Inkjet Bioprinting Research Articles

Bioprinting of Human Musculoskeletal Interface

Human musculoskeletal interface (MI) refer to biofunctional and engineering similarities enabling smooth connections through muscular and skeletal attachments. MI is commonly involved in musculoskeletal injuries and degenerative diseases, while the key problem to achieve biological integration with the surrounding host tissues of MI is fabricating substitution with precisely structural and material distribution. Bioprinting has made it possible to achieve artificial tissues with spatial controlled heterogeneity of physical properties and bioactive composition similar to native MI tissues. In this review, emphasis is put on detailed introduction of structural and biofunctional properties of MI tissues, as well as recent MI bioprinting application of microextrusion and inkjet bioprinting approaches. Specially, by combining 3D printing and bioprinting approaches, hybrid bioprinting approaches of reinforced MI constructs are retrospected. Other issues of biomaterials, bioprinting methods and relevant regulations are discussed. Future challenges in engineering, material science and clinical transformation are also summarized. Taken together, bioprinting of MI tissues can be of great interest in future development of tissue engineering and regenerative medicine.

Advancing Frontiers in Bone Bioprinting.

Three-dimensional (3D) bioprinting of cell-laden biomaterials is used to fabricate constructs that can mimic the structure of native tissues. The main techniques used for 3D bioprinting include microextrusion, inkjet, and laser-assisted bioprinting. Bioinks used for bone bioprinting include hydrogels loaded with bioactive ceramics, cells, and growth factors. In this review, a critical overview of the recent literature on various types of bioinks used for bone bioprinting is presented. Major challenges, such as the vascularity, clinically relevant size, and mechanical properties of 3D printed structures, that need to be addressed to successfully use the technology in clinical settings, are discussed. Emerging approaches to solve these problems are reviewed, and future strategies to design customized 3D printed structures are proposed.

Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers

Successful tissue engineering requires the generation of human scale implants that mimic the structure, composition and mechanical properties of native tissues. Here, we report a novel biofabrication strategy that enables the engineering of structurally organised tissues by guiding the growth of cellular spheroids within arrays of 3D printed polymeric microchambers. With the goal of engineering stratified articular cartilage, inkjet bioprinting was used to deposit defined numbers of mesenchymal stromal cells (MSCs) and chondrocytes into pre-printed microchambers. These jetted cell suspensions rapidly underwent condensation within the hydrophobic microchambers, leading to the formation of organised arrays of cellular spheroids. The microchambers were also designed to provide boundary conditions to these spheroids, guiding their growth and eventual fusion, leading to the development of stratified cartilage tissue with a depth-dependant collagen fiber architecture that mimicked the structure of native articular cartilage. Furthermore, the composition and biomechanical properties of the bioprinted cartilage was also comparable to the native tissue. Using multi-tool biofabrication, we were also able to engineer anatomically accurate, human scale, osteochondral templates by printing this microchamber system on top of a hypertrophic cartilage region designed to support endochondral bone formation and then maintaining the entire construct in long-term bioreactor culture to enhance tissue development. This bioprinting strategy provides a versatile and scalable approach to engineer structurally organised cartilage tissues for joint resurfacing applications.

Effective bioprinting resolution in tissue model fabrication.

Recent advancements in bioprinting techniques have enabled convenient fabrication of micro-tissues in organ-on-a-chip platforms. In a sense, the success of bioprinted micro-tissues depends on how close their architectures are to the anatomical features of their native counterparts. The bioprinting resolution largely relates to the technical specifications of the bioprinter platforms and the physicochemical properties of the bioinks. In this article, we compare inkjet, extrusion, and light-assisted bioprinting technologies for fabrication of micro-tissues towards construction of biomimetic organ-on-a-chip platforms. Our theoretical analyses reveal that for a given printhead diameter, surface contact angle dominates inkjet bioprinting resolution, while nozzle moving speed and the nonlinearity of viscosity for bioinks regulate extrusion bioprinting resolution. The resolution of light-assisted bioprinting is strongly affected by the photocrosslinking behavior and light characteristics. Our tutorial guideline for optimizing bioprinting resolution would potentially help model the complex microenvironment of biological tissues in organ-on-a-chip platforms.

