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

This study offers a novel 3D bioprinting method based on hollow calcium alginate filaments by using a coaxial nozzle, in which high strength cell-laden hydrogel 3D structures with built-in microchannels can be fabricated by controlling the crosslinking time to realize fusion of adjacent hollow filaments. A 3D bioprinting system with a Z-shape platform was used to realize layer-by-layer fabrication of cell-laden hydrogel structures. Curving, straight, stretched or fractured filaments can be formed by changes to the filament extrusion speed or the platform movement speed. To print a 3D structure, we first adjusted the concentration and flow rate of the sodium alginate and calcium chloride solution in the crosslinking process to get partially crosslinked filaments. Next, a motorized XY stages with the coaxial nozzle attached was used to control adjacent hollow filament deposition in the precise location for fusion. Then the Z stage attached with a Z-shape platform moved down sequentially to print layers of structure. And the printing process always kept the top two layers fusing and the below layers solidifying. Finally, the Z stage moved down to keep the printed structure immersed in the CaCl2 solution for complete crosslinking. The mechanical properties of the resulting fused structures were investigated. High-strength structures can be formed using higher concentrations of sodium alginate solution with smaller distance between adjacent hollow filaments. In addition, cell viability of this method was investigated, and the findings show that the viability of L929 mouse fibroblasts in the hollow constructs was higher than that in alginate structures without built-in microchannels. Compared with other bioprinting methods, this study is an important technique to allow easy fabrication of lager-scale organs with built-in microchannels. 

Coaxial nozzle-assisted 3D bioprinting with built-in microchannels for nutrients delivery.

Desktop three-dimensional (3D) printers (D3DPs) have become a popular tool for fabricating personalized consumer products, favored for low cost, easy operation, and other advantageous qualities. This study focused on the potential for using D3DPs to successfully, rapidly, and economically print customized implants at medical clinics. An experiment was conducted on a D3DP-printed anterior cruciate ligament surgical implant using a rabbit model. A well-defined, orthogonal, porous PLA screw-like scaffold was printed, then coated with hydroxyapatite (HA) to improve its osteoconductivity. As an internal fixation as well as an ideal cell delivery system, the osteogenic scaffold loaded with mesenchymal stem cells (MSCs) were evaluated through both in vitro and in vivo tests to observe bone-ligament healing via cell therapy. The MSCs suspended in Pluronic F-127 hydrogel on PLA/HA screw-like scaffold showed the highest cell proliferation and osteogenesis in vitro. In vivo assessment of rabbit anterior cruciate ligament models for 4 and 12 weeks showed that the PLA/HA screw-like scaffold loaded with MSCs suspended in Pluronic F-127 hydrogel exhibited significant bone ingrowth and bone-graft interface formation within the bone tunnel. Overall, the results of this study demonstrate that fabricating surgical implants at the clinic (fab@clinic) with D3DPs can be feasible, effective, and economical. 

3D Printing Surgical Implants at the clinic: A Experimental Study on Anterior Cruciate Ligament Reconstruction.

As the biocompatible materials, hydrogels have been widely used in three- dimensional (3D) bioprinting/organ printing to load cell for tissue engineering. It is important to precisely control hydrogels deposition during printing the mimic organ structures. However, the printability of hydrogels about printing parameters is seldom addressed. In this paper, we systemically investigated the printability of hydrogels from printing lines (one dimensional, 1D structures) to printing lattices/films (two dimensional, 2D structures) and printing 3D structures with a special attention to the accurate printing. After a series of experiments, we discovered the relationships between the important factors such as air pressure, feedrate, or even printing distance and the printing quality of the expected structures. Dumbbell shape was observed in the lattice structures printing due to the hydrogel diffuses at the intersection. Collapses and fusion of adjacent layer would result in the error accumulation at Z direction which was an important fact that could cause printing failure. Finally, we successfully demonstrated a 3D printing hydrogel scaffold through harmonize with all the parameters. The cell viability after printing was compared with the casting and the results showed that our bioprinting method almost had no extra damage to the cells.

Research on the printability of hydrogels in 3D bioprinting.

