3D Bioprinting of Acellular Corneal Stromal Scaffolds with a Low Cost Modified 3D Printer: A Feasibility Study.

PURPOSE: Loss of corneal transparency is one of the major causes of visual loss, generating a considerable health and economic burden globally. Corneal transplantation is the leading treatment procedure, where the diseased cornea is replaced by donated corneal tissue. Despite the rise of cornea donations in the past decade, there is still a huge gap between cornea supply and demand worldwide. 3D bioprinting is an emerging technology that can be used to fabricate tissue equivalents that resemble the native tissue, which holds great potential for corneal tissue engineering application. This study evaluates the manufacturability of 3D bioprinted acellular corneal grafts using low-cost equipment and software, not necessarily designed for bioprinting applications. This approach allows access to 3D printed structures where commercial 3D bioprinters are cost prohibitive and not readily accessible to researchers and clinicians. METHODS: Two extrusion-based methods were used to 3D print acellular corneal stromal scaffolds with collagen, alginate, and alginate-gelatin composite bioinks from a digital corneal model. Compression testing was used to determine moduli. RESULTS: The printed model was visually transparent with tunable mechanical properties. The model had central radius of curvature of 7.4 mm, diameter of 13.2 mm, and central thickness of 0.4 mm. The compressive secant modulus of the material was 23.7 ± 1.7 kPa at 20% strain. 3D printing into a concave mold had reliability advantages over printing into a convex mold. CONCLUSIONS: The printed corneal models exhibited visible transparency and a dome shape, demonstrating the potential of this process for the preparation of acellular partial thickness corneal replacements. The modified printing process presented a low-cost option for corneal bioprinting.

3D Bioprinting of Diatom-Laden Living Materials for Water Quality Assessment.

Diatoms have long been used as living biological indicators for the assessment of water quality in lakes and rivers worldwide. While this approach benefits from the great diversity of these unicellular algae, established protocols are time-consuming and require specialized equipment. Here, this work 3D prints diatom-laden hydrogels that can be used as a simple multiplex bio-indicator for water assessment. The hydrogel-based living materials are created with the help of a desktop extrusion-based printer using a suspension of diatoms, cellulose nanocrystals (CNC) and alginate as bio-ink constituents. Rheology and mechanical tests are employed to establish optimum bio-ink formulations, whereas cell culture experiments are utilized to evaluate the proliferation of the entrapped diatoms in the presence of selected water contaminants. Bioprinting of diatom-laden hydrogels is shown to be an enticing approach to generate living materials that can serve as low-cost bio-indicators for water quality assessment.

Traction of 3D and 4D Printing in the Healthcare Industry: From Drug Delivery and Analysis to Regenerative Medicine.

Three-dimensional (3D) printing and 3D bioprinting are promising technologies for a broad range of healthcare applications from frontier regenerative medicine and tissue engineering therapies to pharmaceutical advancements yet must overcome the challenges of biocompatibility and resolution. Through comparison of traditional biofabrication methods with 3D (bio)printing, this review highlights the promise of 3D printing for the production of on-demand, personalized, and complex products that enhance the accessibility, effectiveness, and safety of drug therapies and delivery systems. In addition, this review describes the capacity of 3D bioprinting to fabricate patient-specific tissues and living cell systems (e.g., vascular networks, organs, muscles, and skeletal systems) as well as its applications in the delivery of cells and genes, microfluidics, and organ-on-chip constructs. This review summarizes how tailoring selected parameters (i.e., accurately selecting the appropriate printing method, materials, and printing parameters based on the desired application and behavior) can better facilitate the development of optimized 3D-printed products and how dynamic 4D-printed strategies (printing materials designed to change with time or stimulus) may be deployed to overcome many of the inherent limitations of conventional 3D-printed technologies. Comprehensive insights into a critical perspective of the future of 4D bioprinting, crucial requirements for 4D printing including the programmability of a material, multimaterial printing methods, and precise designs for meticulous transformations or even clinical applications are also given.

Regenerative treatments for kidney diseases: The closest and fastest strategies to solving related medical and economic problems.