Electro-Assisted Bioprinting of Low-Concentration GelMA Microdroplets.

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.

Advances in 3D Bioprinting for Neural Tissue Engineering

AbstractCurrent therapies for nerve regeneration within injured tissues have had limited success due to complicated neural anatomy and inhibitory barriers in situ. Recent advancements in 3D bioprinting technologies have enabled researchers to develop novel 3D scaffolds with complex architectures in an effort to mitigate the challenges that beset reliable and defined neural tissue regeneration. Among several possible neuroregenerative treatment approaches that are being explored today, 3D bioprinted scaffolds have the unique advantage of being highly modifiable, which promotes greater resemblance to the native biological architecture of in vivo systems. This high architectural similarity between printed constructs and in vivo structures is thought to facilitate a greater capacity for repair of damaged nerve tissues. In this review, advances of several 3D bioprinting methods are introduced, including laser bioprinting, inkjet bioprinting, and extrusion‐based printing. In addition, the emergence of 4D printing is discussed, which adds a dimension of transformation over time to traditional 3D printing. Finally, an overview of emerging trends in advanced bioprinting materials is provided and their therapeutic potential for application in neural tissue regeneration is evaluated in both the central nervous system and the peripheral nervous system.

Paper-based inkjet bioprinting to detect fluorescence resonance energy transfer for the assessment of anti-inflammatory activity

For the first time, a paper-based fluorescence resonance energy transfer (FRET) determination with cyclic AMP (cAMP)-specific phosphodiesterase 4B (PDE4B) inhibitory assay using an inkjet-printing technique is proposed. Non-fabricated parchment paper is found to constitute a unique substrate to measure fluorescent energy transfer, due to its insignificant self-absorption, and enables efficient sample interaction. Here, we report the responsive FRET signals generated on paper, upon sequentially printing reaction components on parchment paper using a conventional inkjet printer equipped with four cartridges. After printing, the energy emitted by Eu chelate was transferred by FRET to ULight molecule on paper, detected at 665 nm. In the absence of free cAMP, a maximum FRET signal was achieved on paper, while a decrease in FRET signals was recorded when free cAMP produced by PDE4B inhibitors compete with Eu-cAMP, binding with ULight-mAb. The IM50 value was determined as 2.46 × 10−13 mole for roliparm and 1.86 × 10−13 mole for roflumilast, to effectively inhibit PDE4B activity. Inkjet printing-based FRET signal determination utilizes components that are less than the femtomole range, which was four-orders less than the standard assay method. The methodology reported here constitutes an innovative approach towards the determination of FRET signals generated on paper.

Drop-On-Drop Multimaterial 3D Bioprinting Realized by Peroxidase-Mediated Cross-Linking.

A cytocompatible inkjet bioprinting approach that enables the use of a variety of bioinks to produce hydrogels with a wide range of characteristics is developed. Stabilization of bioinks is caused by horseradish peroxidase (HRP)-catalyzed cross-linking consuming hydrogen peroxide (H2 O2 ). 3D cell-laden hydrogels are fabricated by the sequential dropping of a bioink containing polymer(s) cross-linkable through the enzymatic reaction and H2 O2 onto droplets of another bioink containing the polymer, HRP, and cells. The ≈95% viability of enclosed mouse fibroblasts and subsequent elongation of the cells in a bioprinted hydrogel consisting of gelatin and hyaluronic acid derivatives suggest the high cytocompatibility of the developed printing approach. The existence of numerous polymers, including derivatives of polysaccharides, proteins, and synthetic polymers, cross-linkable through the HRP-catalyzed reaction, means the current approach shows great promise for biofabrication of functional and structurally complex tissues.

In Situ Printing-then-Mixing for Biological Structure Fabrication Using Intersecting Jets.

