Electro-Biofabrication. Coupling Electrochemical and Biomolecular Methods to Create Functional Bio-Based Hydrogels.

Twenty years ago, this journal published a review entitled "Biofabrication with Chitosan" based on the observations that (i) chitosan could be electrodeposited using low voltage electrical inputs (typically less than 5 V) and (ii) the enzyme tyrosinase could be used to graft proteins (via accessible tyrosine residues) to chitosan. Here, we provide a progress report on the coupling of electronic inputs with advanced biological methods for the fabrication of biopolymer-based hydrogel films. In many cases, the initial observations of chitosan's electrodeposition have been extended and generalized: mechanisms have been established for the electrodeposition of various other biological polymers (proteins and polysaccharides), and electrodeposition has been shown to allow the precise control of the hydrogel's emergent microstructure. In addition, the use of biotechnological methods to confer function has been extended from tyrosinase conjugation to the use of protein engineering to create genetically fused assembly tags (short sequences of accessible amino acid residues) that facilitate the attachment of function-conferring proteins to electrodeposited films using alternative enzymes (e.g., transglutaminase), metal chelation, and electrochemically induced oxidative mechanisms. Over these 20 years, the contributions from numerous groups have also identified exciting opportunities. First, electrochemistry provides unique capabilities to impose chemical and electrical cues that can induce assembly while controlling the emergent microstructure. Second, it is clear that the detailed mechanisms of biopolymer self-assembly (i.e., chitosan gel formation) are far more complex than anticipated, and this provides a rich opportunity both for fundamental inquiry and for the creation of high performance and sustainable material systems. Third, the mild conditions used for electrodeposition allow cells to be co-deposited for the fabrication of living materials. Finally, the applications have been expanded from biosensing and lab-on-a-chip systems to bioelectronic and medical materials. We suggest that electro-biofabrication is poised to emerge as an enabling additive manufacturing method especially suited for life science applications and to bridge communication between our biological and technological worlds.

Single Step Assembly of Janus Porous Biomaterial by Sub-Ambient Temperature Electrodeposition.

Janus porous biomaterials are gaining increasing attention and there are considerable efforts to develop simple, rapid, and scalable methods capable of tuning micro- and macro-structures. Here, a single-step electro-fabrication method to create a Janus porous film by the electrodeposition of the amino-polysaccharide chitosan is reported. Specifically, a Janus structure emerges spontaneously when electrodeposition is performed at sub-ambient temperature (0-5 °C). Sub-ambient temperature electrodeposition experiments show that: a Janus microstructure emerges (potentially as the result of a subtle alteration of the intermolecular interactions responsible for self-assembly); important microstructural features (pore size, porosity, and thicknesses) can be tuned by conditions; and this method is readily scalable (vs serial printing) and can yield complex tubular structures with Janus faces. In vitro studies demonstrate anisotropic cell guidance, and in vivo studies using a rat calvarial defect model further confirm the beneficial features of such Janus porous film for guided bone regeneration. In summary, these results further demonstrate that electro-fabrication provides a simple and scalable platform technology for the controlled functional structures of soft matter for applications in regenerative medicine.

Electrical signals triggered controllable formation of calcium-alginate film for wound treatment.

Wound dressings play important roles in the management of wounds, and calcium cross-linked alginate (Ca2+-Alg) is a commonly used hydrogel that is adapted for wound treatment. However, conventional methods for fabricating Ca2+-Alg hydrogels can be tedious and difficult to control because of the rapid Ca2+-induced gelation of alginate. In this study, An electrodeposition method was used to rapidly and controllably fabricate Ca2+-Alg films for wound treatment. Several measures of film growth (e.g., thickness and mass) are shown to linearly correlate to the imposed charge transfer at the electrode. Similarly, this charge transfer was also observed to control important physicochemical wound healing properties such as water uptake and retention capacity. Furthermore, a wound healing animal test was performed to evaluate the performance of this electro-fabricated calcium alginate film for wound treatment. This in vivo study demonstrated that wounds dressed with an electro-fabricated Ca2+-Alg film closed faster than that of untreated wounds. Further, the new dermis tissue that formed was composed of reorganized and stratified epithelial layer, with fully developed connective tissue, hair follicle, sebaceous glands as well as aligned collagen. Therefore, our study indicates that this electrofabrication method for the rapid and controlled preparation of alginate film could provide exciting opportunities for wound treatment. More broadly, this study demonstrates the potential of electrochemistry for the fabrication of high performance polymeric materials. Here we report a rapid and controllable fabrication of free-standing alginate films by coupling anodic electrodeposition with subsequent peeling of deposited materials for wound dressing.

Programmable Electro-Assembly of Collagen: Constructing Porous Janus Films with Customized Dual Signals for Immunomodulation and Tissue Regeneration in Periodontitis Treatment.