Recent advances in developmental biology and stem cell biology have led to the increased availability of extrarenal stem cells, including mesenchymal/stromal stem cells (MSCs), renal stem or progenitor cells isolated from embryonic and adult kidneys, and kidney lineage cells or tissues generated from human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human-induced pluripotent stem cells. Regenerative medicine strategies for kidney diseases are largely categorized into the transplantation of reconstructed kidney organs and cell therapies. Reconstruction is being attempted by hPSC-derived kidney lineage cells with various strategies, such as self-organization, interspecies blastocyst complementation, utilization of a xenogeneic organ niche, decellularization and repopulation, and 3D bioprinting. However, cell therapies using extrarenal stem cells, such as MSCs, and renal stem or progenitor cells derived from embryonic and adult kidneys or differentiated from hPSCs have been investigated in animal models of both acute kidney injury and chronic kidney disease. Indeed, multiple clinical trials using MSCs, bone marrow stem cells, and kidney-derived cells have already been carried out. This review summarizes the current status and future perspective of kidney regenerative medicine strategies and discusses the closest and fastest strategies to solving the medical and economic problems associated with kidney diseases.

Cell Processing for 3D Bioprinting: Quality Requirements for Quality Assurance in Fundamental Research and Translation.

Bioprinting is an additive manufacturing process where biomaterials-based inks are printed layer-by-layer to create three-dimensional (3D) structures that mimic natural tissues. Quality assurance for 3D bioprinting is paramount to undertaking fundamental research and preclinical and clinical product development. It forms part of quality management and is vital to reproducible and safe tissue fabrication, function, and regulatory approval for translational application. This chapter seeks to place the implementation of quality practices in 3D bioprinting front-of-mind, with emphasis on cell processing, although important to all components and procedures of the printing pipeline.

Assessment methodologies for extrusion-based bioink printability.

Extrusion-based bioprinting is one of the leading manufacturing techniques for tissue engineering and regenerative medicine. Its primary limitation is the lack of materials, known as bioinks, which are suitable for the bioprinting process. The degree to which a bioink is suitable for bioprinting has been described as its 'printability.' However, a lack of clarity surrounding the methodologies used to evaluate a bioink's printability, as well as the usage of the term itself, have hindered the field. This article presents a review of measures used to assess the printability of extrusion-based bioinks in an attempt to assist researchers during the bioink development process. Many different aspects of printability exist and many different measurements have been proposed as a consequence. Researchers often do not evaluate a new bioink's printability at all, while others simply do so qualitatively. Several quantitative measures have been presented for the extrudability, shape fidelity, and printing accuracy of bioinks. Different measures have been developed even within these aspects, each testing the bioink in a slightly different way. Additionally, other relevant measures which had little or no examples of quantifiable methods are also to be considered. Looking forward, further work is needed to improve upon current assessment methodologies, to move towards a more comprehensive view of printability, and to standardize these printability measurements between researchers. Better assessment techniques will naturally lead to a better understanding of the underlying mechanisms which affect printability and better comparisons between bioinks. This in turn will help improve upon the bioink development process and the bioinks available for use in bioprinting.

Using Sacrificial Cell Spheroids for the Bioprinting of Perfusable 3D Tissue and Organ Constructs: A Computational Study.

A long-standing problem in tissue engineering is the biofabrication of perfusable tissue constructs that can be readily connected to the patient's vasculature. It was partially solved by three-dimensional (3D) printing of sacrificial material (e.g., hydrogel) strands: upon incorporation in another cell-laden hydrogel, the strands were removed, leaving behind perfusable channels. Their complexity, however, did not match that of the native vasculature. Here, we propose to use multicellular spheroids as a sacrificial material and investigate their potential benefits in the context of 3D bioprinting of cell aggregates and/or cell-laden hydrogels. Our study is based on computer simulations of postprinting cellular rearrangements. The computational model of the biological system is built on a cubic lattice, whereas its evolution is simulated using the Metropolis Monte Carlo algorithm. The simulations describe structural changes in three types of tissue constructs: a tube made of a single cell type, a tube made of two cell types, and a cell-laden hydrogel slab that incorporates a branching tube. In all three constructs, the lumen is obtained after the elimination of the sacrificial cell population. Our study suggests that sacrificial cell spheroids (sacrospheres) enable one to print tissue constructs outfitted with a finer and more complex network of channels than the ones obtained so far. Moreover, cellular interactions might give rise to a tissue microarchitecture that lies beyond the bioprinter's resolution. Although more expensive than inert materials, sacrificial cells have the potential to bring further progress towards the biofabrication of fully vascularized tissue substitutes.

Translating Biofabrication to the Market.

Biofabrication holds great potential to revolutionize important industries in the health, food, and textile sectors, but its translation to market is still challenging. I analyze the current state of innovation and commercialization in biofabrication and try to assess its limitations, strengths, and future progress.

