Bioprinting has attracted increasing attention in the tissue engineering field and has been touted to potentially become the leading technology to fabricate, and regenerate, tissues and organs. Bioprinting is derived from well-known additive manufacturing (AM) technology, which features layered deposition of materials into complex three-dimensional geometries that are difficult to fabricate using conventional manufacturing methods. Unlike the conventional thermoplastics used in desktop, AM bioprinting uses cell-laden hydrogel materials, also known as bioinks, to construct complex living biological model systems. Inkjet, stereolithography, laser-induced forward transfer, and extrusion are the four main methods in bioprinting, with extrusion being the most commonly used. In extrusion-based bioprinting, soft materials are loaded into the cartridges and extruded from the nozzle via pneumatic or mechanical actuation. Multiple materials can be printed into the same structure resulting in heterogeneous models. In this focused review, we first review the different methods to describe the physical mechanisms of the extrusion process, followed by the commonly employed bioprintable soft materials with their mechanical and biochemical properties and finally reviewing the up-to-date heterogeneous in vitro models afforded via bioprinting.
Tunable bioprinting materials are capable of creating a broad spectrum of physiological mimicking 3D models enabling in vitro studies that more accurately resemble in vivo conditions. Tailoring the material properties of the bioink such that it achieves both bioprintability and biomimicry remains a key challenge. Here we report the development of engineered composite hydrogels consisting of gelatin and alginate components. The composite gels are demonstrated as a cell-laden bioink to build 3D bioprinted in vitro breast tumor models. The initial mechanical characteristics of each composite hydrogel are correlated to cell proliferation rates and cell spheroid morphology spanning month long culture conditions. MDA-MB-231 breast cancer cells show gel formulation-dependency on the rates and frequency of self-assembly into multicellular tumor spheroids (MCTS). Hydrogel compositions comprised of decreasing alginate concentrations, and increasing gelatin concentrations, result in gels that are mechanically soft and contain a greater number of cell-adhesion moieties driving the development of large MCTS; conversely gels containing increasing alginate, and decreasing gelatin concentrations are mechanically stiffer, with fewer cell-adhesion moieties present in the composite gels yielding smaller and less viable MCTS. These composite hydrogels can be used in the biofabrication of tunable in vitro systems that mimic both the mechanical and biochemical properties of the native tumor stroma.
Three-dimensional (3D) printing is an emerging technology in the field of dentistry. It uses a layer-by-layer manufacturing technique to create scaffolds that can be used for dental tissue engineering applications. While several 3D printing methodologies exist, such as selective laser sintering or fused deposition modeling, this paper will review the applications of 3D printing for craniofacial tissue engineering; in particular for the periodontal complex, dental pulp, alveolar bone, and cartilage. For the periodontal complex, a 3D printed scaffold was attempted to treat a periodontal defect; for dental pulp, hydrogels were created that can support an odontoblastic cell line; for bone and cartilage, a polycaprolactone scaffold with microspheres induced the formation of multiphase fibrocartilaginous tissues. While the current research highlights the development and potential of 3D printing, more research is required to fully understand this technology and for its incorporation into the dental field.
Reinforced extracellular matrix (ECM)-based hydrogels recapitulate several mechanical and biochemical features found in the tumor microenvironment (TME) in vivo. While these gels retain several critical structural and bioactive molecules that promote cell–matrix interactivity, their mechanical properties tend toward the viscous regime limiting their ability to retain ordered structural characteristics when considered as architectured scaffolds. To overcome this limitation characteristic of pure ECM hydrogels, we present a composite material containing alginate, a seaweed-derived polysaccharide, and gelatin, denatured collagen, as rheological modifiers which impart mechanical integrity to the biologically active decellularized ECM (dECM). After an optimization process, the reinforced gel proposed is mechanically stable and bioprintable and has a stiffness within the expected physiological values. Our hydrogel’s elastic modulus has no significant difference when compared to tumors induced in preclinical xenograft head and neck squamous cell carcinoma (HNSCC) mouse models. The bioprinted cell-laden model is highly reproducible and allows proliferation and reorganization of HNSCC cells while maintaining cell viability above 90% for periods of nearly 3 weeks. Cells encapsulated in our bioink produce spheroids of at least 3000 μm2 of cross-sectional area by day 15 of culture and are positive for cytokeratin in immunofluorescence quantification, a common marker of HNSCC model validation in 2D and 3D models. We use this in vitro model system to evaluate the standard-of-care small molecule therapeutics used to treat HNSCC clinically and report a 4-fold increase in the IC50 of cisplatin and an 80-fold increase for 5-fluorouracil compared to monolayer cultures. Our work suggests that fabricating in vitro models using reinforced dECM provides a physiologically relevant system to evaluate malignant neoplastic phenomena in vitro due to the physical and biological features replicated from the source tissue microenvironment.
Hydrogels consisting of controlled fractions of alginate, gelatin, and Matrigel enable the development of patient-derived bioprinted tissue models that support cancer spheroid growth and expansion. These engineered models can be dissociated to be then reintroduced to new hydrogel solutions and subsequently reprinted to generate multigenerational models. The process of harvesting cells from 3D bioprinted models is possible by chelating the ions that crosslink alginate, causing the gel to weaken. Inclusion of the gelatin and Matrigel fractions to the hydrogel increases the bioactivity by providing cell-matrix binding sites and promoting cross-talk between cancer cells and their microenvironment. Here we show that immortalized triple-negative breast cancer cells (MDA-MB-231) and patient-derived gastric adenocarcinoma cells can be reprinted for at least three 21 d culture cycles following bioprinting in the alginate/gelatin/Matrigel hydrogels. Our drug testing results suggest that our 3D bioprinted model can also be used to recapitulate in vivo patient drug response. Furthermore, our results show that iterative bioprinting techniques coupled with alginate biomaterials can be used to maintain and expand patient-derived cancer spheroid cultures for extended periods without compromising cell viability, altering division rates, or disrupting cancer spheroid formation.
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