Fish Gelatin-Nanoclay Composite Film. Mechanical and Physical Properties, Effect of Enzyme Cross-Linking, and as a Functional Film Layer

The effect of clay content, homogenization RPM, and pH on the mechanical and barrier properties of fish gelatin/nanoclay composite films was investigated. The addition of clay increased the tensile strength (TS) from 30.31±2.37 MPa to 40.71±3.30 MPa. The nanoclay composite film had improved oxygen and water barrier properties when compared to neat fish gelatin film. Oxygen permeability decreased from 0.0004028±0.0000007 gm/m2dayatm to 0.0001144±0.0000162 gm/m2dayatm and the water vapor permeability decreased from 0.0312±0.0016 ngm/m2sPa to 0.0081±0.0001 ngm/m2sPa. The Small angle x-ray scattering (SAXS) and Transmission electron microscopy (TEM) observations confirmed that the ultrasonification treatment (30 min at 40% output) resulted in exfoliation of the silicates. Intercalation was achieved within the composite film without the ultrasonification treatment. The fish gelatin solution was cross-linked by the addition of Microbial transglutaminase (MTGase) in an effort to measure the effect on film mechanical and barrier properties. The viscosity of the MTGase treated gelatin solution (2% w/w) increased from 86.25±1.77 cp (0 min) to 243±12.37 cp (80 min). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the molecular weight of fish gelatin solutions increased after treatment with MTGase. The increase of molecular weight imparted steric hindrance to intercalation, which also resulted in a marked decrease of intercalation. The tensile strength decreased from 61.30±1.90 MPa (0 min) to 57.36±4.97 MPa (50 min), and the elongation at break (EB) decreased from 16.73±4.47% (0 min) to 13.34±5.13% (50 min) at 2% (w/w) MTGase concentration. The oxygen permeability and water vapor permeability were not significantly different as a function of treatment time at 2% (w/w) MTGase concentration. The incorporation of nanoclay to the MTGase treated film decreased oxygen permeability. The SAXS and TEM results suggested that the nanoclay was exfoliated in the MTGase treated fish gelatin film. A three layer laminant film, utilizing the fish gelatin-nanoclay composite film as the functional barrier, was produced using a pilot scale laminator. The laminant film structure was low density polyethylene (LDPE), fish gelatin-nanoclay composite film, and polyester (PET). The fish gelatin-nanoclay laminant film showed excellent oxygen barrier (0~50% RH) when compared to a similar laminant structure utilizing an industry standard ethylene vinyl alcohol (EVOH) film as the barrier layer. In addition, the fish gelatin-nanoclay composite film exhibited sufficient bond strength (greater than 500 gf) to both the LDPE and the PET. Therefore, the fish gelatin-nanoclay barrier film has the potential to be used as a functional biopolymer barrier in laminant film structures for various food packaging applications

Microscale Strategies for Generating Cell-Encapsulating Hydrogels

Hydrogels in which cells are encapsulated are of great potential interest for tissue engineering applications. These gels provide a structure inside which cells can spread and proliferate. Such structures benefit from controlled microarchitectures that can affect the behavior of the enclosed cells. Microfabrication-based techniques are emerging as powerful approaches to generate such cell-encapsulating hydrogel structures. In this paper we introduce common hydrogels and their crosslinking methods and review the latest microscale approaches for generation of cell containing gel particles. We specifically focus on microfluidics-based methods and on techniques such as micromolding and electrospinning.National Science Foundation (U.S.) (DMR0847287)National Institutes of Health (U.S.) (EB008392)National Institutes of Health (U.S.) (DE019024)National Institutes of Health (U.S.) (HL099073)National Institutes of Health (U.S.) (AR057837)National Institutes of Health (U.S.) (HL092836)United States. Army Research Office (contract W911NF-07-D-0004)United States. Army Research Office (Institute for Soldier Nanotechnology)United States. Army. Corps of EngineersInnovative Med Tech (Postdoctoral fellowship

Bioprinting and biomaterials for dental alveolar tissue regeneration

Three dimensional (3D) bioprinting is a powerful tool, that was recently applied to tissue engineering. This technique allows the precise deposition of cells encapsulated in supportive bioinks to fabricate complex scaffolds, which are used to repair targeted tissues. Here, we review the recent developments in the application of 3D bioprinting to dental tissue engineering. These tissues, including teeth, periodontal ligament, alveolar bones, and dental pulp, present cell types and mechanical properties with great heterogeneity, which is challenging to reproduce in vitro. After highlighting the different bioprinting methods used in regenerative dentistry, we reviewed the great variety of bioink formulations and their effects on cells, which have been established to support the development of these tissues. We discussed the different advances achieved in the fabrication of each dental tissue to provide an overview of the current state of the methods. We conclude with the remaining challenges and future needsThis work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Numbers 22K18936 and 21K04852); AMED (Grant Number JP21gm1310001); The JST Adaptable and Seamless Technology Transfer Program through Target-driven R&D (Grant Number JPMJTM22BD), CASIO SCIENCE PROMOTION FOUNDATION, and by the Research Center for Biomedical Engineering at Tokyo Medical and Dental University, Japan

DNA directed self-assembly of shape-controlled hydrogels

Using DNA as programmable, sequence specific ‘glues’, shape-controlled hydrogel units are self-assembled into prescribed structures. Here we report that aggregates are produced using hydrogel cubes with edge length ranging from 30 micrometers to 1 millimeter, demonstrating assembly across scales. In a simple one-pot agitation reaction, 25 dimers are constructed in parallel from 50 distinct hydrogel cube species, demonstrating highly multiplexed assembly. Using hydrogel cuboids displaying face-specific DNA glues, diverse structures are achieved in aqueous and in interfacial agitation systems. These include dimers, extended chains, and open network structures in an aqueous system; and dimers, chains of fixed length, T-junctions, and square shapes in the interfacial system, demonstrating the versatility of the assembly system

