Inkjet Bioprinting Research Articles

Bio-Printing of Materials for Bone Tissue Engineering

Abstract: The complicated internal mechanical and structural qualities of normal bone tissue still prevent the development of effective therapeutic procedures for major bone lesions. It is still difficult to use tissue engineering to return damaged bones back to how they were originally intended. Due to recent advances in 3D printing, together with the introduction of new materials and technological assistance, the basis for BTE has been established. Biological 3D biomaterials have cells inside them, which allows for the creation of structures that mimic real tissues. Microextrusion, inkjet, and laser-assisted bioprinting are the three primary methods used in 3D bioprinting manufacturing. Hydrogels packed with cells, growth hormones, and bioactive ceramics are among the bioinks utilized in bone bioprinting. With the use of magnetic resonance imaging or computed tomography scanning, 3D printing offers substantial benefits for tailored treatment by enabling the creation of scaffolds with the right structural qualities, form, and dimensions. Three-dimensional (3D) bioprinting is a cutting-edge technique that has been utilized recently to create multicellular, biomimetic tissues with layers upon layers of intricate tissue microenvironment printing. We approached the use of hydrogels with great strength in 3D printing for BTE with an emphasis on first providing a thorough study about the development of 3D printing, printing techniques, and ink selection in this review. A brief prediction on how 3D printing would advance in the future was made.

Multi-physical field control piezoelectric inkjet bioprinting for 3D tissue-like structure manufacturing

Multi-physical field control piezoelectric inkjet bioprinting for 3D tissue-like structure manufacturing

The significance of bioprinting methods in bioprinting for tissue engineering

Bioprinting has been a trending technology in recent years. It is a combination of engineering and biology. It can be used in the fields of tissue engineering and drug screening. It can be used to print 3D structures using 3D modeling software and to print with cultured cells. Bioink should have cells with the following characteristics: cell viability, cell differentiation, degradation, biocompatibility, and printability. Bioink contains biomaterials, living cells, and growth factors that are used to grow the cells. The replaced cells should possess chemical, physical, and mechanical properties. There are several methods to print biological organs, including microextrusion, inkjet bioprinting, and stereolithography.

3D printing dielectric elastomers for advanced functional structures: A mini‐review

AbstractThree‐dimensional (3D) printed bionic products play an important role in intelligent robotics, microelectronics, and polymers. The printing and manufacturing process of 3D printers is conducive to obtaining soft structures that meet specific requirements, and saves time and cost. Soft intelligent robotics, an emerging research field, has always been developed based on soft materials and actuators with their biological properties. This article reviews the current understanding of 3D bioprinting technologies for dielectric elastomers (DEs), DE actuators (DEAs) and soft robots, such as inkjet, extrusion, laser‐induced and stereolithography bioprinting. 3D printers for fabricating soft materials are presented and classified. The approaches to exploit 3D bioprinters for DEs/DEAs are as follows: (1) 3D printing DEAs utilize ionic hydrogel–elastomer hybrids that are analogous to human muscles, and the DEAs usually have flexible structures and large deformations with multiple functionalities. (2) An electrohydrodynamic (EHD) 3D printer confers high printing resolution and high production efficiency, which offers advantages such as full automation and flexible design. The optimal printing conditions are mainly determined by the effects of printing voltages and ink properties, which are related to the formation of the liquid cone and the printed line width. Furthermore, the advantages of 3D bioprinting technologies have accelerated their development and applications.

Applications of Light-Based 3D Bioprinting and Photoactive Biomaterials for Tissue Engineering

The emergence of additive manufacturing, commonly referred to as 3D printing, has led to a revolution in the field of biofabrication. Numerous types of 3D bioprinting, including extrusion bioprinting, inkjet bioprinting, and lithography-based bioprinting, have been developed and have played pivotal roles in driving a multitude of pioneering breakthroughs in the fields of tissue engineering and regenerative medicine. Among all the 3D bioprinting methods, light-based bioprinting utilizes light to crosslink or solidify photoreactive biomaterials, offering unprecedented spatiotemporal control over biomaterials and enabling the creation of 3D structures with extremely high resolution and precision. However, the lack of suitable photoactive biomaterials has hindered the application of light-based bioprinting in tissue engineering. The development of photoactive biomaterials has only recently been expanded. Therefore, this review summarizes the latest advancements in light-based 3D bioprinting technologies, including the development of light-based bioprinting techniques, photo-initiators (PIs), and photoactive biomaterials and their corresponding applications. Moreover, the challenges facing bioprinting are discussed, and future development directions are proposed.