Currently available guided bone regeneration (GBR) films lack active immunomodulation and sufficient osteogenic ability- in the treatment of periodontitis, leading to unsatisfactory treatment outcomes. Challenges remain in developing simple, rapid, and programmable manufacturing methods for constructing bioactive GBR films with tailored biofunctional compositions and microstructures. Herein, the controlled electroassembly of collagen under the salt effect is reported, which enables the construction of porous films with precisely tunable porous structures (i.e., porosity and pore size). In particular, bioactive salt species such as the anti-inflammatory drug diclofenac sodium (DS) can induce and customize porous structures while enabling the loading of bioactive salts and their gradual release. Sequential electro-assembly under pre-programmed salt conditions enables the manufacture of a Janus composite film with a dense and DS-containing porous layer capable of multiple functions in periodontitis treatment, which provides mechanical support, guides fibrous tissue growth, and acts as a barrier preventing its penetration into bone defects. The DS-containing porous layer delivers dual bio-signals through its morphology and the released DS, inhibiting inflammation and promoting osteogenesis. Overall, this study demonstrates the potential of electrofabrication as a customized manufacturing platform for the programmable assembly of collagen for tailored functions to adapt to specific needs in regenerative medicine.

Extracellular Matrix-Mimetic Immunomodulatory Hydrogel for Accelerating Wound Healing.

Macrophages play a crucial role in the complete processes of tissue repair and regeneration, and the activation of M2 polarization is an effective approach to provide a pro-regenerative immune microenvironment. Natural extracellular matrix (ECM) has the capability to modulate macrophage activities via its molecular, physical, and mechanical properties. Inspired by this, an ECM-mimetic hydrogel strategy to modulate macrophages via its dynamic structural characteristics and bioactive cell adhesion sites is proposed. The LZM-SC/SS hydrogel is in situ formed through the amidation reaction between lysozyme (LZM), 4-arm-PEG-SC, and 4-arm-PEG-SS, where LZM provides DGR tripeptide for cell adhesion, 4-arm-PEG-SS provides succinyl ester for dynamic hydrolysis, and 4-arm-PEG-SC balances the stability and dynamics of the network. In vitro and subcutaneous tests indicate the dynamic structural evolution and cell adhesion capacity promotes macrophage movement and M2 polarization synergistically. Comprehensive bioinformatic analysis further confirms the immunomodulatory ability, and reveals a significant correlation between M2 polarization and cell adhesion. A full-thickness wound model is employed to validate the induced M2 polarization, vessel development, and accelerated healing by LZM-SC/SS. This study represents a pioneering exploration of macrophage modulation by biomaterials' structures and components rather than drug or cytokines and provides new strategies to promote tissue repair and regeneration.

A novel way to facilely degrade organic pollutants with the tail-gas derived from PHP (phosphoric acid plus hydrogen peroxide) pretreatment of lignocellulose.

The abundantly released tail-gas from lignocellulose pretreatment with phosphoric acid plus hydrogen peroxide (PHP) was found to accelerate the aging of latex/silicone textural accessories of the pretreatment device. Inspired by this, tail-gas was utilized to control organic pollutants. Methylene blue (MB), as a model pollutant, was rapidly decolorized by the tail-gas, and oxidative degradation was substantially proven by full-wavelength scanning with a UV-visible spectrometer. The tail-gas from six typical lignocellulosic feedstocks produced 68.0-98.3% MB degradation, suggesting its wide feedstock compatibility. Three other dyes, including rhodamine B, methyl orange and malachite green, obtained 97.5-99.5% degradation; moreover, tetracycline, resorcinol and hexachlorobenzene achieved 73.8-93.7% degradation, suggesting a superior pollutant compatibility. In a cytotoxicity assessment, the survival rate of the degraded MB was 103.5% compared with 80.4% for the untreated MB, implying almost no cytotoxicity after MB degradation. Mechanism investigations indicated that the self-exothermic reaction in PHP pretreatment drove the self-generated peroxy acids into tail-gas. Moreover, it heated the pollutant solution and thermally activated peroxy acids as free radicals for efficient pollutant degradation. Here, a brand-new technique for degrading organic pollutants with a "Win-Win-Win" concept was purposed for lignocellulose valorization, pollutant control by waste tail-gas, and biofuel production.

Zwitterionic nanogels with temperature sensitivity and redox-degradability for controlled drug release.

Zwitterionic polymers play an attractive role in the application of stealthy nanocarriers for their excellent antifouling property. Herein, a zwitterionic nanogel with temperature sensitivity and redox-responsive degradability prepared by copolymerization of N-vinylcaprolactam (VCL) and 2-(methacryloyloxy) ethyldimethyl-(3-sulfopropyl) ammonium hydroxide (DMAPS) via aqueous precipitation polymerization. The prepared nanogels own ultra-high colloidal stability and non-specific protein adsorption resistance as a result of the incorporation of zwitterionic groups. Meanwhile, they exhibit sensitive temperature-induced swelling/collapse transition in aqueous solution and excellent redox-degradability ascribed to the presence of disulfide bonds. The nanogels loaded with anticancer drug doxorubicin (DOX) exhibit low leakage of DOX under physiological conditions (merely 23.8 % within 24 h), whereas striking release amount of DOX under reducing conditions combined with elevated temperature (93.4 % within 24 h). The measurement of cell viability showed that the cytotoxicity of blank nanogels to tumor cells (HeLa cells) was negligible, while the nanogels loaded with DOX had a prominent inhibitory impact on tumor cells.