Rapid 3D bioprinting of decellularized extracellular matrix with regionally varied mechanical properties and biomimetic microarchitecture.

Hepatocellular carcinoma (HCC), as the fifth most common malignant cancer, develops and progresses mostly in a cirrhotic liver where stiff nodules are separated by fibrous bands. Scaffolds that can provide a 3D cirrhotic mechanical environment with complex native composition and biomimetic architecture are necessary for the development of better predictive tissue models. Here, we developed photocrosslinkable liver decellularized extracellular matrix (dECM) and a rapid light-based 3D bioprinting process to pattern liver dECM with tailorable mechanical properties to serve as a platform for HCC progression study. 3D bioprinted liver dECM scaffolds were able to stably recapitulate the clinically relevant mechanical properties of cirrhotic liver tissue. When encapsulated in dECM scaffolds with cirrhotic stiffness, HepG2 cells demonstrated reduced growth along with an upregulation of invasion markers compared to healthy controls. Moreover, an engineered cancer tissue platform possessing tissue-scale organization and distinct regional stiffness enabled the visualization of HepG2 stromal invasion from the nodule with cirrhotic stiffness. This work demonstrates a significant advancement in rapid 3D patterning of complex ECM biomaterials with biomimetic architecture and tunable mechanical properties for in vitro disease modeling.

Printing of Patterned, Engineered E. coli Biofilms with a Low-Cost 3D Printer.

Biofilms can grow on virtually any surface available, with impacts ranging from endangering the lives of patients to degrading unwanted water contaminants. Biofilm research is challenging due to the high degree of biofilm heterogeneity. A method for the production of standardized, reproducible, and patterned biofilm-inspired materials could be a boon for biofilm research and allow for completely new engineering applications. Here, we present such a method, combining 3D printing with genetic engineering. We prototyped a low-cost 3D printer that prints bioink, a suspension of bacteria in a solution of alginate that solidifies on a calcium-containing substrate. We 3D-printed Escherichia coli in different shapes and in discrete layers, after which the cells survived in the printing matrix for at least 1 week. When printed bacteria were induced to form curli fibers, the major proteinaceous extracellular component of E. coli biofilms, they remained adherent to the printing substrate and stably spatially patterned even after treatment with a matrix-dissolving agent, indicating that a biofilm-mimicking structure had formed. This work is the first demonstration of patterned, biofilm-inspired living materials that are produced by genetic control over curli formation in combination with spatial control by 3D printing. These materials could be used as living, functional materials in applications such as water filtration, metal ion sequestration, or civil engineering, and potentially as standardizable models for certain curli-containing biofilms.

Uso de digitalizador e impressora doméstica em 3 dimensões em rinoplastia / Domestic three-dimensional scanner and printer in patients undergoing rhinoplasty

Os princípios para uma rinoplastia bem-sucedida incluem consulta e planejamento pré-operatório e uma análise clínica abrangente que defina as metas da cirurgia. Mais recentemente, a digitalização e a impressão doméstica em 3 dimensões tornaram-se disponíveis. O objetivo deste estudo é descrever um método de digitalização em 3 dimensões e de impressão doméstica da anatomia real do paciente para ser usada como ajuda intraoperatória. Nós apresentamos uma forma de uso desta tecnologia no transoperatório, auxiliando o cirurgião a comparar os resultados obtidos após suas manobras, verificar a sua adesão ao plano cirúrgico previamente estabelecido e melhorar a sua tomada de decisão durante a cirurgia. Em conclusão, a aplicação da impressão doméstica em 3 dimensões demonstra um efeito positivo sobre o tratamento de alterações estéticas do nariz.

The principles for a successful rhinoplasty include preoperative consultation and planning, as well as a comprehensive clinical analysis and defining rhinoplasty goals. Three-dimensional domestic scanning and printing have recently become available. We sought to objectively describe this method as an intraoperative aid in patients' anatomy. This method can be used trans-operatively to help surgeons compare the results of his or her technique, check adherence to the surgical plan, and improve his or her surgical decision-making. We found that the application of 3-dimensional printing had a positive effect on the treatment of patients with aesthetic nose disorders.

Assessment of hydrogels for bioprinting of endothelial cells.

