Nonadjacent Wireless Electrotherapy for Tissue Repair by a 3D-Printed Bioresorbable Fully Soft Triboelectric Nanogenerator.

Electrotherapy is a promising tissue repair technique. However, electrotherapy devices are frequently complex and must be placed adjacent to injured tissue, thereby limiting their clinical application. Here, we propose a general strategy to facilitate tissue repair by modulating endogenous electric fields with nonadjacent (approximately 44 mm) wireless electrotherapy through a 3D-printed entirely soft and bioresorbable triboelectric nanogenerator based stimulator, without any electrical accessories, which has biomimetic mechanical properties similar to those of soft tissue. In addition, the feasibility of using the stimulator to construct an electrical double layer with tissue for nonadjacent wireless electrotherapy was demonstrated by skin and muscle injury models. The treated groups showed significantly improved tissue repair compared with the control group. In conclusion, we developed a promising electrotherapy strategy and may inspire next-generation electrotherapy for tissue repair.

An Encapsulation-Free and Hierarchical Porous Triboelectric Scaffold with Dynamic Hydrophilicity for Efficient Cartilage Regeneration.

Tissue engineering and electrotherapy are two promising methods to promote tissue repair. However, their integration remains an underexplored area, because their requirements on devices are usually distinct. Triboelectric nanogenerators (TENGs) have shown great potential to develop self-powered devices. However, due to their susceptibility to moisture, TENGs have to be encapsulated in vivo. Therefore, existing TENGs cannot be employed as tissue engineering scaffolds, which require direct interaction with surrounding cells. Here, the concept of triboelectric scaffolds (TESs) is proposed. Poly(glycerol sebacate), a biodegradable and relatively hydrophobic elastomer, is selected as the matrix of TESs. Each hydrophobic micropore in multi-hierarchical porous TESs efficiently serves as a moisture-resistant working unit of TENGs. Integration of tons of micropores ensures the electrotherapy ability of TESs in vivo without encapsulation. Originally hydrophobic TESs are degraded by surface erosion and transformed into hydrophilic surfaces, facilitating their role as tissue engineering scaffolds. Notably, TESs seeded with chondrocytes obtain dense and large matured cartilages after subcutaneous implantation in nude mice. Importantly, rabbits with osteochondral defects receiving TES implantation show favorable hyaline cartilage regeneration and complete cartilage healing. This work provides a promising electronic biomedical device and will inspire a series of new in vivo applications.

Silane-functionalized polyionenes-coated cotton fabrics with potent antimicrobial and antiviral activities.

Bacterial and viral infections are posing a huge burden on healthcare industry. Existing antimicrobial textiles that are used to prevent infection transmission are lack of durability and antiviral activity. Here, we report on silane-functionalized polyionenes-coated cotton textiles with durable potent antimicrobial and antiviral activities. To obtain silane-functionalized polyionenes, silane group-containing monomers were synthesized and used to polymerize with co-monomers. These polyionenes were then conjugated onto the surface of cotton fabrics via covalent bonds. These polymers demonstrated broad-spectrum antimicrobial activity against various types of pathogenic microbes as evidenced by low effective concentration. The fabrics coated with these polymers exhibited potent bactericidal (>99.999%) and virucidal (7-log PFU reduction) activities. In addition, the antimicrobial efficacy was still more than 92% even after 50 times of washing. Evaluation of cytocompatibility and skin compatibility of the polymer-coated cotton fabrics in mice revealed that they were compatible with cells and mouse skin, and neither erythema nor edema was found in the area that was in contact with the polymer-coated fabrics. The silane-functionalized polyionenes are potentially promising antimicrobial and antiviral coating materials for textiles and other applications to prevent microbial and viral infections.

Structural characterization and directed modification of Sus scrofa SAMHD1 reveal the mechanism underlying deoxynucleotide regulation.

Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is an intrinsic antiviral restriction factor known to play a vital role in preventing multiple viral infections and in the control of the cellular deoxynucleoside triphosphate (dNTP) pool. Human and mouse SAMHD1 have both been extensively studied; however, our knowledge on porcine SAMHD1 is limited. Here, we report our findings from comprehensive structural and functional studies on porcine SAMHD1. We determined the crystal structure of porcine SAMHD1 and showed that it forms a symmetric tetramer. Moreover, we modified the deoxynucleotide triphosphohydrolase (dNTPase) activity of SAMHD1 by site-directed mutagenesis based on the crystal structure, and obtained an artificial dimeric enzyme possessing high dNTPase activity. Taken together, our results define the mechanism underlying dNTP regulation and provide a deeper understanding of the regulation of porcine SAMHD1 functions. Directed modification of key residues based on the protein structure enhances the activity of the enzyme, which will be beneficial in the search for new antiviral strategies and for future translational applications.

Large-Scale and Rapid Preparation of Nanofibrous Meshes and Their Application for Drug-Loaded Multilayer Mucoadhesive Patch Fabrication for Mouth Ulcer Treatment.

Electrospinning provides a simple and convenient method to fabricate nanofibrous meshes. However, the nanofiber productivity is often limited to the laboratory scale, which cannot satisfy the requirements of practical application. In this study, we developed a novel needleless electrospinning spinneret based on a double-ring slit to fabricate drug-loaded nanofibrous meshes. In contrast to the conventional single-needle electrospinning spinneret, our needless spinneret can significantly improve nanofiber productivity due to the simultaneous formation of multiple jets during electrospinning. Curcumin-loaded poly(l-lactic acid) (PLLA) nanofiber meshes with various concentrations and on the large scale were manufactured by employing our developed needleless spinneret-based electrospinning device. We systematically investigated the drug release behaviors, antioxidant properties, anti-inflammatory attributes, and cytotoxicity of the curcumin-loaded PLLA nanofibrous meshes. Furthermore, a bilayer nanofibrous composite mesh was successfully generated by electrospinning curcumin-loaded PLLA solution and diclofenac sodium loaded poly(ethylene oxide) solution in a predetermined time sequence, which revealed potent antibacterial properties. Subsequently, novel mucoadhesive patches were assembled by combining the bilayer composite nanofibrous meshes with (hydroxypropyl)methyl cellulose based mucoadhesive film. The multilayered mucoadhesive patch has excellent adhesion properties on the porcine buccal mucosa. Overall, our double-ring slit spinneret can provide a novel method to rapidly produce large-scale drug-loaded nanofibrous meshes to fabricate mucoadhesive patches. The multiple-layered mucoadhesive patches enable the incorporation of multiple drugs with different targets of action, such as analgesic, anti-inflammatory, and antimicrobial compounds, for mouth ulcer or other oral disease treatments.

Hydrophilic and degradable polyesters based on l-aspartic acid with antibacterial properties for potential application in hernia repair.

A polyester hernia patch has received extensive attention in mesh hernia repair. However, it is still a challenge to develop polyester-based implants with inherent antibacterial properties due to the lack of active functional groups. In this study, poly(butylene succinate-co-butylene aspartate) (PBSA) was constructed by introducing aspartic acid on a polybutylene succinate (PBS) polyester chain (PBSA). Antimicrobial treatment was conducted by grafting levofloxacin (Lv) on the surface of a PBSA polymer (PBSA-g-Lv). In vitro antibacterial test results showed that PBSA-g-Lv had sufficient local antimicrobiotic effects against Staphylococcus aureus and Escherichia coli and no side effect on L929 cells was observed. Furthermore, almost no change was observed in the thermodynamic properties of PBS and PBSA; in vivo tests demonstrated that this contact-active antibacterial PBSA-g-Lv nanofiber is a promising material to fulfill the dual functions of promoting tissue regeneration and preventing bacterial infection. The presented data confirmed that an antibiotic surface modification of PBSA polyesters was expected to be used as hernia repair materials.

Synthesis and characterization of arginine-NIPAAm hybrid hydrogel as wound dressing: In vitro and in vivo study.

