Highly conductive, stretchable, and biocompatible graphene oxide biocomposite hydrogel for advanced tissue engineering.

The importance of hydrogels in tissue engineering cannot be overemphasized due to their resemblance to the native extracellular matrix. However, natural hydrogels with satisfactory biocompatibility exhibit poor mechanical behavior, which hampers their application in stress-bearing soft tissue engineering. Here, we describe the fabrication of a double methacrylated gelatin bioink covalently linked to graphene oxide (GO) via a zero-length crosslinker, digitally light-processed (DLP) printable into 3D complex structures with high fidelity. The resultant natural hydrogel (GelGOMA) exhibits a conductivity of 15.0 S m-1as a result of the delocalization of theπ-orbital from the covalently linked GO. Furthermore, the hydrogel shows a compressive strength of 1.6 MPa, and a 2.0 mm thick GelGOMA can withstand a 1.0 kg ms-1momentum. The printability and mechanical strengths of GelGOMAs were demonstrated by printing a fish heart with a functional fluid pumping mechanism and tricuspid valves. Its biocompatibility, electroconductivity, and physiological relevance enhanced the proliferation and differentiation of myoblasts and neuroblasts and the contraction of human-induced pluripotent stem cell-derived cardiomyocytes. GelGOMA demonstrates the potential for the tissue engineering of functional hearts and wearable electronic devices.

A digital light processing 3D-printed artificial skin model and full-thickness wound models using silk fibroin bioink.

A three-dimensional (3D) artificial skin model offers diverse platforms for skin transplantation, disease mechanisms, and biomaterial testing for skin tissue. However, implementing physiological complexes such as the neurovascular system with living cells in this stratified structure is extremely difficult. In this study, full-thickness skin models were fabricated from methacrylated silk fibroin (Silk-GMA) and gelatin (Gel-GMA) seeded with keratinocytes, fibroblasts, and vascular endothelial cells representing the epidermis and dermis layers through a digital light processing (DLP) 3D printer. Printability, mechanical properties, and cell viability of the skin hydrogels fabricated with different concentrations of Silk-GMA and Gel-GMA were analyzed to find the optimal concentrations for the 3D printing of the artificial skin model. After the skin model was DLP-3D printed using Gel-GMA 15% + Silk-GMA 5% bioink, cultured, and air-lifted for four weeks, well-proliferated keratinocytes and fibroblasts were observed in histological analysis, and increased expressions of Cytokeratin 13, Phalloidin, and CD31 were noted in immunofluorescence staining. Furthermore, full-thickness skin wound models were 3D-printed to evaluate the wound-healing capabilities of the skin hydrogel. When the epidermal growth factor (EGF) was applied, enhanced wound healing in the epidermis and dermis layer with the proliferation of keratinocytes and fibroblasts was observed. Also, the semi-quantitative reverse transcription-polymerase chain reaction revealed increased expression of Cytokeratin 13, fibroblast growth factor, and CD31 in the EGF-treated group relative to the control group. The DLP 3D-printed artificial skin model was mechanically stable and biocompatible for more than four weeks, demonstrating the potential for application in skin tissue engineering. STATEMENT OF SIGNIFICANCE: A full-thickness artificial skin model was 3D-printed in this study with a digital light processing technique using silk fibroin and gelatin, which mimics the structural and cellular compositions of the human skin. The 3D-printed skin hydrogel ensured the viability of the cells in the skin layers that proliferated well after air-lifting cultivation, shown in the histological analysis and immunofluorescence stainings. Furthermore, full-thickness skin wound models were 3D-printed to evaluate the wound healing capabilities of the skin hydrogel, which demonstrated enhanced wound healing in the epidermis and dermis layer with the application of epidermal growth factor on the wound compared to the control. The bioengineered hydrogel expands the applicability of artificial skin models for skin substitutes, wound models, and drug testing.

Fluidic integrated 3D bioprinting system to sustain cell viability towards larynx fabrication.

