garnered great attention as emerging materials for bioelectrode applications because they can permit intimate/conformal contact with living tissues and tissue-like softness. However, administration and control over the in vivo lifetime of bioelectrodes remain challenges. Here, injectable conductive hydrogels (ICHs) with tunable degradability as implantable bioelectrodes are developed. ICHs were constructed via thiol-ene reactions using poly(ethylene glycol)-tetrathiol and thiol-functionalized reduced graphene oxide with either hydrolyzable poly(ethylene glycol)-diacrylate or stable poly(ethylene glycol)-dimaleimide, the resultant hydrogels of which are degradable and nondegradable, respectively. The ICH electrodes had conductivities of 21-22 mS cm-1 and Young's moduli of 15-17 kPa, and showed excellent cell and tissue compatibility. The hydrolyzable conductive hydrogels disappeared 3 days after in vivo administration, while the stable conductive hydrogels maintained their shapes for up to 7 days. Our proof-of-concept studies reveal that electromyography signals with significantly improved sensitivity from rats could be obtained from the injected ICH electrodes compared to skin electrodes and injected nonconductive hydrogel electrodes. The ICHs, offering convenience in use, controllable degradation and excellent signal transmission, will have great potential to develop various bioelectronics devices.

High-Performance Implantable Bioelectrodes with Immunocompatible Topography for Modulation of Macrophage Responses.

Implantable bioelectrodes enable precise recording or stimulation of electrical signals with living tissues in close contact. However, their performance is frequently compromised owing to inflammatory tissue reactions, which macrophages either induce or resolve by polarizing to an inflammatory (M1) or noninflammatory (M2) phenotype, respectively. Thus, we aimed to fabricate biocompatible and functional implantable conductive polymer bioelectrodes with optimal topography for the modulation of macrophage responses. To this end, we produced heparin-doped polypyrrole (PPy/Hep) electrodes of different surface roughness, with Ra values from 5.5 to 17.6 nm, by varying the charge densities during electrochemical synthesis. In vitro culture revealed that macrophages on rough PPy/Hep electrodes preferentially polarized to noninflammatory phenotypes. In particular, PPy/Hep-900 (Ra = 14 nm) was optimal with respect to electrochemical properties and the suppression of inflammatory M1 polarization. In vivo implantation indicated that PPy/Hep-900 significantly reduced macrophage recruitment, suppressed inflammatory polarization, and mitigated fibrotic tissue formation. In addition, the implanted PPy/Hep-900 electrodes could successfully record electrocardiographic signals for up to 10 days without substantial decreases in sensitivity, while other electrodes substantially lost their signal sensitivity during implantation. Altogether, we demonstrate that modulating the surface features of PPy/Hep can benefit the design and applications of high-performance and high-biocompatibility bioelectrodes.

Towards the translation of electroconductive organic materials for regeneration of neural tissues.

Carbon-based conductive and electroactive materials (e.g., derivatives of graphene, fullerenes, polypyrrole, polythiophene, polyaniline) have been studied since the 1970s for use in a broad range of applications. These materials have electrical properties comparable to those of commonly used metals, while providing other benefits such as flexibility in processing and modification with biologics (e.g., cells, biomolecules), to yield electroactive materials with biomimetic mechanical and chemical properties. In this review, we focus on the uses of these electroconductive materials in the context of the central and peripheral nervous system, specifically recent studies in the peripheral nerve, spinal cord, brain, eye, and ear. We also highlight in vivo studies and clinical trials, as well as a snapshot of emerging classes of electroconductive materials (e.g., biodegradable materials). We believe such specialized electrically conductive biomaterials will clinically impact the field of tissue regeneration in the foreseeable future. STATEMENT OF SIGNIFICANCE: This review addresses the use of conductive and electroactive materials for neural tissue regeneration, which is of significant interest to a broad readership, and of particular relevance to the growing community of scientists, engineers and clinicians in academia and industry who develop novel medical devices for tissue engineering and regenerative medicine. The review covers the materials that may be employed (primarily focusing on derivatives of fullerenes, graphene and conjugated polymers) and techniques used to analyze materials composed thereof, followed by sections on the application of these materials to nervous tissues (i.e., peripheral nerve, spinal cord, brain, optical, and auditory tissues) throughout the body.

