4D-Printable Photocrosslinkable Polyurethane-Based Inks for Tissue Scaffold and Actuator Applications.

4D printing recently emerges as an exciting evolution of conventional 3D printing, where a printed construct can quickly transform in response to a specific stimulus to switch between a temporary variable state and an original state. In this work, a photocrosslinkable polyethylene-glycol polyurethane ink is synthesized for light-assisted 4D printing of smart materials. The molecular weight distribution of the ink monomers is tunable by adjusting the copolymerization reaction time. Digital light processing (DLP) technique is used to program a differential swelling response in the printed constructs after humidity variation. Bioactive microparticles are embedded into the ink and the improvement of biocompatibility of the printed constructs is demonstrated for tissue engineering applications. Cell studies reveal above 90% viability in 1 week and ≈50% biodegradability after 4 weeks. Self-folding capillary scaffolds, dynamic grippers, and film actuators are made and activated in a humid environment. The approach offers a versatile platform for the fabrication of complex constructs. The ink can be used in tissue engineering and actuator applications, making the ink a promising avenue for future research.

Cell encapsulation in gelatin methacryloyl bioinks impairs microscale diffusion properties.

Light-assisted bioprinted gelatin methacryloyl (GelMA) constructs have been used for cell-laden microtissues and organoids. GelMA can be loaded by desired cells, which can regulate the biophysical properties of bioprinted constructs. We study how the degree of methacrylation (MA degree), GelMA mass concentration, and cell density change mass transport properties. We introduce a fluorescent-microscopy-based method of biotransport testing with improved sensitivity compared to the traditional particle tracking methods. The diffusion capacity of GelMA with a higher MA significantly decreased compared to a lower MA. Opposed to a steady range of linear elastic moduli, the diffusion coefficient in GelMA varied when cell densities ranged from 0 to 10 × 106 cells/ml. A comparative study of different cell sizes showed a higher diffusivity coefficient for the case of larger cells. The results of this study can help bioengineers and scientists to better control the biotransport characteristics in light-assisted bioprinted microtissues and organoids.

Ink Material Selection and Optical Design Considerations in DLP 3D Printing.

Digital light processing (DLP) 3D printing has become a powerful manufacturing tool for the fast fabrication of complex functional structures. The rapid progress in DLP printing has been linked to research on optical design factors and ink selection. This critical review highlights the main challenges in the DLP printing of photopolymerizable inks. The kinetics equations of photopolymerization reaction in a DLP printer are solved, and the dependence of curing depth on the process optical parameters and ink chemical properties are explained. Developments in DLP platform design and ink selection are summarized, and the roles of monomer structure and molecular weight on DLP printing resolution are shown by experimental data. A detailed guideline is presented to help engineers and scientists to select inks and optical parameters for fabricating functional structures for multi-material and 4D printing applications.

Nanostructured bioactive glasses: A bird's eye view on cancer therapy.

Bioactive glasses (BGs) arewell known for their successful applications in tissue engineering and regenerative medicine. Recent experimental studies have shown their potential usability in oncology, either alone or in combination with other biocompatible materials, such as biopolymers. Direct contact with BG particles has been found to cause toxicity and death in specific cancer cells (bone-derived neoplastic stromal cells) in vitro. Nanostructured BGs (NBGs) can be doped with anticancer elements, such as gallium, to enhance their toxic effects against tumor cells. However, the molecular mechanisms and intracellular targets for anticancer compositions of NBGs require further clarification. NBGs have been successfully evaluated for use in various well-established cancer treatment strategies, including cancer hyperthermia, phototherapy, and anticancer drug delivery. Existing results indicate that NBGs not only enhance cancer cell death, but can also participate in the regeneration of lost healthy tissues. However, the application of NBGs in oncology is still in its early stages, and numerous unanswered questions must be addressed. For example, the impact of the composition, biodegradation, size, and morphology of NBGs on their anticancer efficacy should be defined for each type of cancer and treatment strategy. Moreover, it should be more clearly assessed whether NBGs can shrink tumors, slow/stop cancer progression, or cure cancer completely. In this regard, the use of computational studies (in silico methods) is highly recommended to design the most effective glass formulations for cancer therapy approaches and to predict, to some extent, the relevant properties, efficacy, and outcomes. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

3D Bioprinted Hydrogel Microfluidic Devices for Parallel Drug Screening.