Herein, we report the first study to create a three-dimensional (3D) bioprinted artificial larynx for whole-laryngeal replacement. Our 3D bio-printed larynx was generated using extrusion-based 3D bioprinter with rabbit's chondrocyte-laden gelatin methacryloyl (GelMA)/glycidyl-methacrylated hyaluronic acid (GMHA) hybrid bioink. We used a polycaprolactone (PCL) outer framework incorporated with pores to achieve the structural strength of printed constructs, as well as to provide a suitable microenvironment to support printed cells. Notably, we established a novel fluidics supply (FS) system that simultaneously supplies basal medium together with a 3D bioprinting process, thereby improving cell survival during the printing process. Our results showed that the FS system enhanced post-printing cell viability, which enabled the generation of a large-scale cell-laden artificial laryngeal framework. Additionally, the incorporation of the PCL outer framework with pores and inner hydrogel provides structural stability and sufficient nutrient/oxygen transport. An animal study confirmed that the transplanted 3D bio-larynx successfully maintained the airway. With further development, our new strategy holds great potential for fabricating human-scale larynxes with in vivo-like biological functions for laryngectomy patients.

Functional Skeletal Muscle Regeneration Using Muscle Mimetic Tissue Fabricated by Microvalve-Assisted Coaxial 3D Bioprinting.

3D-printed artificial skeletal muscle, which mimics the structural and functional characteristics of native skeletal muscle, is a promising treatment method for muscle reconstruction. Although various fabrication techniques for skeletal muscle using 3D bio-printers are studied, it is still challenging to build a functional muscle structure. A strategy using microvalve-assisted coaxial 3D bioprinting in consideration of functional skeletal muscle fabrication is reported. The unit (artificial muscle fascicle: AMF) of muscle mimetic tissue is composed of a core filled with medium-based C2C12 myoblast aggregates as a role of muscle fibers and a photo cross-linkable hydrogel-based shell as a role of connective tissue in muscles that enhances printability and cell adhesion and proliferation. Especially, a microvalve system is applied for the core part with even cell distribution and strong cell-cell interaction. This system enhances myotube formation and consequently shows spontaneous contraction. A multi-printed AMF (artificial muscle tissue: AMT) as a piece of muscle is implanted into the anterior tibia (TA) muscle defect site of immunocompromised rats. As a result, the TA-implanted AMT responds to electrical stimulation and represents histologically regenerated muscle tissue. This microvalve-assisted coaxial 3D bioprinting shows a significant step forward to mimicking native skeletal muscle tissue.

Biocompatible fluorescent silk fibroin bioink for digital light processing 3D printing.

Chemically modified silk fibroin (SF) bioink has been used for three-dimensional (3D) bioprinting in tissue engineering because of its biocompatibility and printability. Also, fluorescent silk fibroin (FSF) from transgenic silkworms has been recently applied in biomedicine because of its fluorescence property. However, the fabrication of fluorescent hydrogel from FSF has not been elucidated. In this study, we showed the fabrication of a digital light processing (DLP) printable bioink from a chemically modified FSF. This bioink was fabricated by covalent conjugation of FSF and glycidyl methacrylate (GMA) and can be printed into various structures, such as the brain, ear, hand, lung, and internal organs. The physical properties of glycidyl methacrylated fluorescent silk fibroin (FSGMA) hydrogel was like the glycidyl methacrylated non-fluorescent silk fibroin (SGMA) hydrogel. The FSGMA hydrogel significantly retains its fluorescence property and has excellent biocompatibility. All these properties make FSGMA hydrogel a potent tool in encapsulated cell tracking and observing the scaffolds' degradation in vivo. This study suggested that our 3D DLP printable FSF bioink could play a promising role in the biomedical field.

Silk Fibroin-Based Biomaterials for Hemostatic Applications.

Hemostasis plays an essential role in all surgical procedures. Uncontrolled hemorrhage is the primary cause of death during surgeries, and effective blood loss control can significantly reduce mortality. For modern surgeons to select the right agent at the right time, they must understand the mechanisms of action, the effectiveness, and the possible adverse effects of each agent. Over the past decade, various hemostatic agents have grown intensely. These agents vary from absorbable topical hemostats, including collagen, gelatins, microfibrillar, and regenerated oxidized cellulose, to biologically active topical hemostats such as thrombin, biological adhesives, and other combined agents. Commercially available products have since expanded to include topical hemostats, surgical sealants, and adhesives. Silk is a natural protein consisting of fibroin and sericin. Silk fibroin (SF), derived from silkworm Bombyx mori, is a fibrous protein that has been used mostly in fashion textiles and surgical sutures. Additionally, SF has been widely applied as a potential biomaterial in several biomedical and biotechnological fields. Furthermore, SF has been employed as a hemostatic agent in several studies. In this review, we summarize the several morphologic forms of SF and the latest technological advances on the use of SF-based hemostatic agents.

3D bioprinted silk fibroin hydrogels for tissue engineering.