Perspectives on the Current State of Bioprinted Skin Substitutes for Wound Healing.

Human skin is particularly vulnerable to external damaging influences such as irradiation, extreme temperatures, chemical trauma, and certain systemic diseases, which reduce the skin's capacity for regeneration and restoration and can possibly lead to large-scale skin defects. To restore skin continuity in severe cases, surgical interventions such as the transplantation of autologous tissue are needed. Nevertheless, the coverage of larger skin defects caused by severe third-grade burns or extensive irradiation therapy is limited due to the depletion of uninjured autologous tissue. In such cases, many of the patient's epidermal cells can become available using biofabricated skin grafts, thereby restoring the skin's vital functions. Given the limited availability of autologous skin grafts for restoring integrity in large-scale defects, using bioprinted constructs as skin graft substitutes could offer an encouraging therapeutic alternative to conventional therapies for large-scale wounds, such as the transplantation of autologous tissue. Using layer-by-layer aggregation or volumetric bioprinting, inkjet bioprinting, laser-assisted bioprinting, or extrusion-based bioprinting, skin cells are deposited in a desired pattern. The resulting constructs may be used as skin graft substitutes to accelerate wound healing and reconstitute the physiological functions of the skin. In this review, we aimed to elucidate the current state of bioprinting within the context of skin tissue engineering and introduce and discuss different bioprinting techniques, possible approaches and materials, commonly used cell types, and strategies for graft vascularization for the production of bioprinted constructs for use as skin graft substitutes.

Inkjet-based bioprinting for tissue engineering

Inkjet bioprinting, a derivative of traditional inkjet technology, is gaining recognition in the fields of life sciences and tissue engineering due to its ability to produce picoliter volume droplets at high speeds in a non-contact fashion. This method has impressively evolved from enabling the production of 2-dimensional (2D) prints to complex 3-dimensional (3D) structures, and is increasingly being used in the manufacturing of electronic components. More recently, this technology has been effectively adapted for a variety of medical applications, such as cell patterning, scaffold construction, and 3D tissue fabrication. In this review, we delve into the principles and biological uses of inkjet technology. We provide an in-depth discussion on the latest developments in inkjet bioprinting, with a focus on cell patterning and 3D fabrication of tissue models, including multilayered lung, bladder, and skin. We also explore the potential of high-throughput 3D-bioprinted tissue models in toxicology and drug efficacy testing.

Splashing of droplet under the vibration effect of flexible membrane

The impact dynamics of a droplet falling onto a substrate is significant in various applications such as inkjet printing, spray coating, and bioprinting. Generally, several factors, such as the fluid properties (e.g. viscosity or surface tension) and substrate characteristics (e.g. surface roughness or flexibility) govern whether falling droplets spread or splash. In this study, we experimentally investigate droplet impact dynamics on a thin, flexible membrane for various Weber numbers (230 < We < 600) and membrane stiffnesses, which can be controlled by the applied tensile force. In addition, the effect of membrane vibration on impact behavior is considered. In the high Weber number regime, splashing is dominant, regardless of the membrane stiffness. In contrast, in the low Weber number regime, a transition between splashing and deposition is observed, depending on the membrane stiffness. In short, flexible membrane vibration induces droplet splashing despite the insufficient kinetic energy of the droplet. Increasing the stiffness of the membrane contributes to the suppression of droplet splashing by minimizing membrane vibration.

A study on cell viability based on thermal inkjet three-dimensional bioprinting

Thermal inkjet three-dimensional (3D) bioprinting (TIJ) is a biological additive manufacturing technology with high cell viability, fast printing speeds, and low costs. It is widely used in biology, chemistry, and pharmaceuticals. In recent years, remarkable results have been achieved in the printing of biological tissues using TIJ. However, few studies have reported on the relationship between TIJ and cell viability. In particular, there have been no reports relating cell viability and the TIJ input energy. In this work, we aim to determine the relationship between the input pulse, printing frequency, and cell viability from the TIJ working principle and find an optimized pulse waveform to improve cell viability. We propose a novel approach to study cell viability. The state of the droplet is observed while controlling the printing pulse and frequency, and then, the corresponding cell viability is determined. The results show that an increase in the pulse increases the shear stress and temperature in the bio-ink, which reduces the viability of the cells. The shear stress and viability of the printed cells show a corresponding piecewise functional relationship. The cell viability is significantly reduced when the ambient temperature is higher than 40 °C. Increasing the printing frequency reduces the rate of printing heat loss, thereby raising the ambient temperature and impairing cell viability. Finally, the optimized input waveform can increase cell viability by up to about 95%.

