Incorporation of Cell-Adhesive Proteins in 3D-Printed Lipoic Acid-Maleic Acid-Poly(Propylene Glycol)-Based Tough Gel Ink for Cell-Supportive Microenvironment.

In extrusion-based 3D printing, the use of synthetic polymeric hydrogels can facilitate fabrication of cellularized and implanted scaffolds with sufficient mechanical properties to maintain the structural integrity and physical stress within the in vivo conditions. However, synthetic hydrogels face challenges due to their poor properties of cellular adhesion, bioactivity, and biofunctionality. New compositions of hydrogel inks have been designed to address this limitation. A viscous poly(maleate-propylene oxide)-lipoate-poly(ethylene oxide) (MPLE) hydrogel is recently developed that shows high-resolution printability, drug-controlled release, excellent mechanical properties with adhesiveness, and biocompatibility. In this study, the authors demonstrate that the incorporation of cell-adhesive proteins like gelatin and albumin within the MPLE gel allows printing of biologically functional 3D scaffolds with rapid cell spreading (within 7 days) and high cell proliferation (twofold increase) as compared with MPLE gel only. Addition of proteins (10% w/v) supports the formation of interconnected cell clusters (≈1.6-fold increase in cell areas after 7-day) and spreading of cells in the printed scaffolds without additional growth factors. In in vivo studies, the protein-loaded scaffolds showed excellent biocompatibility and increased angiogenesis without inflammatory response after 4-week implantation in mice, thus demonstrating the promise to contribute to the printable tough hydrogel inks for tissue engineering.

Control of maleic acid-propylene diepoxide hydrogel for 3D printing application for flexible tissue engineering scaffold with high resolution by end capping and graft polymerization.

BACKGROUND: Control of 3D printing of highly tough hydrogel inks with adequate printability, scaffold fidelity and mechanical properties are highly desirable for biomedical and tissue engineering applications. However, developing a biocompatible tough ink with high-resolution printability, biodegradability, self-healing, adhesion, and integration with surrounding tissues is a big challenge in 3D printing. The aim of this study was to develop extrusion-based 3D printing of viscous hydrogel composing of maleic acid and propylene diepoxide by controlling continuous mechanisms of condensation and radical polymerization. METHODS: The molecular weight of highly adhesive propagating poly(malate-co-propylene oxide) copolymer was controlled by capping its growing chain with mono-functional lipoic acid with different compositions during condensation reaction to form lipoic acid capped gel (LP-capped gel). Poly(ethylene oxide)-diacrylate, PEGDA, is graft-polymerized to the LP-capped backbone polymer (MPLE gel) by UV irradiation during 3D printing process to control the properties of gel printability, mechanical properties, and cell adhesiveness and post-printing fidelity of the printed scaffolds with high resolution and mechanical properties (MPLE scaffold). The scaffolds in complex geometries have been printed out in diverse forms with addition of model drugs with different molecular weights and chemical structures. Both the highly adhesive LP-capped gel and printing-controlled MPLE gel/scaffolds are diversely characterized and compared with for their applications to the extrusion-based printability, including biocompatibility, self-healing, drug releasing, adhesiveness, multi-layered high-resolution printing. Further in vitro/in vivo tests were done to observe cytotoxicity, immune response and tissue formation by using different cells in mice model. RESULTS: LP-capped hydrogel from maleic acid and propylene diepoxide gel showed control of gel properties with lipoic acid with one function group of thiol during condensation reaction, and the ratio at 1:0.3 (w/v) between LP-capped gel and PEGDA was chosen for the optimal results during radical polymerization process for 3D printing at high resolution (90-140 µm in strut thickness) with various complex geometries (lattice, rhombus, and honeycomb). The hydrogel showed excellent properties of self-healing, mechanical strength, biocompatibility, etc. In addition, the long-term release profiles of bioactive molecules were well-controlled by incorporating drugs of high molecular bovine serum albumin (BSA, 21 days, 98.4 ± 0.69%), or small molecule ornidazole (ORN, 14 days, 97.1 ± 1.98%) into the MPLE gel scaffolds for the tests of potential therapeutic applications. More importantly, the MPLE gels represents excellent in vitro cyto-compatibility against osteoblast-like cells (MC3T3) with viability value at 96.43% ± 7.48% over 7 culturing days. For in-vivo studies, the flexible MPLE scaffolds showed significant improvement on angiogenesis with minor inflammatory response after 4-week implantation in mice. CONCLUSION: The MPLE gel inks was well-controlled for the fabrication of flexible complex tissue engineering scaffold with high resolutions, shear-thinning, 3D printability and post-printing fidelity, by modulating the composition of the highly adhesive LP-capped gel and inert PEGDA as well as end capping of lipoic acid to the propagating poly(malate-co-propylene oxide) copolymer. The gel ink demonstrated its excellent printability, in vitro/in vivo biocompatibility and mechanical properties as well as sustained drug release from the gel.