Biomimetic nonbiofouling polypyrrole electrodes grafted with zwitterionic polymer using gamma rays.

Bioelectrodes, including metallic and conductive polymer (CP) bioelectrodes, often suffer from biofouling by contamination from microbacteria and/or biomolecules in biological systems, which can cause substantial impairment of biofunctionality and biocompatibility. Herein, we have employed an in situ polymerization of methacryloyloxyethyl phosphorylcholine (MPC) by gamma radiation to introduce fouling-resistant properties onto the surface of the conductive polymer, polypyrrole (PPy). The concentrations of an MPC monomer were varied during the grafting. PPy electrodes modified with MPC (PPy-g-MPC) revealed excellent anti-biofouling properties, as demonstrated by multiple analyses, such as serum protein adsorption, fibroblast adhesion, bacteria adhesion, and scar tissue formation in vivo. Importantly, PPy-g-MPC, which was modified with 0.2 M MPC using gamma radiation, exhibited electrical properties similar to unmodified PPy electrodes, indicating that our MPC grafting strategies did not cause impairment of electrical/electrochemical properties of the original PPy electrodes while successfully introducing anti-biofouling properties. Zwitterionic MPC polymer grafting on PPy electrodes by in situ polymerization with gamma radiation will benefit the development of highly biocompatible and functional bioelectrodes, such as neural electrodes, stimulators, and biosensors.

Universal surface modification using dopamine-hyaluronic acid conjugates for anti-biofouling.

Various materials are used to make implantable devices such as pumps, catheters, and stimulators. However, such implants inevitably undergo biofouling, which is associated with non-specific proteins and cell adhesion on the material surfaces. Severe biofouling often leads to adverse tissue reactions, such as foreign body responses, and thick scar tissue formation around the implantation, which in turn hampers the function and performance of the implants. Herein, a simple and effective surface modification method using dopamine-hyaluronic acid conjugate (DA-HA) is demonstrated to implement anti-biofouling in various biomaterials. DA-HA was synthesized and utilized as a coating material and five commonly used implantable substrates (i.e., polyimide (PI), gold (Au), poly(methyl methacrylate) (PMMA), polytetrafluoroethylene (PTFE), and polyurethane (PU)) were tested for modification with DA-HA. Highly hydrophilic HA chains were immobilized on the substrate surfaces through self-polymerization of DA-HA at an alkaline pH. The DA-HA-modified substrates displayed significant resistance to both non-specific protein adsorption and cell adhesion on the surfaces. In addition, a reduction in scar tissue formation was observed in the DA-HA-coated substrates compared to the unmodified bare substrates after a 4-week subcutaneous implantation. This universal surface coating can be further implemented in various biomedical applications in which severe biofouling is highly undesirable.

A Novel Conductive and Micropatterned PEG-Based Hydrogel Enabling the Topographical and Electrical Stimulation of Myoblasts.

In this study, we designed a cell-adhesive poly(ethylene glycol) (PEG)-based hydrogel that simultaneously provides topographical and electrical stimuli to C2C12 myoblasts. Specifically, PEG hydrogels with microgroove structures of 3 µm ridges and 3 µm grooves were prepared by micromolding; in situ polymerization of poly(3,4-ethylenedioxythiophene) (PEDOT) was then performed within the micropatterned PEG hydrogels to create a microgrooved conductive hydrogel (CH/P). The CH/P had clear replica patterns of the silicone mold and a conductivity of 2.49 × 10-3 S/cm, with greater than 85% water content. In addition, the CH exhibited Young's modulus (45.84 ± 7.12 kPa) similar to that of a muscle tissue. The surface of the CH/P was further modified via covalent bonding with cell-adhesive peptides to facilitate cell adhesion without affecting conductivity. An in vitro cell assay revealed that the CH/P was cytocompatible and enhanced the cell alignment and elongation of C2C12 myoblasts. The microgrooves and conductivity of the CH/P had the greatest positive effect on the myogenesis of C2C12 myoblasts compared to the other PEG hydrogel samples without conductivity or/and microgrooves, even in the absence of electrical stimulation. Electrical stimulation studies indicated that the combination of topographical and electrical cues maximized the differentiation of C2C12 myoblasts into myotubes, confirming the synergetic effect of incorporating microgroove surface features and a conductive PEDOT component into hydrogels.