The development of biocompatible and precisely printable bioink addresses the growing demand for three-dimensional (3D) bioprinting applications in the field of tissue engineering. We developed a methacrylated photocurable silk fibroin (SF) bioink for digital light processing 3D bioprinting to generate structures with high mechanical stability and biocompatibility for tissue engineering applications. Procedure 1 describes the synthesis of photocurable methacrylated SF bioink, which takes 2 weeks to complete. Digital light processing is used to fabricate 3D hydrogels using the bioink (1.5 h), which are characterized in terms of methacrylation, printability, mechanical and rheological properties, and biocompatibility. The physicochemical properties of the bioink can be modulated by varying photopolymerization conditions such as the degree of methacrylation, light intensity, and concentration of the photoinitiator and bioink. The versatile bioink can be used broadly in a range of applications, including nerve tissue engineering through co-polymerization of the bioink with graphene oxide, and for wound healing as a sealant. Procedure 2 outlines how to apply 3D-printed SF hydrogels embedded with chondrocytes and turbinate-derived mesenchymal stem cells in one specific in vivo application, trachea tissue engineering, which takes 2-9 weeks.

A digital light processing 3D printed magnetic bioreactor system using silk magnetic bioink.

Among various bioreactors used in the field of tissue engineering and regenerative medicine, a magnetic bioreactor is more capable of providing steady force to the cells while avoiding direct manipulation of the materials. However, most of them are complex and difficult to fabricate, with drawbacks in terms of consistency and biocompatibility. In this study, a magnetic bioreactor system and a magnetic hydrogel were manufactured by single-stage three-dimensional (3D) printing with digital light processing (DLP) technique for differentiation of myoblast cells. The hydrogel was composed of a magnetic part containing iron oxide and glycidyl-methacrylated silk fibroin, and a cellular part printed by adding mouse myoblast cell (C2C12) to gelatin glycidyl methacrylate, that was placed in the magnetic bioreactor system to stimulate the cells in the hydrogel. The composite hydrogel was steadily printed by a one-stage layering technique using a DLP printer. The magnetic bioreactor offered mechanical stretching of the cells in the hydrogel in 3D ways, so that the cellular differentiation could be executed in three dimensions just like the human environment. Cell viability, as well as gene expression using quantitative reverse transcription-polymerase chain reaction, were assessed after magneto-mechanical stimulation of the myoblast cell-embedded hydrogel in the magnetic bioreactor system. Comparison with the control group revealed that the magnetic bioreactor system accelerated differentiation of mouse myoblast cells in the hydrogel and increased myotube diameter and lengthin vitro. The DLP-printed magnetic bioreactor and the hydrogel were simply manufactured and easy-to-use, providing an efficient environment for applying noninvasive mechanical force via FDA-approved silk fibroin and iron oxide biocomposite hydrogel, to stimulate cells without any evidence of cytotoxicity, demonstrating the potential for application in muscle tissue engineering.

Treatment of Fungal-Infected Diabetic Wounds with Low Temperature Plasma.

Diabetes mellitus renders patients susceptible to chronic wounds and various infections. Regarding the latter, fungal infections are of particular concern since, although they are the source of significant morbidity and mortality in immunocompromised patients, they are generally resistant to conventional treatment and a definite treatment strategy has not yet been established. Herein, we report the treatment of skin wounds in a diabetic rat model, infected by Candida albicans, with low temperature helium plasma generated in a hand-held atmospheric jet device. A fungal infection was induced on two dorsal skin wounds of the diabetic rats, and one wound was treated with the plasma jet whereas the other served as a control. Histological analysis revealed accelerated skin wound healing and decreased evidence of fungal infection in the plasma-treated group, as compared to the control group. Regeneration of the epidermis and dermis, collagen deposition, and neovascularization were all observed as a result of plasma treatment, but without wound contraction, scar formation or any evidence of thermal damage to the tissue. These findings demonstrate that the He plasma jet is remarkably effective in diabetic skin wounds infected by Candida albicans, thereby providing a promising medical treatment option for diabetes mellitus patients with skin wound and fungal infections.

Reinforced-hydrogel encapsulated hMSCs towards brain injury treatment by trans-septal approach.