Thermal inkjet bioprinting drastically alters cell phenotype

Since the first description of inkjet bioprinting of cells in 2003, quantifying the input and measuring the output of the printers has been the hallmark of the field of bioprinting, as it is virtually impossible to characterize cells that are inside the printing orifices or extrusion needles. We will describe here some recent discoveries of cell behavior due to inkjet bioprinting. Primary and immortalized adult dermal fibroblasts were expanded for 2–3 passages upon receiving. The cells were harvested, resuspended in PBS, and bioprinted into a 96-well plate with pluriSTEM media. Cells were then transferred either into precoated 96-well plates or 20 µl drops were pipetted for hanging drop culture. IPC differentiation protocols were applied and the induction was begun approximately 45 min after printing. When differentiating aggregates, the initiation happened 45 min after the aggregates were transferred into the 96 wells. Standard immunostaining and RNA sequencing (RNA-Seq) were used to analyze the cell phenotypes. Preliminary results indicate that all cells expressed the three pluripotency markers oct-4, nanog, and sox-2. After applying a cardiomyocyte differentiation protocol, the cells stained positively for troponin-3. The cells also elongated and became more cardiomyocyte-like in their morphology. We analyzed bulk RNA seq data and our preliminary results show upregulation of some genes that have been implicated as stem cell markers: EPCAM, LEFTY1, ZFP42, and TEX19. In addition, differential expression of genes associated with pluripotency-relevant pathways shows some pathways are off like the MAPK/p38, MAPK/JNK1-3 which is expected for a pluripotent state. We also have data supporting the activation of the hippo pathway with transcriptional co-activator with PDZ binding motif (TAZ) highly upregulated and yes-associated protein staining the cell body. In addition, GSK3B is off and TGFB1, LIF/PIK3, and AKT1 are on as expected for pluripotency. Examining the gene network of upregulated genes, one can clearly distinguish the pivotal role of FOS, FOXO1, and PIK3 all related to pluripotency. Bioprinted fibroblasts will at least temporarily adopt a more primitive or dedifferentiated state, reminiscent of pluripotency. While immunochemistry shows the classic transcription factors required for pluripotency, gene expression shows a more nuanced picture of the transformations that occur upon printing. Understanding these transformations, even if temporary will be crucial when trying to build tissues using bioprinting technologies.

Understanding droplet jetting on varying substrate for biological applications.

In the inkjet printing process, the droplet experience two phases, namely the jetting and the impacting phases. In this review article, we aim to understand the physics of a jetted ink, which begins during the droplet formation process. Following which, we highlight the different impacts during which the droplet lands on varying substrates such as solid, liquid, and less commonly known viscoelastic material. Next, the article states important process-specific considerations in determining the success of inkjet bioprinted constructs. Techniques to reduce cell deformation throughout the inkjet printing process are highlighted. Modifying postimpact events, such as spreading, evaporation, and absorption, improves cell viability of printed droplet. Last, applications that leverage on the advantage of pixelation in inkjet printing technology have been shown for drug screening and cell-material interaction studies. It is noteworthy that inkjet bioprinting technology has been integrated with other processing technologies to improve the structural integrity and biofunctionality of bioprinted construct.

Chondroitinase ABC Treatment Improves the Organization and Mechanics of 3D Bioprinted Meniscal Tissue.

The meniscus is a fibrocartilage tissue that is integral to the correct functioning of the knee joint. The tissue possesses a unique collagen fiber architecture that is integral to its biomechanical functionality. In particular, a network of circumferentially aligned collagen fibers function to bear the high tensile forces generated in the tissue during normal daily activities. The limited regenerative capacity of the meniscus has motivated increased interest in meniscus tissue engineering; however, the in vitro generation of structurally organized meniscal grafts with a collagen architecture mimetic of the native meniscus remains a significant challenge. Here we used melt electrowriting (MEW) to produce scaffolds with defined pore architectures to impose physical boundaries upon cell growth and extracellular matrix production. This enabled the bioprinting of anisotropic tissues with collagen fibers preferentially oriented parallel to the long axis of the scaffold pores. Furthermore, temporally removing glycosaminoglycans (sGAGs) during the early stages of in vitro tissue development using chondroitinase ABC (cABC) was found to positively impact collagen network maturation. Specially we found that temporal depletion of sGAGs is associated with an increase in collagen fiber diameter without any detrimental effect on the development of a meniscal tissue phenotype or subsequent extracellular matrix production. Moreover, temporal cABC treatment supported the development of engineered tissues with superior tensile mechanical properties compared to empty MEW scaffolds. These findings demonstrate the benefit of temporal enzymatic treatments when engineering structurally anisotropic tissues using emerging biofabrication technologies such as MEW and inkjet bioprinting.