A multi-functional hybrid hydrogel P(M-Arg/NIPAAm) with temperature response, anti-protein adsorption and antibacterial properties was prepared and applied as wound dressing. The hydrogel was carried out by free radical copolymerization of methacrylate arginine (M-Arg) and N-isopropyl acrylamide (NIPAAm) monomers using N,N'-methylene bisacrylamide as a crosslinker, and ammonium persulfate/N,N,N', N'-tetramethylethylenediamine as the redox initiator. To endow the antimicrobial property, chlorhexidine diacetate (CHX) was preloaded into the hydrogel and polyhexamethylene guanidine phosphate (PHMG) was grafted on the hydrogel surface, respectively. The antimicrobial property of two series of hydrogels was evaluated and compared. The successful synthesis of M-Arg, PHMG and hydrogels was proved by 13C NMR, 1H NMR and FTIR spectroscopy. The hydrogel morphology characterized by scanning electron microscopy confirmed that the homogeneous porous and interconnected structures of the hydrogels. The swelling, protein adsorption property, in vitro release of CHX, antimicrobial assessment, cell viability as well as in vivo wound healing in a mouse model were studied. The results showed the nontoxicity and antimicrobial P(M-Arg/NIPAAm) hydrogel accelerated the full-thickness wound healing process and had the potential application in wound dressing. STATEMENT OF SIGNIFICANCE: Despite the zwitterionic characteristic and biocompatible property of arginine based hydrogels, the brittle behavior and non-transparency still remain as a significant problem for wound dressing. Furthermore promoting the antibacterial property of the zwitterionic hydrogel is also necessary to prevent the bacterial colonization and subsequent wound infection. Therefore, we created a hybrid hydrogel combined methacrylate arginine (M-Arg) and N-isopropyl acrylamide (NIPAAm). NIPAAm improves transparency and mechanical property as well as acts as a temperature-response drug release system. Additionally, chlorhexidine (CHX) was preloaded into the hydrogels and polyhexamethylene guanidine phosphate (PHMG) was grafted on the hydrogel surface, respectively, which make the hydrogel useful as a favorable antibacterial dressing. The hybrid hydrogel has a combination effect of biocompatibility, environmentally responsive transformation behavior, biodegradability, anti-protein adsorption and antimicrobial properties. This report proposes the preparation of P(M-Arg/NIPAAm) hydrogel that has a great potential for wound healing.

Nanofiber-structured hydrogel yarns with pH-response capacity and cardiomyocyte-drivability for bio-microactuator application.

Polymeric hydrogels have great potential in soft biological micro-actuator applications. However, inappropriate micro-architecture, non-anisotropy, weak biomechanics, and inferior response behaviors limit their development. In this study, we designed and manufactured novel polyacrylonitrile (PAN)-based hydrogel yarns composed with uniaxially aligned nanofibers. The nanofibrous hydrogel yarns possessed anisotropic architecture and robust mechanical properties with flexibility, and could be assembled into defined scaffold structures by subsequent processes. The as-prepared hydrogel yarns showed excellent pH response behaviors, with around 100% maximum length and 900% maximum diameter changes, and the pH response was completed within several seconds. Moreover, the hydrogel yarns displayed unique cell-responsive abilities to promote the cell adhesion, proliferation, and smooth muscle differentiation of human adipose derived mesenchymal stem cells (HADMSC). Chicken cardiomyocytes were further seeded onto our nanofibrous hydrogel yarns to engineer living cell-based microactuators. Our results demonstrated that the uniaxially aligned nanofibrous networks within the hydrogel yarns were the key characteristics leading to the anisotropic organization of cardiac cells, and improved sarcomere organization, mimicking the cardiomyocyte bundles in the native myocardium. The construct is capable of sustaining spontaneous cardiomyocyte pumping behaviors for 7days. Our PAN-based nanofibrous hydrogel yarns are attractive for creating linear microactuators with pH-response capacity and biological microactuators with cardiomyocyte-drivability. STATEMENT OF SIGNIFICANCE: A mechanically robust polyacrylonitrile-based nanofibrous hydrogel yarn is fabricated by using a modified electrospinning setup in combination with chemical modification processes. The as-prepared hydrogel yarn possesses a uniaxially aligned nanofiber microarchitecture and supports a rapid, pH-dependent expansion/contraction response within a few seconds. Embryonic cardiomyocytes-seeded hydrogel yarn improves the sarcomere organization and mimics the cardiomyocyte bundles in the native myocardium, which sustains spontaneous cardiomyocyte pumping behaviors. The nanofibrous hydrogel yarn has several advantages over traditional bulk hydrogel scaffolds in terms of robust biomechanics, anisotropic aligned architecture, and superior pH response behaviors. Our nanofibrous hydrogel yarn holds the potential to be developed into novel linear and biological microactuators for various biomedical applications.