Now, more and more patients are suffering cerebral aneurysm. However, long training time limits the rapid growth of cerebrovascular neurosurgeons. Here we developed a novel cerebral aneurysm simulator which can be better represented the dynamic bulging process of cerebral aneurysm The proposed simulator features the integration of a hollow elastic vascular model, a skull model and a brain model, which can be affordably fabricated at the clinic (Fab@Clinic), under $25.00 each with the help of a low-cost desktop 3D printer. Moreover, the clinical blood flow and pulsation pressure similar to the human can be well simulated, which can be used to train the neurosurgical residents how to clip aneurysms more effectively.

Fabrication of cerebral aneurysm simulator with a desktop 3D printer.

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.

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.

Hydrogel structures equipped with internal microchannels offer more in vivo-relevant models for construction of tissues and organs in vitro. However, currently used microfabrication methods of constructing microfluidic devices are not suitable for the handling of hydrogel. This study presents a novel method of fabricating hydrogel-based microfluidic chips by combining the casting and bonding processes. A twice cross-linking strategy is designed to obtain a bonding interface that has the same strength with the hydrogel bulk, which can be applied to arbitrary combinations of hydrogels. It is convenient to achieve the construction of hydrogel structures with channels in branched, spiral, serpentine, and multilayer forms. The experimental results show that the combination of gelatin and gelatin methacrylate (GelMA) owns the best biocompatibility and can promote cell functionalization. Based on these, a vessel-on-a-chip system with vascular function in both physiological and pathological situations is established, providing a promising model for further investigations such as vascularization, vascular inflammation, tissue engineering, and drug development. Taken together, a facile and cytocompatible approach is developed for engineering a user-defined hydrogel-based chip that can be potentially useful in developing vascularized tissue or organ models.

Vessel-on-a-chip with Hydrogel-based Microfluidics.

As a pump-free and lightweight analytical tool, paper-based microfluidic analytical devices (μPADs) attract more and more interest. If the flow speed of μPAD can be programmed, the analytical sequences could be designed and they will be more popular. This reports presents a novel μPAD, driven by the capillary force of cellulose powder, printed by a desktop three-dimensional (3D) printer, which has some promising features, such as easy fabrication and programmable flow speed. First, a suitable size-scale substrate with open microchannels on its surface is printed. Next, the surface of the substrate is covered with a thin layer of polydimethylsiloxane (PDMS) to seal the micro gap caused by 3D printing. Then, the microchannels are filled with a mixture of cellulose powder and deionized water in an appropriate proportion. After drying in an oven at 60 °C for 30 min, it is ready for use. As the different channel depths can be easily printed, which can be used to achieve the programmable capillary flow speed of cellulose powder in the microchannels. A series of microfluidic analytical experiments, including quantitative analysis of nitrite ion and fabrication of T-sensor were used to demonstrate its capability. As the desktop 3D printer (D3DP) is very cheap and accessible, this device can be rapidly printed at the test field with a low cost and has a promising potential in the point-of-care (POC) system or as a lightweight platform for analytical chemistry.

3D Printed Paper-Based Microfluidic Analytical Devices.

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?

Applying ultrasound for scoliosis assessment has been an attractive topic over the past decade. This study proposed a new fast 3-D ultrasound projection imaging method to evaluate the spine deformity. A narrow-band rendering method was used to generate the coronal images based on B-mode images and their corresponding positional data. The non-planar reslicing method, which followed the natural spine curve, was used to project the complete spine data into the coronal image. The repeatability of the new method was tested. A comparison experiment on the reconstructed images and the processing time between the conventional 3-D rendering method and the developed projection imaging method was also performed among 70 patients with scoliosis. The intra- and inter-operator tests results demonstrated very good repeatability (ICC ≥ 0.90). The mean processing times for the developed projection method and conventional rendering method were 15.07 ± 0.03 s and 130.31 ± 35.07 s, respectively. The angle measurement results showed a high correlation (y = 0.984x, r = 0.954) between the images obtained using the two methods. The above results indicated that the developed projection imaging method could greatly decrease the processing time while preserving the comparative image quality. It can be expected that this novel method may help to provide fast 3-D ultrasound diagnosis of scoliosis in clinics.

A fast 3-D ultrasound projection imaging method for scoliosis assessment.