In tissue engineering applications, vascularization can be accomplished by coimplantation of tissue forming cells and endothelial cells (ECs), whereby the latter are able to form functional blood vessels. The use of three-dimensional (3D) bioprinting technologies has the potential to improve the classical tissue engineering approach because these will allow the generation of scaffolds with high spatial control of endothelial cell allocation. This study focuses on a side by side comparison of popular commercially available bioprinting hydrogels (Matrigel, fibrin, collagen, gelatin, agarose, Pluronic F-127, alginate, and alginate/gelatin) in the context of their physicochemical parameters, their swelling/degradation characteristics, their biological effects on vasculogenesis-related EC parameters and their printability. The aim of this study was to identify the most suitable hydrogel or hydrogel combination for inkjet printing of ECs to build prevascularized tissue constructs. Most tested hydrogels displayed physicochemical characteristics suitable for inkjet printing. However, Pluronic F-127 and the alginate/gelatin blend were rapidly degraded when incubated in cell culture medium. Agarose, Pluronic F-127, alginate and alginate/gelatin hydrogels turned out to be unsuitable for bioprinting of ECs because of their non-adherent properties and/or their incapability to support EC proliferation. Gelatin was able to support EC proliferation and viability but was unable to support endothelial cell sprouting. Our experiments revealed fibrin and collagen to be most suitable for bioprinting of ECs, because these hydrogels showed acceptable swelling/degradation characteristics, supported vasculogenesis-related EC parameters and showed good printability. Moreover, ECs in constructs of preformed spheroids survived the printing process and formed capillary-like cords. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 935-947, 2018.

Bioprinting Pattern-Dependent Electrical/Mechanical Behavior of Cardiac Alginate Implants: Characterization and Ex Vivo Phase-Contrast Microtomography Assessment.

Three-dimensional (3D)-bioprinting techniques may be used to modulate electrical/mechanical properties and porosity of hydrogel constructs for fabrication of suitable cardiac implants. Notably, characterization of these properties after implantation remains a challenge, raising the need for the development of novel quantitative imaging techniques for monitoring hydrogel implant behavior in situ. This study aims at (i) assessing the influence of hydrogel bioprinting patterns on electrical/mechanical behavior of cardiac implants based on a 3D-printing technique and (ii) investigating the potential of synchrotron X-ray phase-contrast imaging computed tomography (PCI-CT) for estimating elastic modulus/impedance/porosity and microstructural features of 3D-printed cardiac implants in situ via an ex vivo study. Alginate laden with human coronary artery endothelial cells was bioprinted layer by layer, forming cardiac constructs with varying architectures. The elastic modulus, impedance, porosity, and other structural features, along with the cell viability and degradation of printed implants were examined in vitro over 25 days. Two selected cardiac constructs were surgically implanted onto the myocardium of rats and 10 days later, the rat hearts with implants were imaged ex vivo by means of PCI-CT at varying X-ray energies and CT-scan times. The elastic modulus/impedance, porosity, and structural features of the implant were inferred from the PCI-CT images by using statistical models and compared with measured values. The printing patterns had significant effects on implant porosity, elastic modulus, and impedance. A particular 3D-printing pattern with an interstrand distance of 900 µm and strand alignment angle of 0/45/90/135° provided relatively higher stiffness and electrical conductivity with a suitable porosity, maintaining high cell viability over 7 days. The X-ray photon energy of 30-33 keV utilizing a CT-scan time of 1-1.2 h resulted in a low-dose PCI-CT, which provided a good visibility of the low-X-ray absorbent alginate implants. After 10 days postimplantation, the PCI-CT provided a reasonably accurate estimation of implant strand thickness and alignment, pore size and interconnectivity, porosity, elastic modulus, and impedance, which were consistent with our measurements. Findings from this study suggest that 3D-printing patterns can be used to modulate electrical/mechanical behavior of alginate implants, and PCI-CT can be potentially used as a 3D quantitative imaging tool for assessing structural and electrical/mechanical behavior of hydrogel cardiac implants in small animal models.

Printed organo-functionalized graphene for biosensing applications.