Living nano-micro fibrous woven fabric/hydrogel composite scaffolds for heart valve engineering.

Regeneration and repair of injured or diseased heart valves remains a clinical challenge. Tissue engineering provides a promising treatment approach to facilitate living heart valve repair and regeneration. Three-dimensional (3D) biomimetic scaffolds that possess heterogeneous and anisotropic features that approximate those of native heart valve tissue are beneficial to the successful in vitro development of tissue engineered heart valves (TEHV). Here we report the development and characterization of a novel composite scaffold consisting of nano- and micro-scale fibrous woven fabrics and 3D hydrogels by using textile techniques combined with bioactive hydrogel formation. Embedded nano-micro fibrous scaffolds within hydrogel enhanced mechanical strength and physical structural anisotropy of the composite scaffold (similar to native aortic valve leaflets) and also reduced its compaction. We determined that the composite scaffolds supported the growth of human aortic valve interstitial cells (HAVIC), balanced the remodeling of heart valve ECM against shrinkage, and maintained better physiological fibroblastic phenotype in both normal and diseased HAVIC over single materials. These fabricated composite scaffolds enable the engineering of a living heart valve graft with improved anisotropic structure and tissue biomechanics important for maintaining valve cell phenotypes. STATEMENT OF SIGNIFICANCE: Heart valve-related disease is an important clinical problem, with over 300,000 surgical repairs performed annually. Tissue engineering offers a promising strategy for heart valve repair and regeneration. In this study, we developed and tissue engineered living nano-micro fibrous woven fabric/hydrogel composite scaffolds by using textile technique combined with bioactive hydrogel formation. The novelty of our technique is that the composite scaffolds can mimic physical structure anisotropy and the mechanical strength of natural aortic valve leaflet. Moreover, the composite scaffolds prevented the matrix shrinkage, which is major problem that causes the failure of TEHV, and better maintained physiological fibroblastic phenotype in both normal and diseased HAVIC. This work marks the first report of a combination composite scaffold using 3D hydrogel enhanced by nano-micro fibrous woven fabric, and represents a promising tissue engineering strategy to treat heart valve injury.

Evaluation of electrospun biomimetic substrate surface-decorated with nanohydroxyapatite precipitation for osteoblasts behavior.

To engineer bone tissue, it is crucial to design scaffolds with micro- and nano-sized architecture imitating approximate hierarchical structure of native bone, and afford desirable biological properties by introducing biocompatible polymers and bioceramics into the scaffolds. Here, a novel scaffold consisting of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV)/polyaspartic acid (PAA) was fabricated by electrospinning and nano-hydroxyapatite (nHA) was deposited by calcium-phosphate dipping process for bone tissue regeneration. Characterization of the prepared nanofibers revealed the formation of definite nHA crystal, porous structure of membranes, improved wettability with nHA deposition and satisfied mechanical properties. Human fetal osteoblasts were cultured on nanofibers and experienced in vitro evaluations of cell proliferation, adhesion and mineralization confirming the non-cytotoxicity and biocompatibility of scaffolds. Cells proliferation rate and ALP expression on PHBV/PAA-nHA were 36.40% and 40.14% higher than on PHBV/PAA, respectively. The utmost significance of this study is introducing bioactive PAA-nHA on polymeric nanofibers to regulate and improve specific cells adhesion, proliferation and mineralization of osteoblasts. All results indicate PHBV/PAA-nHA nanofibrous scaffolds can be applied as biomimetic platform for bone tissue repairation with appropriate physico-chemical properties, osteoinductivity and osteoconductivity.

Fabrication of Aligned Nanofiber Polymer Yarn Networks for Anisotropic Soft Tissue Scaffolds.