Redox-Channeling Polydopamine-Ferrocene (PDA-Fc) Coating To Confer Context-Dependent and Photothermal Antimicrobial Activities.

Microbial disinfection associated with medical device surfaces has been an increasing need, and surface modification strategies such as antibacterial coatings have gained great interest. Here, we report the development of polydopamine-ferrocene (PDA-Fc)-functionalized TiO2 nanorods (Ti-Nd-PDA-Fc) as a context-dependent antibacterial system on implant to combat bacterial infection and hinder biofilm formation. In this work, two synergistic antimicrobial mechanisms of the PDA-Fc coating are proposed. First, the PDA-Fc coating is redox-active and can be locally activated to release antibacterial reactive oxygen species (ROS), especially ·OH in response to the acidic microenvironment induced by bacteria colonization and host immune responses. The results demonstrate that redox-based antimicrobial activity of Ti-Nd-PDA-Fc offers antibacterial efficacy of over 95 and 92% against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), respectively. Second, the photothermal effect of PDA can enhance the antibacterial capability upon near-infrared (NIR) irradiation, with over 99% killing efficacy against MRSA and E. coli, and even suppress the formation of biofilm through both localized hyperthermia and enhanced ·OH generation. Additionally, Ti-Nd-PDA-Fc is biocompatible when tested with model pre-osteoblast MC-3T3 E1 cells and promotes cell adhesion and spreading presumably due to its nanotopographical features. The MRSA-infected wound model also indicates that Ti-Nd-PDA-Fc with NIR irradiation can effectively eliminate bacterial infection and suppress host inflammatory responses. We believe that this study demonstrates a simple means to create biocompatible redox-active coatings that confer context-dependent antibacterial activities to implant surfaces.

Tag-Free Site-Specific BMP-2 Immobilization with Long-Acting Bioactivities via a Simple Sugar-Lectin Interaction.

The construction of a biomaterial matrix with biological properties is of great importance to developing functional materials for clinical use. However, the site-specific immobilization of growth factors to endow materials with bioactivities has been a challenge to date. Considering the wide existence of glycosylation in mammalian proteins or recombinant proteins, we establish a bioaffinity-based protein immobilization strategy (bioanchoring method) utilizing the native sugar-lectin interaction between concanavalin A (Con A) and the oligosaccharide chain on glycosylated bone morphogenetic protein-2 (GBMP-2). The interaction realizes the site-specific immobilization of GBMP-2 to a substrate modified with Con A while preserving its bioactivity in a sustained and highly efficient way, as evidenced by its enhanced ability to induce osteodifferentiation compared with that of the soluble GBMP-2. Moreover, the surface with Con A-bioanchored GBMP-2 can be reused to stimulate multiple batches of C2C12 cells to differentiate almost to the same degree. Even after 4 month storage at 4 °C in phosphate-buffered saline (PBS), the Con A-bioanchored GBMP-2 still maintains the bioactivity to stimulate the differentiation of C2C12 cells. Furthermore, the ectopic ossification test proves the in vivo bioactivity of bioanchored GBMP-2. Overall, our results demonstrate that the tag-free and site (i.e., sugar chain)-specific protein immobilization strategy represents a simple and generic alternative, which is promising to apply for other glycoprotein immobilization and application. It should be noted that although the lectin we utilized can only bind to d-mannose/d-glucose, the diversity of the lectin family assures that a specific lectin could be offered for other sugar types, thus expanding the applicable scope further.

Coupling PEG-LZM polymer networks with polyphenols yields suturable biohydrogels for tissue patching.

Poor mechanical performances severely limit the application of hydrogels in vivo; for example, it is difficult to perform a very common suturing operation on hydrogels during surgery. There is a growing demand to improve the mechanical properties of hydrogels for broadening their clinical applications. Natural polyphenols can match the potential toughening sites in our previously reported PEG-lysozyme (LZM) hydrogel because polyphenols have unique structural units including a hydroxyl group and an aromatic ring that can interact with PEG via hydrogen bonding and form hydrophobic interactions with LZM. By utilizing polyphenols as noncovalent crosslinkers, the resultant PEG-LZM-polyphenol hydrogel presents super toughness and high elasticity in comparison to pristine PEG-LZM with no obvious changes in the initial shape, and it can even withstand the high pressure from sutures. At the same time, the mechanical properties could be widely adjusted by varying the polyphenol concentration. Interestingly, the PEG-LZM-polyphenol hydrogel has a higher water content than other polyphenol-toughened hydrogels, which may better meet the clinical needs for hydrogel materials. Besides, the introduction of polyphenols endows the hydrogel with improved antibacterial and anti-inflammatory abilities. Finally, the PEG-LZM-polyphenol (tannic acid) hydrogel was demonstrated to successfully patch a rabbit myocardial defect by suturing for 4 weeks and improve the wound healing and heart function recovery compared to autologous muscle patches.