3D culture of fibroblasts and neuronal cells on microfabricated free-floating carriers

3D cell culture is a relatively recent trend in biomedical research for artificially mimicking in vivo environment and providing three dimensions for the cells to grow in vitro, particularly with regard to surface-adherent mammalian cells. Different cells and research objectives require different culture conditions which has led to an increase in the diversity of 3D cell culture models. In this study, we show two independent on-carrier 3D cell culture models aimed at two different potential applications. Firstly, micron-scale porous spherical structures fabricated from poly (lactic-co-glycolic acid) or PLGA are used as 3D cell carriers so that the cells do not lose their physiologically relevant spherical shape. Secondly, millimetre-scale silk fibroin structures fabricated by 3D inkjet bioprinting are used as 3D cell carriers to demonstrate cell growth patterning in 3D for use in applications which require directed cell growth. The L929 fibroblasts demonstrated excellent adherence, cell-division and proliferation on the PLGA carriers, while the PC12 neuronal cells showed excellent adherence, proliferation and spread on the fibroin carriers without any evidence of cytotoxicity from the carriers. The present study thus proposes two models for 3D cell culture and demonstrates, firstly, that easily fabricable porous PLGA structures can act as excellent cell carriers for aiding cells easily retain their physiologically relevant 3D spherical shape in vitro, and secondly, that 3D inkjet printed silk fibroin structures can act as geometrically-shaped carriers for 3D cell patterning or directed cell growth in vitro. While the ‘fibroblasts on PLGA carriers’ model will help achieve more accurate results than the conventional 2D culture in cell research, such as drug discovery, and cell proliferation for adoptive cell transfer, such as stem cell therapy, the ‘neuronal cells on silk fibroin carriers’ model will help in research requiring patterned cell growth, such as treatment of neuropathies.

Sonochemical Degradation of Gelatin Methacryloyl to Control Viscoelasticity for Inkjet Bioprinting.

Inkjet printing enables the mimicry of the microenvironment of natural complex tissues by patterning cells and hydrogels at a high resolution. However, the polymer content of an inkjet-printable bioink is limited as it leads to strong viscoelasticity in the inkjet nozzle. Here it is demonstrated that sonochemical treatment controls the viscoelasticity of a gelatin methacryloyl (GelMA) based bioink by shortening the length of polymer chains without causing chemical destruction of the methacryloyl groups. The rheological properties of treated GelMA inks are evaluated by a piezo-axial vibrator over a wide range of frequencies between 10 and 10000Hz. This approach enables to effectively increase the maximum printable polymer concentration from 3% to 10%. Then it is studied how the sonochemical treatment effectively controls the microstructure and mechanical properties of GelMA hydrogel constructs after crosslinking while maintaining its fluid properties within the printable range. The control of mechanical properties of GelMA hydrogels can lead fibroblasts more spreading on the hydrogels. A 3D cell-laden multilayered hydrogel constructs containing layers with different physical properties is fabrictated by using high-resolution inkjet printing. The sonochemical treatment delivers a new path to inkjet bioprinting to build microarchitectures with various physical properties by expanding the range of applicable bioinks.

Bioprinting of structurally organized meniscal tissue within anisotropic melt electrowritten scaffolds.

The meniscus is characterised by an anisotropic collagen fibre network which is integral to its biomechanical functionality. The engineering of structurally organized meniscal grafts that mimic the anisotropy of the native tissue remains a significant challenge. In this study, inkjet bioprinting was used to deposit a cell-laden bioink into additively manufactured scaffolds of differing architectures to engineer fibrocartilage grafts with user defined collagen architectures. Polymeric scaffolds consisting of guiding fibre networks with varying aspect ratios (1:1; 1:4; 1:16) were produced using either fused deposition modelling (FDM) or melt electrowriting (MEW), resulting in scaffolds with different internal architectures and fibre diameters. Scaffold architecture was found to influence the spatial organization of the collagen network laid down by the jetted cells, with higher aspect ratios (1:4 and 1:16) supporting the formation of structurally anisotropic tissues. The MEW scaffolds supported the development of a fibrocartilaginous tissue with compressive mechanical properties similar to that of native meniscus, while the anisotropic tensile properties of these constructs could be tuned by altering the fibre network aspect ratio. This MEW framework was then used to generate scaffolds with spatially distinct fibre patterns, which in turn supported the development of heterogenous tissues consisting of isotropic and anisotropic collagen networks. Such bioprinted tissues could potentially form the basis of new treatment options for damaged and diseased meniscal tissue. STATEMENT OF SIGNIFICANCE: This study describes a multiple tool biofabrication strategy which enables the engineering of spatially organized fibrocartilage tissues. The architecture of MEW scaffolds can be tailored to not only modulate the directionality of the collagen fibres laid down by cells, but also to tune the anisotropic tensile mechanical properties of the resulting constructs, thereby enabling the engineering of biomimetic meniscal-like tissues. Furthermore, the inherent flexibility of MEW enables the development of zonally defined and potentially patient-specific implants.