Encapsulated stem cells in various biomaterials have become a potentially promising cell transplantation strategy in the treatment of various neurologic disorders. However, there is no ideal cell delivery material and method for clinical application in brain diseases. Here we show silk fibroin (SF)-based hydrogel encapsulated engineered human mesenchymal stem cells (hMSCs) to overproduce brain-derived neurotrophic factor (BDNF) (BDNF-hMSC) is an effective approach to treat brain injury through trans-septal cell transplantation in the rat model. In this study, we observed SF induced sustained BDNF production by BDNF-hMSC both in 2D (9.367 ± 1.969 ng/ml) and 3D (7.319 ± 0.1025 ng/ml) culture conditions for 3 days. Through immunohistochemistry using α-tubulin, BDNF-hMSCs showed a significant increased average neurite length of co-cultured neuro 2a (N2a) cells, suggested that BDNF-hMSCs induced neurogenesis in vitro. Encapsulated BDNF-hMSC, pre-labeled with the red fluorescent dye PKH-26, exhibited intense fluorescence up to 14 days trans-septal transplantation, indicated excellent viability of the transplanted cells. Compared to the vehicle-treated, encapsulated BDNF- hMSC demonstrated significantly increased BDNF level both in the sham-operated and injured hippocampus (Hip) through immunoblot analysis after 7 days implantation. Transplantation of the encapsulated BDNF-hMSC promoted neurological functional recovery via significantly reduced neuronal death in the Hip 7 days post-injury. Using magnetic resonance imaging (MRI) analysis, we demonstrated that encapsulated BDNF-hMSC reduced lesion area significantly at 14 and 21 days in the damaged brain following trans-septal implantation. This stem cell transplantation approach represents a critical set up towards brain injury treatment for clinical application.

A 3D Printable Electroconductive Biocomposite Bioink Based on Silk Fibroin-Conjugated Graphene Oxide.

Reduced graphene oxide (rGO) has wide application as a nanofiller in the fabrication of electroconductive biocomposites due to its exceptional properties. However, the hydrophobicity and chemical stability of rGO limit its ability to be incorporated into precursor polymers for physical mixing during biocomposite fabrication. Moreover, until now, no suitable rGO-combining biomaterials that are stable, soluble, biocompatible, and 3D printable have been developed. In this study, we fabricated digital light processing (DLP) printable bioink (SGOB1), through covalent reduction of graphene oxide (GO) by glycidyl methacrylated silk fibroin (SB). Compositional analyses showed that SGOB1 contains approximately 8.42% GO in its reduced state. Our results also showed that the rGO content of SGOB1 became more thermally stable and highly soluble. SGOB1 hydrogels demonstrated superior mechanical, electroconductive, and neurogenic properties than (SB). Furthermore, the photocurable bioink supported Neuro2a cell proliferation and viability. Therefore, SGOB1 could be a suitable biocomposite for neural tissue engineering.

4D-bioprinted silk hydrogels for tissue engineering.

Recently, four-dimensional (4D) printing is emerging as the next-generation biofabrication technology. However, current 4D bioprinting lacks biocompatibility or multi-component printability. In addition, suitable implantable targets capable of applying 4D bioprinted products have not yet been established, except theoretical and in vitro study. Herein, we describe a cell-friendly and biocompatible 4D bioprinting system including more than two cell types based on digital light processing (DLP) and photocurable silk fibroin (Sil-MA) hydrogel. The shape changes of 3D printed bilayered Sil-MA hydrogels were controlled by modulating their interior or exterior properties in physiological conditions. We used finite element analysis (FEA) simulations to explore the possible changes in the complex structure. Finally, we made trachea mimetic tissue with two cell types using this 4D bioprinting system and implanted it into a damaged trachea of rabbit for 8 weeks. The implants were integrated with the host trachea naturally, and both epithelium and cartilage were formed at the predicted sites. These findings demonstrate that 4D bioprinting system could make tissue mimetic scaffold biologically and suggest the potential value of the 4D bioprinting system for tissue engineering and the clinical application.

Cytocompatibility of Modified Silk Fibroin with Glycidyl Methacrylate for Tissue Engineering and Biomedical Applications.

Hydrogel with chemical modification has been used for 3D printing in the biomedical field of cell and tissue-based regeneration because it provides a good cellular microenvironment and mechanical supportive ability. As a scaffold and a matrix, hydrogel itself has to be modified chemically and physically to form a ß-sheet crosslinking structure for the strength of the biomaterials. These chemical modifications could affect the biological damage done to encapsulated cells or surrounding tissues due to unreacted chemical residues. Biological assessment, including assessment of the cytocompatibility of hydrogel in clinical trials, must involve testing with cytotoxicity, irritation, and sensitization. Here, we modified silk fibroin and glycidyl methacrylate (Silk-GMA) and evaluated the physical characterizations, residual chemical detection, and the biological effect of residual GMA depending on dialysis periods. Silk-GMA depending on each dialysis period had a typical ß-sheet structure in the characterization analysis and residual GMA decreased from dialysis day 1. Moreover, cell proliferation and viability rate gradually increased; additionally, necrotic and apoptotic cells decreased from dialysis day 2. These results indicate that the dialysis periods during chemical modification of natural polymer are important for removing unreacted chemical residues and for the potential application of the manufacturing standardization for chemically modified hydrogel for the clinical transplantation for tissue engineering and biomedical applications.

