protocols-3d-bioprinting-gelatin-methacryloyl-hydrogel-based

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Zhejiang University

To construct a recombinant adenovirus vector of calcitonin gene-related peptide (CGRP) by AdEasy system and to validate its expression in myocardial cells.

[Construction of recombinant adenovirus vector of calcitonin gene-related peptide gene and transfection to neonatal rat cardiomyocytes].

Diabetic hearts are vulnerable to ischemia/reperfusion (I/R) injury. Pretreatment with exogenous calcitonin gene-related peptide (CGRP) exerts a cardioprotective effect against myocardial I/R injury. Our previous study found that the CGRP level was decreased in diabetic hearts. This study aimed to investigate whether up-regulation of CGRP could reduce I/R injury in diabetic hearts.

Up-regulation of calcitonin gene-related peptide protects streptozotocin-induced diabetic hearts from ischemia/reperfusion injury.

The field of how to rapidly assemble microfluidics with modular components continuously attracts researchers' attention, however, extra efforts must be devoted to solving the problems of leaking and aligning between individual modules. This paper presents a novel type of modular microfluidic device, driven by capillary force. There is no necessity for a strict seal or special alignment, and its open structures make it easy to integrate various stents and reactants. The key rationale for this method is to print different functional modules with a low-cost three-dimensional (3D) printer, then fill the channels with capillary materials and assemble them with plugs like Lego bricks. This rapidly reconstructed modular microfluidic device consists of a variety of common functional modules and other personalized modules, each module having a unified standard interface for easy assembly. As it can be printed by a desktop 3D printer, the manufacturing process is simple and efficient, with controllable regulation of the flow channel scale. Through diverse combinations of different modules, a variety of different functions can be achieved, without duplicating the manufacturing process. A single module can also be taken out for testing and analysis. What's more, combined with basic circuit components, it can serve as a low-cost Lego-like modular microfluidic circuits. As a proof of concept, the modular microfluidic device has been successfully demonstrated and used for stent degradation and cell cultures, revealing the potential use of this method in both chemical and biological research.

3D printed Lego-like modular microfluidic devices based on capillary driving.

Hydrogel microspheroids are widely used in tissue engineering, such as injection therapy and 3D cell culture, and among which, heterogeneous microspheroids are drawing much attention as a promising tool to carry multiple cell types in separated phases. However, it is still a big challenge to fabricate heterogeneous microspheroids that can reconstruct built-up tissues' microarchitecture with excellent resolution and spatial organization in limited sizes. Here, a novel airflow-assisted 3D bioprinting method is reported, which can print versatile spiral microarchitectures inside the microspheroids, permitting one-step bioprinting of fascinating hydrogel structures, such as the spherical helix, rose, and saddle. A microfluidic nozzle is developed to improve the capability of intricate cell encapsulation with heterotypic contact. Complex structures, such as a rose, Tai chi pattern, and single cell line can be easily printed in spheroids. The theoretical model during printing is established and process parameters are systematically investigated. As a demonstration, a human multicellular organoid of spirally vascularized ossification is reconstructed with this method, which shows that it is a powerful tool to build mini tissues on microspheroids.

Airflow-Assisted 3D Bioprinting of Human Heterogeneous Microspheroidal Organoids with Microfluidic Nozzle.

The use of microscale fibers could facilitate nutrient diffusion in fiber-based tissue engineering and improve cell survival. However, in order to build a functional mini tissue such as muscle fibers, nerve conduits, and blood vessels, hydrogel microfibers should not only mimic the structural features of native tissues but also offer a cell-favorable environment and sufficient strength for tissue functionalization. Therefore, an important goal is to fabricate morphology-controllable microfibers with appropriate hydrogel materials to mimic the structural and functional complexity of native tissues. Here, gelatin methacrylate (GelMA) is used as the fiber material due to its excellent biological performance, and a novel coaxial bioprinting method is developed to fabricate morphology-controllable GelMA microfibers encapsulated in calcium alginate. By adjusting the flow rates, GelMA microfibers with straight, wavy, and helical morphologies could be obtained. By varying the coaxial nozzle design, more complex GelMA microfibers such as Janus, multilayered, and double helix structures could be fabricated. Using these microfibers, mini tissues containing human umbilical cord vein endothelial cells are built, in which cells gradually migrate and connect to form lumen resembling blood vessels. The merits of cytocompatibility, structural diversity, and mechanical tunability of the versatile microfibers may open more avenues for further biomedical research.

Fiber-Based Mini Tissue with Morphology-Controllable GelMA Microfibers.

Low-concentration gelatin methacryloyl (GelMA) has excellent biocompatibility to cell-laden structures. However, it is still a big challenge to stably fabricate organoids (even microdroplets) using this material due to its extremely low viscosity. Here, a promising electro-assisted bioprinting method is developed, which can print low-concentration pure GelMA microdroplets with low cost, low cell damage, and high efficiency. With the help of electrostatic attraction, uniform GelMA microdroplets measuring about 100 μm are rapidly printed. Due to the application of lower external forces to separate the droplets, cell damage during printing is negligible, which often happens in piezoelectric or thermal inkjet bioprinting. Different printing states and effects of printing parameters (voltages, gas pressure, nozzle size, etc.) on microdroplet diameter are also investigated. The fundamental properties of low-concentration GelMA microspheres are subsequently studied. The results show that the printed microspheres with 5% w/v GelMA can provide a suitable microenvironment for laden bone marrow stem cells. Finally, it is demonstrated that the printed microdroplets can be used in building microspheroidal organoids, in drug controlled release, and in 3D bioprinting as biobricks. This method shows great potential use in cell therapy, drug delivery, and organoid building.