Nanofibrous scaffolds with defined architectures and anisotropic mechanical properties are attractive for many tissue engineering and regenerative medicine applications. Here, a novel electrospinning system is developed and implemented to fabricate continuous processable uniaxially aligned nanofiber yarns (UANY). UANY were processed into fibrous tissue scaffolds with defined anisotropic material properties using various textile-forming technologies, i.e., braiding, weaving, and knitting techniques. UANY braiding dramatically increased overall stiffness and strength compared to the same number of UANY unbraided. Human adipose derived stem cells (HADSC) cultured on UANY or woven and knitted 3D scaffolds aligned along local fiber direction and were >90% viable throughout 21 days. Importantly, UANY supported biochemical induction of HADSC differentiation toward smooth muscle and osteogenic lineages. Moreover, we integrated an anisotropic woven fiber mesh within a bioactive hydrogel to mimic the complex microstructure and mechanical behavior of valve tissues. Human aortic valve interstitial cells (HAVIC) and human aortic root smooth muscle cells (HASMC) were separately encapsulated within hydrogel/woven fabric composite scaffolds for generating scaffolds with anisotropic biomechanics and valve ECM like microenvironment for heart valve tissue engineering. UANY have great potential as building blocks for generating fiber-shaped tissues or tissue microstructures with complex architectures.

Poly-3-hydroxybutyrate-co-3-hydroxyvalerate containing scaffolds and their integration with osteoblasts as a model for bone tissue engineering.

Nano/micro engineered polymeric materials offer expansive scope of biomimetic scaffolds for bone tissue engineering especially those involving electrospun biodegradable nanofibers incorporated with inorganic nanoparticles, thus mimicking the extracellular matrix of bone both structurally and chemically. For the first time, poly-3-hydroxybutyrate-co-3-hydroxyvalerate containing natural poly-(α, ß)-DL-aspartic acid and inorganic hydroxyapatite nanofibers were fabricated using poly-3-hydroxybutyrate-co-3-hydroxyvalerate: poly-(α, ß)-DL-aspartic acid at a ratio of 80:20 (w/w) added with 1% (w/v) of hydroxyapatite, by the process of electrospinning. The surface morphology, chemical, and mechanical properties of electrospun poly-3-hydroxybutyrate-co-3-hydroxyvalerate, poly-3-hydroxybutyrate-co-3-hydroxyvalerate/poly-(α, ß)-DL-aspartic acid, and poly-3-hydroxybutyrate-co-3-hydroxyvalerate/poly-(α, ß)-DL-aspartic acid/hydroxyapatite nanofibers were characterized by using field emission scanning electron microscope, Fourier transform infrared spectroscopy, and tensile tester, respectively. Human fetal osteoblasts were cultured on different nanofibrous scaffolds for evaluating the cell proliferation, alkaline phosphatase activity, and mineralization. Cells on poly-3-hydroxybutyrate-co-3-hydroxyvalerate/poly-(α, ß)-DL-aspartic acid/hydroxyapatite scaffolds demonstrated higher proliferation (30.10%) and mineral deposition (37.60%) than the cells grown on pure poly-3-hydroxybutyrate-co-3-hydroxyvalerate scaffolds. Obtained results highlight the synergistic effect of poly-3-hydroxybutyrate-co-3-hydroxyvalerate, poly-(α, ß)-DL-aspartic acid, and hydroxyapatite towards the enhancement of the osteoinductivity and osteoconductivity of human fetal osteoblasts, demonstrating the appropriate physicochemical and biological properties of poly-3-hydroxybutyrate-co-3-hydroxyvalerate/poly-(α, ß)-DL-aspartic acid/hydroxyapatite nanofibers to function as a substrate for bone tissue regeneration.

Biocomposite scaffolds for bone regeneration: Role of chitosan and hydroxyapatite within poly-3-hydroxybutyrate-co-3-hydroxyvalerate on mechanical properties and in vitro evaluation.