Although traditional three-dimensional bioprinting technology is suitable for many tissue engineering applications, various biomaterials and constructs call for bioprinting innovations. There is a need for the fabrication of complex structures from reactive biomaterials as well as heterogeneous structures with controlled material compositions. In particular, during reactive material printing, reactive solutions/suspensions that undergo changes in rheological properties or cytocompatibility are not printable using traditional bioprinting approaches that require all components of bioinks to be mixed before deposition. The objective of this study is to develop and implement an intersecting jets-based inkjet bioprinting approach, which enables voxel-resolution printing-then-mixing for the fabrication of biological structures using reactive materials as well as structures having a compositional gradient. Inkjetting is implemented herein as a versatile technique to simultaneously deposit droplets of disparate materials at controlled locations where active collision, mixing, and coalescence occur. For reactive material printing, neural stem cell (NSC) spheres are fabricated from reactive PuraMatrix hydrogel solution and physiological cell suspension, and cell-laden alginate structures are also printed in air directly from reactive sodium alginate and calcium chloride solutions. For heterogeneous structure printing, collagen sheets with a hydroxyapatite (HAP) content gradient are fabricated to demonstrate the unique online control of material composition throughout a structure. It is demonstrated that the proposed bioprinting approach is feasible for applications that utilize reactive materials or require heterogeneous compositions.

3D bioprinting using stem cells

Recent advances have allowed for three-dimensional (3D) printing technologies to be applied to biocompatible materials, cells and supporting components, creating a field of 3D bioprinting that holds great promise for artificial organ printing and regenerative medicine. At the same time, stem cells, such as human induced pluripotent stem cells, have driven a paradigm shift in tissue regeneration and the modeling of human disease, and represent an unlimited cell source for tissue regeneration and the study of human disease. The ability to reprogram patient-specific cells holds the promise of an enhanced understanding of disease mechanisms and phenotypic variability. 3D bioprinting has been successfully performed using multiple stem cell types of different lineages and potency. The type of 3D bioprinting employed ranged from microextrusion bioprinting, inkjet bioprinting, laser-assisted bioprinting, to newer technologies such as scaffold-free spheroid-based bioprinting. This review discusses the current advances, applications, limitations and future of 3D bioprinting using stem cells, by organ systems.

Growth-Inhibitory Effect of Chemotherapeutic Drugs Dispensed by Inkjet Bioprinting on Cancer and Non-Cancer Cells

Growth-Inhibitory Effect of Chemotherapeutic Drugs Dispensed by Inkjet Bioprinting on Cancer and Non-Cancer Cells

Growth-Inhibitory Effect of Chemotherapeutic Drugs Dispensed by Inkjet Bioprinting on Cancer and Non-Cancer Cells

Thermal inkjet technology has long been used in the printing industry, but little has been studied on the benefits that it can provide to the drug-screening field. The objective of the work reported here was a proof of concept of using a modified inkjet printer to have a more accessible and miniature cellomic anticancer drug-screening platform. The authors' previous findings have shown that inkjet-based screening can reliably create isolated arrays of spots of living cells and antibiotics at low volume (180 pl) and high throughput (213 spots/sec) [J. I. Rodríguez-Dévora, B. Zhang, D. Reyna, Z. D. Shi, and T. Xu, “High throughput miniature drug-screening platform using bioprinting technology,” Biofabrication 4, 035001 (2012)]. The methodology of the work reported here included using a modified office inkjet printing device; the authors studied the inhibitory effects of dichloroacetate sodium (DCA) over hepatocellular carcinoma (HepG2) and epithelial cells (EpC). A DCA drug concentration gradient was printed over cell cultures to evaluate the drug's cytotoxic effect. Half maximal and ninety percent inhibitory concentrations (IC50, IC90) were obtained from the dose–response curves and compared with concentrations obtained using the traditional micropipetting technique. The resulting inhibitory concentration values obtained by both dispensing techniques fall within the millimolar range. The significance of these finding is that the proposed screening platform closely mimics the traditional screening outcome at a miniaturized volume rate, thus downsizing the screening process from traditional sub-microliter to nano- or even picoliter range. Inkjet technology shows promise in miniaturizing and expediting the drug-screening process. This platform can be used to asses a preliminary dose–response curve in order to improve the treatment modalities using the patient's limited supply of biopsied cells toward personalized medicine. © 2016 Society for Imaging Science and Technology.