Micro/nano devices for integration with human brain organoids.

Brain organoids are powerful experimental models to study fundamental neurodevelopmental processes and the pathology of neurological disorders. Brain organoids can now be generated from human-induced pluripotent stem cells, which pave the way for using them to investigate effective therapies for various neurodegenerative disorders and diseases. However, brain organoids possess complex cellular architecture, various unknown functionalities, and a lack of vascular networks, which have limited their use in biomedicine and clinical research. Micro/nanoscale devices and technologies can help overcome these limitations. This review critically examines recently developed micro/nano devices for integration with brain organoids. The review focuses on devices designed to achieve several key aims: to improve methodologies for in vitro culture; to enable electrophysiological recordings from organoids; to screen drugs for chemotherapy and new treatments; to understand the effects of psychoactive drugs; and to enable development of vascular networks in organoids. Along with the specific device features and their relevance for these applications, we also discuss the current challenges to overcome and future strategies to advance the use of brain organoids in clinical research. The interdisciplinary convergence of brain organoids research with materials science, device engineering, neuroscience, and stem cell biology holds remarkable potential for replicating the human brain in vitro. Micro/nano devices are an important part of realizing this potential that will afford both fundamental insights into the mechanisms underlying brain function and a pathway for developing novel treatments for neurophysiological and neurodegenerative disorders.

Three-Dimensional Printed Design of Antibiotic-Releasing Esophageal Patches for Antimicrobial Activity Prevention.

Pharyngoesophageal defects can cause exposure to various bacterial flora and severe inflammation. We fabricated a biodegradable polycaprolactone (PCL) patch composed of both thin film and three-dimensional (3D) printed lattice, and then investigated the efficacy of pharyngoesophageal reconstruction by using 3D printed antibiotic-releasing PCL patches that inhibited early inflammation by sustained tetracycline (TCN) release from both thin PCL films and printed rods implanted in esophageal partial defects. PCL was 3D printed in lattice form on a presolution casted PCL thin film at ∼100 µm resolution. TCN was loaded onto the PCL-printed patches by 3D printing a mixture of TCN and PCL particles melted at 100°C. TCN exhibited sustained release in vitro for over 1 month. After loading TCN, the patches showed decreased tensile strength and Young's modulus, and less than 20% TCN was slowly released from the 2.5% TCN-loaded PCL patches over 150 days. Cytotoxicity tests of extract solutions from patch samples demonstrated excellent in vitro cell compatibility. Antibiotic-releasing PCL patches were then transplanted into partial esophageal defects in rats. Microcomputed tomography analysis revealed no leak of orally injected contrast agent in the entire esophagus. Tissue remodeling was examined through histological responses of M1 and M2 macrophages. In particular, the 1% and 3% TCN patch groups exhibited significant muscle layer regeneration by desmin immunostaining. Further histological and immunofluorescence analyses revealed that the 1% and 3% TCN patch groups exhibited the best esophageal regeneration according to reepithelialization, neovascularization, and elastin texture around the implanted sites. Our antibiotic-releasing patch successfully consolidates the regenerative potential of esophageal muscle and mucosa and the antibacterial activity of TCN for 3D esophageal reconstruction. Impact statement Anastomosis site leakage and necrosis after pharyngoesophageal transplantation inevitably causes mortality because the mediastinum and neck compartments become contaminated. Herein, we present antibiotic-releasing pharyngoesophageal patch that prevents saliva leakage and has an antimicrobial effect. We have demonstrated antibiotic release profile and mechanical properties for esophageal transplantation. Upon esophageal transplantation of antibiotic-releasing polycaprolactone patches, antimicrobial effects and muscle regeneration around the graft sites were clearly identified in the group containing 1% and 3% of tetracycline. The esophageal graft led to the remarkable recovery throughout reepithelialization, neovascularization, and elastin texture of around the implanted sites. We believe that current system is capable of various applications that require antibacterial in vivo.

Symbiotic culture of nanocellulose pellicle: A potential matrix for 3D bioprinting.