Bio-engineered scaffolds for bone tissue regeneration is an exploding area of research mainly because they can satisfy the essential demands and current challenges in bone replacement therapies, by imitating the extracellular matrix (ECM) of the native bone. We fabricated bio-composite nanofibrous scaffolds with a blend of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), chitosan (CTS) and hydroxyapatite (HA) during this study. Morphological evaluation confirmed the fiber diameters of PHBV, PHBV/CTS (90:10), PHBV/CTS/HA4 (85.5:9.5:5) and PHBV/CTS/HA8 (81:9:10) as 405 ± 74 nm, 334 ± 82 nm, 316 ± 103 nm and 256 ± 110 nm, respectively. The PHBV/CTS/HA4 and PHBV/CTS/HA8 scaffolds were capable of enduring the long term culture of human fetal osteoblasts (hFOB) with ultimate tensile strength of 3.55 ± 0.22 MPa and 4.19 ± 0.19 MPa, respectively. The proliferation of osteoblasts on PHBV/CTS/HA8 scaffold was found 34.10% higher than that on PHBV scaffold on day 20. Cell maturation identified by alkaline phosphatase activity on day 20 was significantly higher on PHBV/CTS/HA8 scaffold than that on PHBV scaffold. The cells on PHBV/CTS/HA8 scaffold also acquired higher mineral deposition (25.79%) than the mineral deposition on PHBV scaffold by day 20, confirmed by EDX analysis. Based on the results, we concluded that the electrospun PHBV/CTS/HA8 scaffolds hold great potential to promote the regeneration of bone tissue due to the synergistic effect of chitosan and HA, whereby chitosan provided cell recognition sites while HA acted as a chelating agent for organizing the apatite-like mineralization.

Guiding the orientation of smooth muscle cells on random and aligned polyurethane/collagen nanofibers.

Fabricating scaffolds that can simulate the architecture and functionality of native extracellular matrix is a huge challenge in vascular tissue engineering. Various kinds of materials are engineered via nano-technological approaches to meet the current challenges in vascular tissue regeneration. During this study, nanofibers from pure polyurethane and hybrid polyurethane/collagen in two different morphologies (random and aligned) and in three different ratios of polyurethane:collagen (75:25; 50:50; 25:75) are fabricated by electrospinning. The fiber diameters of the nanofibrous scaffolds are in the range of 174-453 nm and 145-419 for random and aligned fibers, respectively, where they closely mimic the nanoscale dimensions of native extracellular matrix. The aligned polyurethane/collagen nanofibers expressed anisotropic wettability with mechanical properties which is suitable for regeneration of the artery. After 12 days of human aortic smooth muscle cells culture on different scaffolds, the proliferation of smooth muscle cells on hybrid polyurethane/collagen (3:1) nanofibers was 173% and 212% higher than on pure polyurethane scaffolds for random and aligned scaffolds, respectively. The results of cell morphology and protein staining showed that the aligned polyurethane/collagen (3:1) scaffold promote smooth muscle cells alignment through contact guidance, while the random polyurethane/collagen (3:1) also guided cell orientation most probably due to the inherent biochemical composition. Our studies demonstrate the potential of aligned and random polyurethane/collagen (3:1) as promising substrates for vascular tissue regeneration.

Stem cell differentiation on electrospun nanofibrous substrates for vascular tissue engineering.

Nanotechnology has enabled the engineering of a variety of materials to meet the current challenges and requirements in vascular tissue regeneration. In our study, poly-L-lactide (PLLA) and hybrid PLLA/collagen (PLLA/Coll) nanofibers (3:1 and 1:1) with fiber diameters of 210 to 430 nm were fabricated by electrospinning. Their morphological, chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR), and tensile instrument, respectively. Bone marrow derived mesenchymal stem cells (MSCs) seeded on electrospun nanofibers that are capable of differentiating into vascular cells have great potential for repair of the vascular system. We investigated the potential of MSCs for vascular cell differentiation in vitro on electrospun PLLA/Coll nanofibrous scaffolds using endothelial differentiation media. After 20 days of culture, MSC proliferation on PLLA/Coll(1:1) scaffolds was found 256% higher than the cell proliferation on PLLA scaffolds. SEM images showed that the MSC differentiated endothelial cells on PLLA/Coll scaffolds showed cobblestone morphology in comparison to the fibroblastic type of undifferentiated MSCs. The functionality of the cells in the presence of 'endothelial induction media', was further demonstrated from the immunocytochemical analysis, where the MSCs on PLLA/Coll (1:1) scaffolds differentiated to endothelial cells and expressed the endothelial cell specific proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and Von Willebrand factor (vWF). From the results of the SEM analysis and protein expression studies, we concluded that the electrospun PLLA/Coll nanofibers could mimic the native vascular ECM environment and might be promising substrates for potential application towards vascular regeneration.