Conventional high-throughput screening (HTS) platforms suffer from the need for large cell volumes, high reagent consumption, significant assembly cost, and handling efforts. The assembly of three-dimensional (3D) bioprinted hydrogel-based microfluidic chips within platforms can address these problems. We present a continuous and seamless manufacturing approach to create a bioprinted microfluidic chips with a circular pattern scalable toward HTS platforms. Digital light processing 3D bioprinting is used to tune the local permeability of our chip, made of polyethylene glycol diacrylate and cell-laden gelatin methacryloyl, for creating predefined gradients of biochemical properties. We measured the flow-induced physical characteristics, the mass transport of drug agents, and the biological features of the proposed chip. We measured reactive oxygen species from the encapsulated cells through an integrated process and showed the capacity of the hydrogel-based chip for creating drug/agent gradients. This work introduces a chip design based on a hydrogel that can be changed and could be used for modern HTS platforms such as in vitro organoids.

Digital Light Processing Bioprinting Advances for Microtissue Models.

Digital light processing (DLP) bioprinting has been widely introduced as a fast and robust biofabrication method in tissue engineering. The technique holds a great promise for creating tissue models because it can replicate the resolution and complexity of natural tissues and constructs. A DLP system projects 2D images onto layers of bioink using a digital photomask. The resolution of DLP bioprinting strongly depends on the characteristics of the projected light and the photo-cross-linking response of the bioink microenvironment. In this review, we present a summary of DLP fundamentals with a focus on bioink properties, photoinitiator selection, and light characteristics in resolution of bioprinted constructs. A simple guideline is provided for bioengineers interested in using DLP platforms and customizing technical specifications for its design. The literature review reveals the promising future of DLP bioprinting for disease modeling and biofabrication.

Selection of natural biomaterials for micro-tissue and organ-on-chip models.

The desired organ in micro-tissue models of organ-on-a-chip (OoC) devices dictates the optimum biomaterials, divided into natural and synthetic biomaterials. They can resemble biological tissues' biological functions and architectures by constructing bioactivity of macromolecules, cells, nanoparticles, and other biological agents. The inclusion of such components in OoCs allows them having biological processes, such as basic biorecognition, enzymatic cleavage, and regulated drug release. In this report, we review natural-based biomaterials that are used in OoCs and their main characteristics. We address the preparation, modification, and characterization methods of natural-based biomaterials and summarize recent reports on their applications in the design and fabrication of micro-tissue models. This article will help bioengineers select the proper biomaterials based on developing new technologies to meet clinical expectations and improve patient outcomes fusing disease modeling.

Ink Rheology Regulates Stability of Bioprinted Strands.

Three-dimensional (3D) extrusion bioprinting typically requires an ad hoc trial-and-error optimization of the ink composition toward enhanced resolution. The ink solutions are solidified after leaving cone-shaped or cylindrical nozzles. The presence of ink instability not only hampers the extrusion resolution but also affects the behavior of embedded cellular components. This is a key factor in selecting (bio)inks and bioprinting design parameters for well-established desktop and handheld bioprinters. In this work, we developed an analytical solution for the process of ink deposition and compared its predictions against numerical simulations of the deposition. We estimated the onset of ink instability as a function of ink rheological properties and nozzle geometry. Our analytical results suggest that enhancing the shear-thinning behavior of the ink shortens the toe region of the deposition. Such an extrusion process is often desired, as it leads to faster depositions. However, we demonstrated that such conditions increase the possibility of lateral buckling of the strand once touching the substrate defined as instability in this study. The present study serves as a benchmark for detailed simulations of the extrusion process for optimal bioprinting.

Cancer Stem Cells in Tumor Modeling: Challenges and Future Directions.

Microfluidic tumors-on-chips models have revolutionized anticancer therapeutic research by creating an ideal microenvironment for cancer cells. The tumor microenvironment (TME) includes various cell types and cancer stem cells (CSCs), which are postulated to regulate the growth, invasion, and migratory behavior of tumor cells. In this review, the biological niches of the TME and cancer cell behavior focusing on the behavior of CSCs are summarized. Conventional cancer models such as three-dimensional cultures and organoid models are reviewed. Opportunities for the incorporation of CSCs with tumors-on-chips are then discussed for creating tumor invasion models. Such models will represent a paradigm shift in the cancer community by allowing oncologists and clinicians to predict better which cancer patients will benefit from chemotherapy treatments.

Multi-material digital light processing bioprinting of hydrogel-based microfluidic chips.

Recent advancements in digital-light-processing (DLP)-based bioprinting and hydrogel engineering have enabled novel developments in organs-on-chips. In this work, we designed and developed a multi-material, DLP-based bioprinter for rapid, one-step prototyping of hydrogel-based microfluidic chips. A composite hydrogel bioink based on poly-ethylene-glycol-diacrylate (PEGDA) and gelatin methacryloyl (GelMA) was optimized through varying the bioprinting parameters such as light exposure time, bioink composition, and layer thickness. We showed a wide range of mechanical properties of the microfluidic chips for various ratios of PEGDA:GelMA. Microfluidic features of hydrogel-based chips were then tested using dynamic flow experiments. Human-derived tumor cells were encapsulated in 3D bioprinted structures to demonstrate their bioactivity and cell-friendly environment. Cell seeding experiments then validated the efficacy of the selected bioinks for vascularized micro-tissues. Our biofabrication approach offers a useful tool for the rapid integration of micro-tissue models into organs-on-chips and high-throughput drug screening platforms.