Growth-Inhibitory Effect of Chemotherapeutic Drugs Dispensed by Inkjet Bioprinting on Cancer and Non-Cancer Cells

Thermal inkjet technology has long been used in the printing industry, but little has been studied on the benefits that it can provide to the drug-screening field. The objective of the work reported here was a proof of concept of using a modified inkjet printer to have a more accessible and miniature cellomic anticancer drug-screening platform. The authors’ previous findings have shown that inkjet-based screening can reliably create isolated arrays of spots of living cells and antibiotics at low volume (180 pl) and high throughput (213 spots/sec) [J. I. Rodríguez-Dévora, B. Zhang, D. Reyna, Z. D. Shi, and T. Xu, “High throughput miniature drug-screening platform using bioprinting technology,” Biofabrication 4, 035001 (2012)]. The methodology of the work reported here included using a modified office inkjet printing device; the authors studied the inhibitory effects of dichloroacetate sodium (DCA) over hepatocellular carcinoma (HepG2) and epithelial cells (EpC). A DCA drug concentration gradient was printed over cell cultures to evaluate the drug’s cytotoxic effect. Half maximal and ninety percent inhibitory concentrations (IC50, IC90) were obtained from the dose‐response curves and compared with concentrations obtained using the traditional micropipetting technique. The resulting inhibitory concentration values obtained by both dispensing techniques fall within the millimolar range. The significance of these finding is that the proposed screening platform closely mimics the traditional screening outcome at a miniaturized volume rate, thus downsizing the screening process from traditional sub-microliter to nano- or even picoliter range. Inkjet technology shows promise in miniaturizing and expediting the drug-screening process. This platform can be used to asses a preliminary dose‐response curve in order to improve the treatment modalities using the patient’s limited supply of biopsied cells toward personalized medicine.

A comprehensive review on droplet-based bioprinting: Past, present and future.

Droplet-based bioprinting (DBB) offers greater advantages due to its simplicity and agility with precise control on deposition of biologics including cells, growth factors, genes, drugs and biomaterials, and has been a prominent technology in the bioprinting community. Due to its immense versatility, DBB technology has been adopted by various application areas, including but not limited to, tissue engineering and regenerative medicine, transplantation and clinics, pharmaceutics and high-throughput screening, and cancer research. Despite the great benefits, the technology currently faces several challenges such as a narrow range of available bioink materials, bioprinting-induced cell damage at substantial levels, limited mechanical and structural integrity of bioprinted constructs, and restrictions on the size of constructs due to lack of vascularization and porosity. This paper presents a first-time review of DBB and comprehensively covers the existing DBB modalities including inkjet, electrohydrodynamic, acoustic, and micro-valve bioprinting. The recent notable studies are highlighted, the relevant bioink biomaterials and bioprinters are expounded, the application areas are presented, and the future prospects are provided to the reader.

Biodegradable Polymers and Stem Cells for Bioprinting.

It is imperative to develop organ manufacturing technologies based on the high organ failure mortality and serious donor shortage problems. As an emerging and promising technology, bioprinting has attracted more and more attention with its super precision, easy reproduction, fast manipulation and advantages in many hot research areas, such as tissue engineering, organ manufacturing, and drug screening. Basically, bioprinting technology consists of inkjet bioprinting, laser-based bioprinting and extrusion-based bioprinting techniques. Biodegradable polymers and stem cells are common printing inks. In the printed constructs, biodegradable polymers are usually used as support scaffolds, while stem cells can be engaged to differentiate into different cell/tissue types. The integration of biodegradable polymers and stem cells with the bioprinting techniques has provided huge opportunities for modern science and technologies, including tissue repair, organ transplantation and energy metabolism.

Cost effective open source microvalve bioprinting of therapeutic cells

Cost effective open source microvalve bioprinting of therapeutic cells

Improved properties of bone and cartilage tissue from 3D inkjet-bioprinted human mesenchymal stem cells by simultaneous deposition and photocrosslinking in PEG-GelMA.