Graphene is a highly promising material for biosensors due to its excellent physical and chemical properties which facilitate electron transfer between the active locales of enzymes or other biomaterials and a transducer surface. Printing technology has recently emerged as a low-cost and practical method for fabrication of flexible and disposable electronics devices. The combination of these technologies is promising for the production and commercialization of low cost sensors. In this review, recent developments in organo-functionalized graphene and printed biosensor technologies are comprehensively covered. Firstly, various methods for printing graphene-based fluids on different substrates are discussed. Secondly, different graphene-based ink materials and preparation methods are described. Lastly, biosensing performances of printed or printable graphene-based electrochemical and field effect transistor sensors for some important analytes are elaborated. The reported printed graphene based sensors exhibit promising properties with good reliability suitable for commercial applications. Among most reports, only a few printed graphene-based biosensors including screen-printed oxidase-functionalized graphene biosensor have been demonstrated. The technology is still at early stage but rapidly growing and will earn great attention in the near future due to increasing demand of low-cost and disposable biosensors.

Accessible bioprinting: adaptation of a low-cost 3D-printer for precise cell placement and stem cell differentiation.

The precision and repeatability offered by computer-aided design and computer-numerically controlled techniques in biofabrication processes is quickly becoming an industry standard. However, many hurdles still exist before these techniques can be used in research laboratories for cellular and molecular biology applications. Extrusion-based bioprinting systems have been characterized by high development costs, injector clogging, difficulty achieving small cell number deposits, decreased cell viability, and altered cell function post-printing. To circumvent the high-price barrier to entry of conventional bioprinters, we designed and 3D printed components for the adaptation of an inexpensive 'off-the-shelf' commercially available 3D printer. We also demonstrate via goal based computer simulations that the needle geometries of conventional commercially standardized, 'luer-lock' syringe-needle systems cause many of the issues plaguing conventional bioprinters. To address these performance limitations we optimized flow within several microneedle geometries, which revealed a short tapered injector design with minimal cylindrical needle length was ideal to minimize cell strain and accretion. We then experimentally quantified these geometries using pulled glass microcapillary pipettes and our modified, low-cost 3D printer. This systems performance validated our models exhibiting: reduced clogging, single cell print resolution, and maintenance of cell viability without the use of a sacrificial vehicle. Using this system we show the successful printing of human induced pluripotent stem cells (hiPSCs) into Geltrex and note their retention of a pluripotent state 7 d post printing. We also show embryoid body differentiation of hiPSC by injection into differentiation conducive environments, wherein we observed continuous growth, emergence of various evaginations, and post-printing gene expression indicative of the presence of all three germ layers. These data demonstrate an accessible open-source 3D bioprinter capable of serving the needs of any laboratory interested in 3D cellular interactions and tissue engineering.

The Application of Ultrasound in 3D Bio-Printing.

Three-dimensional (3D) bioprinting is an emerging and promising technology in tissue engineering to construct tissues and organs for implantation. Alignment of self-assembly cell spheroids that are used as bioink could be very accurate after droplet ejection from bioprinter. Complex and heterogeneous tissue structures could be built using rapid additive manufacture technology and multiple cell lines. Effective vascularization in the engineered tissue samples is critical in any clinical application. In this review paper, the current technologies and processing steps (such as printing, preparation of bioink, cross-linking, tissue fusion and maturation) in 3D bio-printing are introduced, and their specifications are compared with each other. In addition, the application of ultrasound in this novel field is also introduced. Cells experience acoustic radiation force in ultrasound standing wave field (USWF) and then accumulate at the pressure node at low acoustic pressure. Formation of cell spheroids by this method is within minutes with uniform size and homogeneous cell distribution. Neovessel formation from USWF-induced endothelial cell spheroids is significant. Low-intensity ultrasound could enhance the proliferation and differentiation of stem cells. Its use is at low cost and compatible with current bioreactor. In summary, ultrasound application in 3D bio-printing may solve some challenges and enhance the outcomes.

A Review of 3D Printing Techniques and the Future in Biofabrication of Bioprinted Tissue.

3D printing has been around in the art, micro-engineering, and manufacturing worlds for decades. Similarly, research for traditionally engineered skin tissue has been in the works since the 1990s. As of recent years, the medical field also began to take advantage of the untapped potential of 3D printing for the biofabrication of tissue. To do so, researchers created a set of goals for fabricated tissues based on the characteristics of natural human tissues and organs. Fabricated tissue was then measured against this set of standards. Researchers were interested in not only creating tissue that functioned like natural tissues but in creating techniques for 3D printing that would print tissues quickly, efficiently, and ultimately result in the ability to mass produce fabricated tissues. Three promising methods of 3D printing emerged from their research: thermal inkjet printing with bioink, direct-write bioprinting, and organ printing using tissue spheroids. This review will discuss all three printing techniques, as well as their advantages, disadvantages, and the possibility of future advancements in the field of tissue fabrication.