Electro-Assisted Bioprinting of Low-Concentration GelMA Microdroplets.

Photo-crosslinkable gelatin methacrylate (GelMA) has become an attractive ink in 3D printing due to its excellent biological performance. However, limited by low viscosity and long cross-linking time, it is still a challenge to directly print GelMA by extrusion-based 3D printing. Here, to balance the printability and biocompatibility, biomaterial ink composed of GelMA and nanoclay was specially designed. Using this ink, complex scaffolds with high shape fidelity can be easily printed based on the thixotropic property of nanoclay. In this study, we tried to answer some basic printing-required questions of this ink, including the printability window, general properties (porosity, mechanical strength, et al), and biocompatibility. We found that the GelMA/Nanoclay ink enabled printing complex 3D scaffolds, such as a bionic ear and a branched vessel. Furthermore, the addition of nanoclay improved the porosity, increased the mechanical strength, reduced the degradation ratio, and maintained a good biocompatibility of the printed scaffolds. Therefore, this method offers an easy way to print complex scaffolds with good shape fidelity and biological performance, and it might open up new potential applications for the customized therapy of tissue defects.

3D printing of complex GelMA-based scaffolds with nanoclay.

Hydrogel microfibers have many fascinating applications as microcarriers for drugs, factors, and cells, such as 3D cell culture, building micro-organoids, and transplantation therapy due to their simple structures. It is unknown whether cell-laden fiber can become a standard-use product like woundplast. Here, from the technical and practical view, the elements required for user-oriented microfibers are first discussed: i) the materials used should promote cell functionalization and be easily processed; ii) follow a manufacturing method for mass fabrication; iii) have the ability to be stored long-term and be available for immediate use. Here, it is demonstrated that bioactive microfibers can be simply fabricated with coaxial bioprinting using gelatin methacrylate due to its tunable biological and mechanical properties. Additionally, programmed microfibers and 3D constructs with controllable composition can also be fabricated. These microfibers can be used to directly build organoids and complex co-culture tissue models. In the present study, vascular organoid, angiogenic sprouts, and tumor angiogenesis are demonstrated. It is also demonstrated, for the first time, that the cell-laden microfibers can be stored long-term via cryopreservation. These results show that cell-laden structures can be developed as a novel type of organoid product, which will open more avenues for tissue engineering and clinical organ repair.

Bioprinting of Cell-Laden Microfiber: Can It Become a Standard Product?

Photogenerated nonequilibrium hot carriers play a key role in graphene's intriguing optoelectronic properties. Compared to conventional photoexcitation, plasmon excitation can be engineered to enhance and control the generation and dynamics of hot carriers. Here, we report an unusual negative differential photoresponse of plasmon-induced "ultrahot" electrons in a graphene-boron nitride-graphene tunneling junction. We demonstrate nanocrescent gold plasmonic nanostructures that substantially enhance the absorption of long-wavelength photons whose energy is greatly below the tunneling barrier and significantly boost the electron thermalization in graphene. We further analyze the generation and transfer of ultrahot electrons under different bias and power conditions. We find that the competition among thermionic emission, the carrier-cooling effect, and the field effect results in a hitherto unusual negative differential photoresponse in the photocurrent-bias plot. Our results not only exemplify a promising platform for detecting low-energy photons, enhancing the photoresponse, and reducing the dark current but also reveal the critically coupled pathways for harvesting ultrahot carriers.

Article	Year
[Construction of recombinant adenovirus vector of calcitonin gene-related peptide gene and transfection to neonatal rat cardiomyocytes]. Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences\| PubMed ID: 20544995	2010
Up-regulation of calcitonin gene-related peptide protects streptozotocin-induced diabetic hearts from ischemia/reperfusion injury. International journal of cardiology\| PubMed ID: 21546103	2012
3D printed Lego-like modular microfluidic devices based on capillary driving. Biofabrication\| PubMed ID: 29417931	2018
Airflow-Assisted 3D Bioprinting of Human Heterogeneous Microspheroidal Organoids with Microfluidic Nozzle. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 30133151	2018
Fiber-Based Mini Tissue with Morphology-Controllable GelMA Microfibers. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 30253060	2018
Electro-Assisted Bioprinting of Low-Concentration GelMA Microdroplets. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 30569632	2019
3D printing of complex GelMA-based scaffolds with nanoclay. Biofabrication\| PubMed ID: 30836349	2019
Bioprinting of Cell-Laden Microfiber: Can It Become a Standard Product? Advanced healthcare materials\| PubMed ID: 30866173	2019
Plasmon Excited Ultrahot Carriers and Negative Differential Photoresponse in a Vertical Graphene van der Waals Heterostructure. Nano letters\| PubMed ID: 31025869	2019

Lei Shao

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