Embryoid body size-mediated differential endodermal and mesodermal differentiation using polyethylene glycol (PEG) microwell array

Embryoid bodies have a number of similarities with cells in gastrulation, which provides useful biological information about embryonic stem cell differentiation. Extensive research has been done to study the control of embryoid body-mediated embryonic stem cell differentiation in various research fields. Recently, microengineering technology has been used to control the size of embryoid bodies and to direct lineage specific differentiation of embryonic stem cells. However, the underlying biology of developmental events in the embryoid bodies of different sizes has not been well elucidated. In this study, embryoid bodies with different sizes were generated within microfabricated PEG microwell arrays, and a series of gene and molecular expressions related to early developmental events was investigated to further elucidate the size-mediated differentiation. The gene and molecular expression profile suggested preferential visceral endoderm formation in 450 μm embryoid bodies and preferential lateral plate mesoderm formation in 150 μm embryoid bodies. These aggregates resulted in higher cardiac differentiation in 450 μm embryoid bodies and higher endothelial differentiation in 150 μm embryoid bodies, respectively. Our findings may provide further insight for understanding embryoid body size-mediated developmental progress.National Science Foundation (U.S.) (CAREER Award DMR0847287)United States. Office of Naval Research (Naval Research Young National Investigator Award)National Institutes of Health (U.S.) (HL092836, EB02597, AR057837

Hybrid hydrogels containing vertically aligned carbon nanotubes with anisotropic electrical conductivity for muscle myofiber fabrication

Biological scaffolds with tunable electrical and mechanical properties are of great interest in many different fields, such as regenerative medicine, biorobotics, and biosensing. In this study, dielectrophoresis (DEP) was used to vertically align carbon nanotubes (CNTs) within methacrylated gelatin (GelMA) hydrogels in a robust, simple, and rapid manner. GelMA-aligned CNT hydrogels showed anisotropic electrical conductivity and superior mechanical properties compared with pristine GelMA hydrogels and GelMA hydrogels containing randomly distributed CNTs. Skeletal muscle cells grown on vertically aligned CNTs in GelMA hydrogels yielded a higher number of functional myofibers than cells that were cultured on hydrogels with randomly distributed CNTs and horizontally aligned CNTs, as confirmed by the expression of myogenic genes and proteins. In addition, the myogenic gene and protein expression increased more profoundly after applying electrical stimulation along the direction of the aligned CNTs due to the anisotropic conductivity of the hybrid GelMA-vertically aligned CNT hydrogels. We believe that platform could attract great attention in other biomedical applications, such as biosensing, bioelectronics, and creating functional biomedical devices

Carbon-Nanotube-Embedded Hydrogel Sheets for Engineering Cardiac Constructs and Bioactuators

We engineered functional cardiac patches by seeding neonatal rat cardiomyocytes onto carbon nanotube (CNT)-incorporated photo-cross-linkable gelatin methacrylate (GelMA) hydrogels. The resulting cardiac constructs showed excellent mechanical integrity and advanced electrophysiological functions. Specifically, myocardial tissues cultured on 50 μm thick CNT-GelMA showed 3 times higher spontaneous synchronous beating rates and 85% lower excitation threshold, compared to those cultured on pristine GelMA hydrogels. Our results indicate that the electrically conductive and nanofibrous networks formed by CNTs within a porous gelatin framework are the key characteristics of CNT-GelMA leading to improved cardiac cell adhesion, organization, and cell–cell coupling. Centimeter-scale patches were released from glass substrates to form 3D biohybrid actuators, which showed controllable linear cyclic contraction/extension, pumping, and swimming actuations. In addition, we demonstrate for the first time that cardiac tissues cultured on CNT-GelMA resist damage by a model cardiac inhibitor as well as a cytotoxic compound. Therefore, incorporation of CNTs into gelatin, and potentially other biomaterials, could be useful in creating multifunctional cardiac scaffolds for both therapeutic purposes and in vitro studies. These hybrid materials could also be used for neuron and other muscle cells to create tissue constructs with improved organization, electroactivity, and mechanical integrity.United States. Army Research Office. Institute for Soldier NanotechnologiesNational Institutes of Health (U.S.) (HL092836)National Institutes of Health (U.S.) (EB02597)National Institutes of Health (U.S.) (AR057837)National Institutes of Health (U.S.) (HL099073)National Science Foundation (U.S.) (DMR0847287)United States. Office of Naval Research (ONR PECASE Award)United States. Office of Naval Research (Young Investigator award)National Research Foundation of Korea (grant (NRF-2010-220-D00014)

Stretchable zein-coated alginate fiber for aligning muscle cells to artificially produce cultivated meat

Abstract Numerous studies have explored the cultivation of muscle cells using non-animal materials for cultivated meat production. Achieving muscle cell proliferation and alignment using 3D scaffolds made from plant-based materials remains challenging. This study introduces a technique to culture and align muscle cells using only plant-based materials, avoiding toxic chemical modifications. Zein-alginate fibers (ZA fibers) were fabricated by coating zein protein onto alginate fibers (A fibers). Zein’s excellent cell compatibility and biodegradability enable high cell adhesion and proliferation rates, and the good ductility of the ZA fibers enable a high strain rate (>75%). We demonstrate mature and aligned myotube formation in ZA fibers, providing a simple way to align muscle cells using plant-based materials. Additionally, cultivated meat was constructed by assembling muscle, fat, and vessel fibers. This method holds promise for the future mass production of cultivated meat