Anti-oxidant activity reinforced reduced graphene oxide/alginate microgels: Mesenchymal stem cell encapsulation and regeneration of infarcted hearts.

Mesenchymal stem cell (MSC) transplantation is promising for repairing heart tissues post myocardial infarction (MI). In particular, paracrine effects of the transplanted MSCs have been highlighted to play major roles in heart regeneration by secreting multiple growth factors and immune-modulatory cytokines. Nevertheless, its therapeutic efficacy still remains low, which is strongly associated with low viability and activity of the transplanted stem cells, because the transplanted MSCs are exposed to high shear stress during injection and harsh environments (e.g., high oxidative stress and host immune reactions) post injection. In this study, we aimed to develop novel injectable MSC-delivery microgel systems possessing high anti-oxidant activities. Specifically, we encapsulated MSCs in graphene oxide (GO)/alginate composite microgels by electrospraying. To further enhance the anti-oxidizing activities of the gels, we developed reduced MSC-embedded GO/alginate microgels (i.e., r(GO/alginate)), which have the potential to protect MSCs from the abovementioned harsh environments within MI tissues. Our in vitro studies demonstrated that the MSCs encapsulated in the r(GO/alginate) microgels showed increased viability under oxidative stress conditions with H2O2. Furthermore, cardiomyocytes (CMs), co-cultured with the encapsulated MSCs in transwells with H2O2 treatment, showed higher cell viability and cardiac maturation compared to monolayer cultured CMs, likely due to ROS scavenging by the gels and positive paracrine signals from the encapsulated MSCs. In vivo experiments with acute MI models demonstrated improved therapeutic efficacy of MSC delivery in r(GO/alginate) microgels, exhibiting significant decreases in the infarction area and the improvement of cardiac function. We believe that our novel MSC encapsulation system with GO, alginate, and mild reduction, which exhibits high cell protection capacity (e.g., anti-oxidant activity), will serve as an effective platform for the delivery of stem cells and other therapeutic cell types to treat various injuries and diseases, including MI.

Micropatterned conductive hydrogels as multifunctional muscle-mimicking biomaterials: Graphene-incorporated hydrogels directly patterned with femtosecond laser ablation.