Digital light processing 3D printed silk fibroin hydrogel for cartilage tissue engineering.

Three-dimensional printing with Digital Lighting Processing (DLP) printer has come into the new wave in the tissue engineering for regenerative medicine. Especially for the clinical application, it needs to develop of bio-ink with biocompatibility, biodegradability and printability. Therefore, we demonstrated that Silk fibroin as a natural polymer fabricated with glycidyl-methacrylate (Silk-GMA) for DLP 3D printing. The ability of chondrogenesis with chondrocyte-laden Silk-GMA evaluated in vitro culture system and applied in vivo. DLP 3D printing system provided 3D product with even cell distribution due to rapid printing speed and photopolymerization of DLP 3D printer. Up to 4 weeks in vitro cultivation of Silk-GMA hydrogel allows to ensure of viability, proliferation and differentiation to chondrogenesis of encapsulated cells. Moreover, in vivo experiments against partially defected trachea rabbit model demonstrated that new cartilage like tissue and epithelium found surrounding transplanted Silk-GMA hydrogel. This study promises the fabricated Silk GMA hydrogel using DLP 3D printer could be applied to the fields of tissue engineering needing mechanical properties like cartilage regeneration.

Recirculating peritoneal dialysis system using urease-fixed silk fibroin membrane filter with spherical carbonaceous adsorbent.

The chronic kidney disease (CKD) patients are undergoing continuous ambulatory peritoneal dialysis (CAPD). However, there are some constraints, the frequent exchange of the dialysate and limitation of outside activity, associated with CAPD remain to be solved. In this study, we designed the wearable artificial kidney (WAK) system for peritoneal dialysis (PD) using urease-immobilized silk fibroin (SF) membrane and polymer-based spherical carbonaceous adsorbent (PSCA). We evaluated this kit's removal abilities of uremic toxins such as urea, creatinine, uric acid, phosphorus, and ß2-microglobulin from the dialysate of end-stage renal disease (ESRD) patients in vitro. The uremic toxins including urea, creatinine, uric acid, and phosphorus were removed about 99% by immobilized SF membrane and PSCA filter after 24 h treatment. However, only 50% of ß2-microglobulin was removed by this filtering system after 24 h treatment. In vivo study result shows that our filtering system has more uremic toxins removal efficiency than exchanged dialysate at every 6 h. We suggest that recirculating PD system using urease-immobilized SF membrane with PSCA could be more efficient than traditional dialysate exchange system for a WAK for PD.

A 4-Axis Technique for Three-Dimensional Printing of an Artificial Trachea.

BACKGROUND: Several types of three-dimensional (3D)-printed tracheal scaffolds have been reported. Nonetheless, most of these studies concentrated only on application of the final product to an in vivo animal study and could not show the effects of various 3D printing methods, materials, or parameters for creation of an optimal 3D-printed tracheal scaffold. The purpose of this study was to characterize polycaprolactone (PCL) tracheal scaffolds 3D-printed by the 4-axis fused deposition modeling (FDM) method and determine the differences in the scaffold depending on the additive manufacturing method. METHODS: The standard 3D trachea model for FDM was applied to a 4-axis FDM scaffold and conventional FDM scaffold. The scaffold morphology, mechanical properties, porosity, and cytotoxicity were evaluated. Scaffolds were implanted into a 7 × 10-mm artificial tracheal defect in rabbits. Four and 8 weeks after the operation, the reconstructed sites were evaluated by bronchoscopic, radiological, and histological analyses. RESULTS: The 4-axis FDM provided greater dimensional accuracy and was significantly closer to CAD software-based designs with a predefined pore size and pore interconnectivity as compared to the conventional scaffold. The 4-axis tracheal scaffold showed superior mechanical properties. CONCLUSION: We suggest that the 4-axis FDM process is more suitable for the development of an accurate and mechanically superior trachea scaffold.