Plants undergo several but very precise molecular, physiological, and biochemical modulations in response to biotic stresses. Mitogen-activated protein kinase (MAPK) cascades orchestrate multiple cellular processes including plant growth and development as well as plant responses against abiotic and biotic stresses. However, the role of MAPK kinases (MAPKKs/MKKs/MEKs) in the regulation of plant resistance to herbivores has not been extensively investigated. Here, we cloned a rice MKK gene, , and investigated its function. It was observed that mechanical wounding, infestation of brown planthopper (BPH) , and treatment with methyl jasmonate (MeJA) or salicylic acid (SA) could induce the expression of . The over-expression of  (oe-MKK3) increased levels of jasmonic acid (JA), jasmonoyl-L-isoleucine (JA-Ile), and abscisic acid (ABA), and decreased SA levels in rice after BPH attack. Additionally, the preference for feeding and oviposition, the hatching rate of BPH eggs, and BPH nymph survival rate were significantly compromised due to over-expression of . Besides, oe-MKK3 also augmented chlorophyll content but impaired plant growth. We confirm that MKK3 plays a pivotal role in the signaling pathway. It is proposed that  mediated positive regulation of rice resistance to BPH by means of herbivory-induced phytohormone dynamics.

 a Stress-Responsive Protein Kinase, Positively Regulates Rice Resistance to  via Phytohormone Dynamics.

Hydrogels are very popular in biomedical areas for their extraordinary biocompatibility. However, most bio-hydrogels are too brittle to perform micro/nanofabrication. An effective method is cast molding; yet during this process, many defects occur as the excessive demolding stress damages the brittle hydrogels. Here, we propose a brand-new damage-free demolding method and a soft ultrafine fiber mold (SUFM) to replace the traditional mold. Both mechanical and finite element analysis (FEA) reveal that SUFMs have obvious advantages especially when the contact area between hydrogel and mold gets larger. By means of a high-resolution 3D printing called electrohydrodynamic (EHD) printing, SUFMs with various topological structures can be achieved with the fiber diameter ranging from 500 nm to 100 μm at a low cost. Microfluidics and cell patterns are implemented as the demonstration for potential applications. Owing to the tiny scale of microstructures and the hydrophilicity of hydrogels, significant capillary effect occurs which can be utilized to deliver liquid and cells autonomously and to seed cells into those ultrafine channels evenly. The results open up a new avenue for a wider use of hydrogels in biomedical devices, tissue engineering, microfluidics and wearable electronics; the proposed fabrication method also has the potential to become a universal technique for micro/nanofabrication of brittle materials.

Micro/nanofabrication of brittle hydrogels using 3D printed soft ultrafine fiber molds for damage-free demolding.

It is a dilemma that both strength and biocompatibility are requirements for an ideal scaffold in tissue engineering. The normal strategy is mixing or coating another material to improve the biocompatibility. Could we solve this dilemma by simply adjusting the scaffold structure? Here, a novel multi-scale scaffold was designed, in which thick fibers provide sufficient strength for mechanical support while the thin fibers provide a cell-favorable microenvironment to facilitate cell adhesion. Moreover, we developed a promising multi-scale direct writing system (MSDWS) for printing the multi-scale scaffolds. By switching the electrostatic field, scaffolds with fiber diameters from 3 μm to 600 μm were fabricated using one nozzle. Using this method, we proved that PCL scaffolds could also have excellent biocompatibility. BMSCs seeded on the scaffolds readily adhered to the thin fibers and maintained a high proliferation rate. Moreover, the cells bridged across the pores to form a cell sheet and gradually migrated to the thick fibers to cover the entire scaffold. We further combined the scaffolds with hydrogel for 3D cell culture and found that the fibers enhanced the strength and induced cell migration. We believe that the multi-scale scaffolds fabricated by an innovative 3D printing system have great potential for tissue engineering.

3D printed multi-scale scaffolds with ultrafine fibers for providing excellent biocompatibility.