Multifunctional biomaterials that can provide physical, electrical, and structural cues to cells and tissues are highly desirable to mimic the important characteristics of native tissues and efficiently modulate cellular behaviors. Especially, electrically conductive biomaterials can efficiently deliver electrical signals to living systems; however, the production of conductive biomaterials presenting multiple cell interactive cues is still a great challenge. In this study, we fabricafed an electrically conductive, mechanically soft, and topographically active hydrogel by micropatterning a graphene oxide (GO)-incorporated polyacrylamide hydrogel (GO/PAAm) with femtosecond laser ablation (FLA) and subsequent chemical reduction. FLA parameters were optimized to efficiently produce distinct line patterns on GO/PAAm hydrogels to induce myoblast alignment and maturation. The line patterns distances (PD) were varied to have different topographies (20-80 µm PD). In vitro studies with C2C12 myoblasts revealed that the micopatterned hydrogels are superior to the unpatterned substrates in inducing myogenesis and myotube alignment. Reduced GO/PAAm with 50 µm PD, i.e., PD50/r(GO/PAAm), showed the best results among the various features for differentiation and myotube alignment. Electrical stimulation of myoblasts on the micropatterned conductive hydrogels further promoted the differentiation of myoblasts. In vivo implantation studies indicated good tissue compatibility of PD50/r(GO/PAAm) samples. Altogether, we successfully demonstrated that the micropatterned r(GO/PAAm) may offer multiple properties capable of positively affecting myoblast responses. This hydrogel may serve as an effective multifunctional biomaterial, which possesses the topography for cell alignment/maturation, mechanical properties of the native skeletal muscle tissue, and desirable electrical conductivity for delivering electrical signals to cells, for various biomedical applications such as muscle tissue scaffolds. STATEMENT OF SIGNIFICANCE: Micropatterned conductive hydrogels were created by polymerization of a graphene oxide-incorporated polyacrylamide hydrogel, micropatterning with femtosecond laser ablation, and chemical reduction, which can mimic important characteristics of native skeletal muscle tissues. The micropatterned conductive hydro-gels promoted myogenesis/alignment, enabled electrical stimulation of myoblasts, and displayed good tissue compatibility, which can therefore serve as a multifunctional biomaterial that is topographically active, mechanically soft, and electrically conductive for delivering multiple cell stimulating signals for potential skeletal muscle tissue engineering applications.

Electrochemical Co-deposition of Polydopamine/Hyaluronic Acid for Anti-biofouling Bioelectrodes.

Bioelectrodes are key components of electronic devices that efficiently mediate electrical signals in biological systems. However, conventional bioelectrodes often undergo biofouling associated with non-specific proteins and cell adhesion on the electrode surfaces, which leads to seriously degraded electrical and/or electrochemical properties. Hence, a facile and effective method to modify the surface of bioelectrodes is required to introduce anti-biofouling properties and improve performance. Here, we report an electrochemical surface modification of a bioelectrode via co-deposition of hyaluronic acid (HA) and polydopamine (PDA). The electrochemical polymerization and deposition of PDA offered simple and effective incorporation of highly hydrophilic and anti-fouling HA to the electrode surfaces, with no substantial increase in impedance. HA-incorporated PDA (PDA/HA)-modified electrodes displayed significant resistance to non-specific protein adsorption and the adhesion of fibroblasts. In addition, 4-week subcutaneous implantation studies revealed that the modified electrodes attenuated scar tissue formation compared with that induced by unmodified bare electrodes. This simple and effective electrochemical surface modification could be further employed for various implantable bioelectrodes (e.g., prosthetics and biosensors) and could extend their bioelectronic applications.

Studies on the effects of microencapsulated human mesenchymal stem cells in RGD-modified alginate on cardiomyocytes under oxidative stress conditions using in vitro biomimetic co-culture system.

Stem cell therapy has been recognized as a promising approach for myocardium regeneration post myocardial infarction (MI); however, it unfortunately often remains a challenge because of poor survival of transplanted cells and a lack of clear understanding of their interactions with host cells. High oxidative stress at heart tissues post MI is considered one of the important factors damaging transplanted cells and native cells/tissues. Here, we employed an in vitro co-culture system, capable of mimicking cases of stem cell transplantation into the myocardium presenting high oxidative stress, using human mesenchymal stem cells (hMSCs) encapsulated in alginate or cell interactive Arg-Gly-Asp (RGD) peptide-modified alginate micro-hydrogels. Under H2O2-induced oxidative stress conditions, viabilities of hMSCs and CMs were significantly higher in their co-culture than in their individual monolayer cultures. Expression of cardiac muscle markers remained high even with H2O2 treatment when cardiomyocytes (CMs) were co-cultured with hMSCs in RGD-alginate. Higher levels of various growth factors (associated with angiogenesis, cardiac regeneration, and contractility) were found in co-culture (noticeably with RGD-alginate) compared to monolayer cultures of CMs or hMSCs. These results can benefit the study of in vivo MI progression with transplanted stem cells and the development of effective stem cell-based therapeutic strategies for various oxidative stress-related diseases.