Multi-Organs-on-Chips for Testing Small-Molecule Drugs: Challenges and Perspectives.

Organ-on-a-chip technology has been used in testing small-molecule drugs for screening potential therapeutics and regulatory protocols. The technology is expected to boost the development of novel therapies and accelerate the discovery of drug combinations in the coming years. This has led to the development of multi-organ-on-a-chip (MOC) for recapitulating various organs involved in the drug-body interactions. In this review, we discuss the current MOCs used in screening small-molecule drugs and then focus on the dynamic process of drug absorption, distribution, metabolism, and excretion. We also address appropriate materials used for MOCs at low cost and scale-up capacity suitable for high-performance analysis of drugs and commercial high-throughput screening platforms.

Survival and Proliferation under Severely Hypoxic Microenvironments Using Cell-Laden Oxygenating Hydrogels.

Different strategies have been employed to provide adequate nutrients for engineered living tissues. These have mainly revolved around providing oxygen to alleviate the effects of chronic hypoxia or anoxia that result in necrosis or weak neovascularization, leading to failure of artificial tissue implants and hence poor clinical outcome. While different biomaterials have been used as oxygen generators for in vitro as well as in vivo applications, certain problems have hampered their wide application. Among these are the generation and the rate at which oxygen is produced together with the production of the reaction intermediates in the form of reactive oxygen species (ROS). Both these factors can be detrimental for cell survival and can severely affect the outcome of such studies. Here we present calcium peroxide (CPO) encapsulated in polycaprolactone as oxygen releasing microparticles (OMPs). While CPO releases oxygen upon hydrolysis, PCL encapsulation ensures that hydrolysis takes place slowly, thereby sustaining prolonged release of oxygen without the stress the bulk release can endow on the encapsulated cells. We used gelatin methacryloyl (GelMA) hydrogels containing these OMPs to stimulate survival and proliferation of encapsulated skeletal myoblasts and optimized the OMP concentration for sustained oxygen delivery over more than a week. The oxygen releasing and delivery platform described in this study opens up opportunities for cell-based therapeutic approaches to treat diseases resulting from ischemic conditions and enhance survival of implants under severe hypoxic conditions for successful clinical translation.

Layered double hydroxide-based nanocomposite scaffolds in tissue engineering applications.

Layered double hydroxides (LDHs), when incorporated into biomaterials, provide a tunable composition, controllable particle size, anion exchange capacity, pH-sensitive solubility, high-drug loading efficiency, efficient gene and drug delivery, controlled release and effective intracellular uptake, natural biodegradability in an acidic medium, and negligible toxicity. In this review, we study potential applications of LDH-based nanocomposite scaffolds for tissue engineering. We address how LDHs provide new solutions for nanostructure stability and enhance in vivo studies' success.

3D Printing metamaterials towards tissue engineering.

The rapid growth and disruptive potentials of three-dimensional (3D) printing demand further research for addressing fundamental fabrication concepts and enabling engineers to realize the capabilities of 3D printing technologies. There is a trend to use these capabilities to develop materials that derive some of their properties via their structural organization rather than their intrinsic constituents, sometimes referred to as mechanical metamaterials. Such materials show qualitatively different mechanical behaviors despite using the same material composition, such as ultra-lightweight, super-elastic, and auxetic structures. In this work, we review current advancements in the design and fabrication of multi-scale advanced structures with properties heretofore unseen in well-established materials. We classify the fabrication methods as conventional methods, additive manufacturing techniques, and 4D printing. Following a comprehensive comparison of different fabrication methods, we suggest some guidelines on the selection of fabrication parameters to construct meta-biomaterials for tissue engineering. The parameters include multi-material capacity, fabrication resolution, prototyping speed, and biological compatibility.

Cell encapsulation in gelatin bioink impairs 3D bioprinting resolution.

Recent advances in three-dimensional (3D) bioprinting technologies have enabled precise patterning of cellular components along with biomimetic constructs for tissue engineering and regenerative medicine. The viscoelasticity of bioinks regulate printability and the smallest feature size in 3D bioprinted constructs. The impact of cellular components is typically neglected when choosing 3D bioprinting parameters. In this short communication, we quantified the effect of cell densities on the printability of hydrogel bioinks. Unexpectedly, our results show that encapsulated cells reduced the steady shear viscosity of gelatin-based bioinks by approximately 50% and the minimum force for onset of flow by approximately 30%. These results may justify the lower spatial resolution in 3D bioprinted cell-laden hydrogels.