Bioprinting of bone and cartilage suffers from low mechanical properties. Here we have developed a unique inkjet bioprinting approach of creating mechanically strong bone and cartilage tissue constructs using poly(ethylene glycol) dimethacrylate, gelatin methacrylate, and human MSCs. The printed hMSCs were evenly distributed in the polymerized PEG-GelMA scaffold during layer-by-layer assembly. The procedure showed a good biocompatibility with >80% of the cells surviving the printing process and the resulting constructs provided strong mechanical support to the embedded cells. The printed mesenchymal stem cells showed an excellent osteogenic and chondrogenic differentiation capacity. Both osteogenic and chondrogenic differentiation as determined by specific gene and protein expression analysis (RUNX2, SP7, DLX5, ALPL, Col1A1, IBSP, BGLAP, SPP1, Col10A1, MMP13, SOX9, Col2A1, ACAN) was improved by PEG-GelMA in comparison to PEG alone. These observations were consistent with the histological evaluation. Inkjet bioprinted-hMSCs in simultaneously photocrosslinked PEG-GelMA hydrogel scaffolds demonstrated an improvement of mechanical properties and osteogenic and chondrogenic differentiation, suggesting its promising potential for usage in bone and cartilage tissue engineering.

Optimizing cell viability in droplet-based cell deposition.

Biofabrication commonly involves the use of liquid droplets to transport cells to the printed structure. However, the viability of the cells after impact is poorly controlled and understood, hampering applications including cell spraying, inkjet bioprinting, and laser-assisted cell transfer. Here, we present an analytical model describing the cell viability after impact as a function of the cell-surrounding droplet characteristics. The model connects (1) the cell survival as a function of cell membrane elongation, (2) the membrane elongation as a function of the cell-containing droplet size and velocity, and (3) the substrate properties. The model is validated by cell viability measurements in cell spraying, which is a method for biofabrication and used for the treatment of burn wounds. The results allow for rational optimization of any droplet-based cell deposition technology, and we include practical suggestions to improve the cell viability in cell spraying.

Inkjet-bioprinted acrylated peptides and PEG hydrogel with human mesenchymal stem cells promote robust bone and cartilage formation with minimal printhead clogging.

Inkjet bioprinting is one of the most promising additive manufacturing approaches for tissue fabrication with the advantages of high speed, high resolution, and low cost. The limitation of this technology is the potential damage to the printed cells and frequent clogging of the printhead. Here we developed acrylated peptides and co-printed with acrylated poly(ethylene glycol) (PEG) hydrogel with simultaneous photopolymerization. At the same time, the bone marrow-derived human mesenchymal stem cells (hMSCs) were precisely printed during the scaffold fabrication process so the cells were delivered simultaneously with minimal UV exposure. The multiple steps of scaffold synthesis and cell encapsulation were successfully combined into one single step using bioprinting. The resulted peptide-conjugated PEG scaffold demonstrated excellent biocompatibility, with a cell viability of 87.9 ± 5.3%. Nozzle clogging was minimized due to the low viscosity of the PEG polymer. With osteogenic and chondrogenic differentiation, the bioprinted bone and cartilage tissue demonstrated excellent mineral and cartilage matrix deposition, as well as significantly increased mechanical properties. Strikingly, the bioprinted PEG-peptide scaffold dramatically inhibited hMSC hypertrophy during chondrogenic differentiation. Collectively, bioprinted PEG-peptide scaffold and hMSCs significantly enhanced osteogenic and chondrogenic differentiation for robust bone and cartilage formation with minimal printhead clogging.

Inexpensive and Reliable Monitoring of the Microdeposition of Biomolecules

Anti-C-reactive protein antibody is used to quantify C-reactive protein in diagnostic tests for assessing inflammation levels. In order to develop a practical chip for diagnostic tests, anti-C-reactive protein antibody was continuously ejected using a piezoelectric injector and microdeposition was monitored on a shot-by-shot basis using a 30 MHz quartz crystal microbalance. Background noise due to external sources such as air flow and temperature fluctuations were largely eliminated by covering the quartz crystal microbalance with a stainless-steel cover and maintaining a fixed distance between the optical microscope lamp and the piezoelectric injector. Each shot of a 1 mg ml−1 solution of anti-C-reactive protein antibody solution delivered approximately 30 pl to the quartz crystal microbalance. The frequency change per shot was 2.21 ± 0.97 Hz and the injected mass was estimated to be 44.2 pg using the Sauerbrey equation. This approach simplifies the experimental evaluation of inkjet bioprinting by allowing the sensitive determination of shot ejection mass and volume, and realizes label-free detection, continuous monitoring, and simple temperature control. Measurements can be performed at atmospheric pressure and at room temperature. Furthermore, the system uses only commercially available devices.