Nanocellulose pellicle is produced as a byproduct during the symbiotic culture of bacteria and yeast in kombucha. It shows good mechanical strength, biocompatibility and hydrophilicity. However, it has limited application in tissue engineering due to its low processability. In this work, bacterial cellulose-based sustainable kombucha (KBC) sheet has been produced and it was acid-treated to partially hydrolyse. This controlled process improves its extrusion and shape formation ability. The physical, functional and biological properties were studied to assess its potential as a 3D printed scaffold. Two different cell lines (Human dermal fibroblast cells and mouse osteoblast cells) were used to study the cytocompatibility. Both the cell types showed good attachment, growth and proliferation on the pure and treated KBC. They attained almost full confluence within 3 days. This study indicates that the controlled partial hydrolysis of KBC can make it suitable for 3D printing retaining its mechanical strength and cytocompatibility. This sustainable microbial biopolymer shows the possibility to be used as a bioink for 3D bioprinting.

3D printable and injectable lactoferrin-loaded carboxymethyl cellulose-glycol chitosan hydrogels for tissue engineering applications.

In this study, carboxymethyl cellulose (CMC)-glycol chitosan (GC) hydrogel, a potential three-dimensional (3D) printing biomaterial ink for tissue engineering applications was synthesized using simple, biocompatible in situ-gelling Schiff's base reaction and ionic interactions. Different grades of hydrogels (C70G30, C50G50 and C30G70) were synthesized at physiological conditions. The oxidation of CMC and imine bond formation in the hydrogel were confirmed spectroscopically. Scanning electron microscopic images revealed the crosslinked interconnected pores in the cross-sectioned hydrogels (dried). Swelling (equilibrium: 1 h), porosity (~75%), in vitro degradation (>30 days) and thermal gravimetric analyses of the dried gels were studied. Initially, cytotoxicity assay was evaluated using mouse osteoblastic cells (MC3T3). These experiments revealed that CMC-GC gels formed stable hydrogel networks and were biocompatible. Particularly, C50G50 gels showed high printability (continuous extrusion) and post-printing stability (without secondary crosslinking). Gel 3D printing was optimized by varying the air pressure, temperature, needle size and nozzle speed, to obtain stable lattice structures (2 to 16 layers). The printed (2 and 5 layers) hydrogels showed high stability in phosphate buffer saline (PBS) solution (1 h), under UV light (1 h) and after autoclaving. The strut dimensions and porosity of the printed gels before and after the stability tests were analyzed. The hydrogel stability may be attributed to both the imine bond and ionic interaction between the cationic and anionic polymer side chains. Lactoferrin (glycoprotein) incorporated C50G50 gels showed sustained release up to 21 days in PBS (pH 7.4) solution and demonstrated increased biocompatibility (>80%) during in vitro cytotoxicity assays (MC3T3 cells and bone marrow mesenchymal stem cells) and Live/Dead assay (MC3T3 cells). A higher number of live osteoblast cells on the C50G50 hydrogels with increasing lactoferrin concentration was observed. These results show that the CMC-GC gels are promising bio-ink candidates for 3D printing and loading proteins or drugs for tissue engineering applications.

3D printable hyaluronic acid-based hydrogel for its potential application as a bioink in tissue engineering.

BACKGROUND: After recognition of 3D printing and injectable hydrogel as a critical issue in tissue/organ engineering and regenerative medicine society, many hydrogels as bioinks have been developed worldwide by using polymeric biomaterials such as gelatin, alginate, hyaluronic acid and others. Even though some gels have shown good performances in 3D bioprinting, still their performances do not meet the requirements enough to be used as a bioink in tissue engineering. METHOD: In this study, a hydrogel consisting of three biocompatible biomaterials such as hyaluronic acid (HA), hydroxyethyl acrylate (HEA) and gelatin-methacryloyl, i.e. HA-g-pHEA-gelatin gel, has been evaluated for its possibility as a bioprinting gel, a bioink. Hydrogel synthesis was obtained by graft polymerization of HEA to HA and then grafting of gelatin- methacryloyl via radical polymerization mechanism. Physical and biological properties of the HA-based hydrogels fabricated with different concentrations of methacrylic anhydride (6 and 8%) for gelatin-methacryloylation have been evaluated such as swelling, rheology, morphology, cell compatibility, and delivery of small molecular dimethyloxalylglycine. Printings of HA-g-pHEA-Gelatin gel and its bioink with bone cell loaded in lattice forms were also evaluated by using home-built multi-material (3D bio-) printing system. CONCLUSION: The experimental results demonstrated that the HA-g-pHEA-gelatin hydrogel showed both stable rheology properties and excellent biocompatibility, and the gel showed printability in good shape. The bone cells in bioinks of the lattice-printed scaffolds were viable. This study showed HA-g-pHEA-Gelatin gel's potential as a bioink or its tissue engineering applications in injectable and 3D bioprinting forms.