Bioprinters for organs-on-chips.

Recent advances in bioprinting technologies have enabled rapid manufacturing of organ-on-chip models along with biomimetic tissue microarchitectures. Bioprinting techniques can be used to integrate microfluidic channels and flow connections in organ-on-chip models. We review bioprinters in two categories of nozzle-based and optical-based methods, and then discuss their fabrication parameters such as resolution, replication fidelity, fabrication time, and cost for micro-tissue models and microfluidic applications. The use of bioprinters has shown successful replicates of functional engineered tissue models integrated within a desired microfluidic system, which facilitates the observation of metabolism or secretion of models and sophisticated control of a dynamic environment. This may provide a wider order of tissue engineering fabrication in mimicking physiological conditions for enhancing further applications such as drug development and pathological studies.

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.

Cardiac Fibrotic Remodeling on a Chip with Dynamic Mechanical Stimulation.

Cardiac tissue is characterized by being dynamic and contractile, imparting the important role of biomechanical cues in the regulation of normal physiological activity or pathological remodeling. However, the dynamic mechanical tension ability also varies due to extracellular matrix remodeling in fibrosis, accompanied with the phenotypic transition from cardiac fibroblasts (CFs) to myofibroblasts. It is hypothesized that the dynamic mechanical tension ability regulates cardiac phenotypic transition within fibrosis in a strain-mediated manner. In this study, a microdevice that is able to simultaneously and accurately mimic the biomechanical properties of the cardiac physiological and pathological microenvironment is developed. The microdevice can apply cyclic compressions with gradient magnitudes (5-20%) and tunable frequency onto gelatin methacryloyl (GelMA) hydrogels laden with CFs, and also enables the integration of cytokines. The strain-response correlations between mechanical compression and CFs spreading, and proliferation and fibrotic phenotype remolding, are investigated. Results reveal that mechanical compression plays a crucial role in the CFs phenotypic transition, depending on the strain of mechanical load and myofibroblast maturity of CFs encapsulated in GelMA hydrogels. The results provide evidence regarding the strain-response correlation of mechanical stimulation in CFs phenotypic remodeling, which can be used to develop new preventive or therapeutic strategies for cardiac fibrosis.

Multiscale bioprinting of vascularized models.

A basic prerequisite for the survival and function of three-dimensional (3D) engineered tissue constructs is the establishment of blood vessels. 3D bioprinting of vascular networks with hierarchical structures that resemble in vivo structures has allowed blood circulation within thick tissue constructs to accelerate vascularization and enhance tissue regeneration. Successful rapid vascularization of tissue constructs requires synergy between fabrication of perfusable channels and functional bioinks that induce angiogenesis and capillary formation within constructs. Combinations of 3D bioprinting techniques and four-dimensional (4D) printing concepts through patterning proangiogenic factors may offer novel solutions for implantation of thick constructs. In this review, we cover current bioprinting techniques for vascularized tissue constructs with vasculatures ranging from capillaries to large blood vessels and discuss how to implement these approaches for patterning proangiogenic factors to maintain long-term, stimuli-controlled formation of new capillaries.

Aqueous Two-Phase Emulsion Bioink-Enabled 3D Bioprinting of Porous Hydrogels.

3D bioprinting technology provides programmable and customizable platforms to engineer cell-laden constructs mimicking human tissues for a wide range of biomedical applications. However, the encapsulated cells are often restricted in spreading and proliferation by dense biomaterial networks from gelation of bioinks. Herein, a cell-benign approach is reported to directly bioprint porous-structured hydrogel constructs by using an aqueous two-phase emulsion bioink. The bioink, which contains two immiscible aqueous phases of cell/gelatin methacryloyl (GelMA) mixture and poly(ethylene oxide) (PEO), is photocrosslinked to fabricate predesigned cell-laden hydrogel constructs by extrusion bioprinting or digital micromirror device-based stereolithographic bioprinting. The porous structure of the 3D-bioprinted hydrogel construct is formed by subsequently removing the PEO phase from the photocrosslinked GelMA hydrogel. Three different cell types (human hepatocellular carcinoma cells, human umbilical vein endothelial cells, and NIH/3T3 mouse embryonic fibroblasts) within the 3D-bioprinted porous hydrogel patterns show enhanced cell viability, spreading, and proliferation compared to the standard (i.e., nonporous) hydrogel constructs. The 3D bioprinting strategy is believed to provide a robust and versatile platform to engineer porous-structured tissue constructs and their models for a variety of applications in tissue engineering, regenerative medicine, drug development, and personalized therapeutics.