Hierarchical tissues composed of spheroid and fiber structures such as tumors, embryos and glomeruli widely exist in organisms. Methods have been developed to build spheroid and fiber structures, independently, as tissue models in vitro. However, it is still a challenge to print them simultaneously and integrated for effectively mimicking the complicated situations in vivo. Here, we propose a novel 3D cell co-culturing system, "microfiber-laden minispheroid", applying two fluidic phenomena, namely the "rope coiling effect" and "electrohydrodynamics", with a co-axial bioprinting nozzle and high voltage system. Gelatin methacryloyl (GelMA) hydrogels were extruded from the outer nozzle and separated by electrical attraction to form spheroids (∼1800 μm). GelMA mixed with sodium alginate was extruded from the inner nozzle to form fibers (∼180 μm) inside spheroids, whose morphology could be controlled by the ratio of inner and outer nozzle extruding flow rates. We analyzed the fabrication process and the material system in detail, verifying the fabrication feasibility and suitable microenvironment. The encapsulated cells possessed high viabilities. Importantly, the actin of human umbilical vein endothelial cells tended to elongate towards the co-cultured tumor cells, in contrast to the HUVECs cultured alone. We believe that "microfiber-laden minispheroids" could be a potential system for 3D cell co-culturing research in the future.

3D biofabrication of microfiber-laden minispheroids: a facile 3D cell co-culturing system.

Maintaining an adequate supply of nutrients/oxygen is a major challenge in the biofabrication of large tissue constructs. However, building preformed nutrient networks may be an effective strategy for engineering thick tissues. Here, a novel way for bioprinting large-scale tissue constructs with intentional nutrient networks is presented. A special nozzle is developed which can print bioink and sacrificial ink half and half synchronously in a single filament. Nutrient networks of these bioprinted constructs are formed by subsequently dissolving away gelatin, which allows for effective oxygen, nutrient, and waste diffusion, facilitating the cell activity and the generation of functional tissues. Due to the cell-laden bioink and sacrificial ink working together and promoting each other's printability to support themselves, complex soft cell-laden constructs with nutrient networks can easily be printed. Furthermore, two different cell types (osteoblast, human umbilical vein endothelial cells) encapsulated in the bioprinted large-scale constructs (≥1 cm) with nutrient networks show enhanced cell viability and spreading within a period of culture. It is envisioned that the advanced bioprinting technology may have significant potentials in facilitating the engineering of complex structures for tissue-specific needs, and bioprinting large-scale tissue constructs with nutrient networks toward applications in organ transplantation and repair.

Article	Year
Coaxial nozzle-assisted 3D bioprinting with built-in microchannels for nutrients delivery. Biomaterials\| PubMed ID: 26004235	2015
3D Printing Surgical Implants at the clinic: A Experimental Study on Anterior Cruciate Ligament Reconstruction. Scientific reports\| PubMed ID: 26875826	2016
Research on the printability of hydrogels in 3D bioprinting. Scientific reports\| PubMed ID: 27436509	2016
Fabrication of cerebral aneurysm simulator with a desktop 3D printer. Scientific reports\| PubMed ID: 28513626	2017
3D printed Lego-like modular microfluidic devices based on capillary driving. Biofabrication\| PubMed ID: 29417931	2018
Fiber-Based Mini Tissue with Morphology-Controllable GelMA Microfibers. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 30253060	2018
Vessel-on-a-chip with Hydrogel-based Microfluidics. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 30307698	2018
3D Printed Paper-Based Microfluidic Analytical Devices. Micromachines\| PubMed ID: 30404282	2016
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
A fast 3-D ultrasound projection imaging method for scoliosis assessment. Mathematical biosciences and engineering : MBE\| PubMed ID: 30947409	2019
a Stress-Responsive Protein Kinase, Positively Regulates Rice Resistance to via Phytohormone Dynamics. International journal of molecular sciences\| PubMed ID: 31226870	2019
Micro/nanofabrication of brittle hydrogels using 3D printed soft ultrafine fiber molds for damage-free demolding. Biofabrication\| PubMed ID: 31726448	2019
3D printed multi-scale scaffolds with ultrafine fibers for providing excellent biocompatibility. Materials science & engineering. C, Materials for biological applications\| PubMed ID: 31761213	2020
3D biofabrication of microfiber-laden minispheroids: a facile 3D cell co-culturing system. Biomaterials science\| PubMed ID: 31761908	2019
Synchronous 3D Bioprinting of Large-Scale Cell-Laden Constructs with Nutrient Networks. Advanced healthcare materials\| PubMed ID: 31846229	2019

Qing Gao

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