Influence of Process Parameters on the Characteristics of Additively Manufactured Parts Made from Advanced Biopolymers.

Over the past few decades, additive manufacturing (AM) has become a reliable tool for prototyping and low-volume production. In recent years, the market share of such products has increased rapidly as these manufacturing concepts allow for greater part complexity compared to conventional manufacturing technologies. Furthermore, as recyclability and biocompatibility have become more important in material selection, biopolymers have also become widely used in AM. This article provides an overview of AM with advanced biopolymers in fields from medicine to food packaging. Various AM technologies are presented, focusing on the biopolymers used, selected part fabrication strategies, and influential parameters of the technologies presented. It should be emphasized that inkjet bioprinting, stereolithography, selective laser sintering, fused deposition modeling, extrusion-based bioprinting, and scaffold-free printing are the most commonly used AM technologies for the production of parts from advanced biopolymers. Achievable part complexity will be discussed with emphasis on manufacturable features, layer thickness, production accuracy, materials applied, and part strength in correlation with key AM technologies and their parameters crucial for producing representative examples, anatomical models, specialized medical instruments, medical implants, time-dependent prosthetic features, etc. Future trends of advanced biopolymers focused on establishing target-time-dependent part properties through 4D additive manufacturing are also discussed.

Application of Hydrogels as Three-Dimensional Bioprinting Ink for Tissue Engineering.

The use of three-dimensional bioprinting technology combined with the principle of tissue engineering is important for the construction of tissue or organ regeneration microenvironments. As a three-dimensional bioprinting ink, hydrogels need to be highly printable and provide a stiff and cell-friendly microenvironment. At present, hydrogels are used as bioprinting inks in tissue engineering. However, there is still a lack of summary of the latest 3D printing technology and the properties of hydrogel materials. In this paper, the materials commonly used as hydrogel bioinks; the advanced technologies including inkjet bioprinting, extrusion bioprinting, laser-assisted bioprinting, stereolithography bioprinting, suspension bioprinting, and digital 3D bioprinting technologies; printing characterization including printability and fidelity; biological properties, and the application fields of bioprinting hydrogels in bone tissue engineering, skin tissue engineering, cardiovascular tissue engineering are reviewed, and the current problems and future directions are prospected.

Inkjet 3D bioprinting for tissue engineering and pharmaceutics

Inkjet 3D bioprinting for tissue engineering and pharmaceutics

Inkjet 3D bioprinting for tissue engineering and pharmaceutics

Inkjet 3D bioprinting for tissue engineering and pharmaceutics

Simulation of a Hemispherical Chamber for Thermal Inkjet Printing.

It is crucial to improve printing frequency and ink droplet quality in thermal inkjet printing. This paper proposed a hemispherical chamber, and we used the CFD (computational fluid dynamics model) to simulate the inkjet process. During the whole simulation process, we first researched the hemispherical chamber's inkjet state equipped with straight, conical shrinkage, and conical diffusion nozzles. Based on the broken time and volume of the liquid column, the nozzle geometry of the hemispherical chamber was determined to be a conical shrinkage nozzle with a specific size of 15 µm in height and 15 µm in diameter at the top, and 20 µm in diameter at the bottom. Next, we researched the inkjet performance of the square chamber, the round chamber, and the trapezoidal chamber. The round chamber showed the best inkjet performance using 1.8 µs as the driving time and 10 MPa as the maximum bubble pressure. After that, we compared the existing thermal inkjet printing heads. The results showed that the hemispherical chamber inkjet head had the best performance, achieving 30 KHz high-frequency printing and having the most significant volume ratio of droplet to the chamber, reaching 14.9%. As opposed to the current 15 KHz printing frequency of the thermal inkjet heads, the hemispherical chamber inkjet head has higher inkjet performance, and the volume ratio between the droplet and the chamber meets the range standard of 10-15%. The hemispherical chamber structure can be applied to thermal inkjet printing, office printing, 3D printing, and bio-printing.