Bioprinting of Collagen: Considerations,Potentials, and Applications

Collagen is the most abundant extracellular matrix protein that is widely used in tissue engineering (TE). There is little research done on printing pure collagen. To understand the bottlenecks in printing pure collagen, it is imperative to understand collagen from a bottom‐up approach. Here it is aimed to provide a comprehensive overview of collagen printing, where collagen assembly in vivo and the various sources of collagen available for TE application are first understood. Next, the current printing technologies and strategy for printing collagen‐based materials are highlighted. Considerations and key challenges faced in collagen printing are identified. Finally, the key research areas that would enhance the functionality of printed collagen are presented.     

Simple,Rapid, and Large-Scale Fabrication of Multi-Branched Hydrogels Based on Viscous Fingering for Cell Culture Applications

Hydrogels are widely used in cell culture applications. For fabricating tissues and organs, it is essential to produce hydrogels with specific structures. For instance, multiple-branched hydrogels are desirable for the development of network architectures that resemble the biological vascular network. However, existing techniques are inefficient and time-consuming for this application. To address this issue, a simple, rapid, and large-scale fabrication method based on viscous fingering is proposed. This approach utilizes only two plates. To produce a thin solution, a high-viscosity solution is introduced into the space between the plates, and one of the plates is peeled off. During this procedure, the solution's high viscosity results in the formation of multi-branched structures. Using this strategy, 180 mm × 200 mm multi-branched Pluronic F-127 hydrogels are successfully fabricated within 1 min. These structures are used as sacrificial layers for the fabrication of polydimethylsiloxane channels for culturing human umbilical vein endothelial cells (HUVECs). Similarly, multi-branched Matrigel and calcium (Ca)-alginate hydrogel structures are fabricated, and HUVECs are successfully cultured inside the hydrogels. Also, the hydrogels are collected from the plate, while maintaining their structures. The proposed fabrication technique will contribute to the development of network architectures such as vascular structures in tissue engineering.     

4D Bioprinting: Technological Advances in Biofabrication

The development of the three‐dimensional (3D) printer has resulted in significant advances in a number of fields, including rapid prototyping and biomedical devices. For 3D structures, the inclusion of dynamic responses to stimuli is added to develop the concept of four‐dimensional (4D) printing. Typically, 4D printing is useful for biofabrication by reproducing a stimulus‐responsive dynamic environment corresponding to physiological activities. Such a dynamic environment can be precisely designed with an understanding of shape‐morphing effects (SMEs), which enables mimicking the functionality or intricate geometry of tissues. Here, 4D bioprinting is investigated for clinical use, for example, in drug delivery systems, tissue engineering, and surgery in vivo. This review presents the concept of 4D bioprinting and smart materials defined by SMEs and stimulus‐responsive mechanisms. Then, biomedical smart materials and applications are discussed along with future perspectives.     

Biofabrication of Cell‐Loaded 3D Spider Silk Constructs

Biofabrication is an emerging and rapidly expanding field of research in which additive manufacturing techniques in combination with cell printing are exploited to generate hierarchical tissue‐like structures. Materials that combine printability with cytocompatibility, so called bioinks, are currently the biggest bottleneck. Since recombinant spider silk proteins are non‐immunogenic, cytocompatible, and exhibit physical crosslinking, their potential as a new bioink system was evaluated. Cell‐loaded spider silk constructs can be printed by robotic dispensing without the need for crosslinking additives or thickeners for mechanical stabilization. Cells are able to adhere and proliferate with good viability over at least one week in such spider silk scaffolds. Introduction of a cell‐binding motif to the spider silk protein further enables fine‐tuned control over cell–material interactions. Spider silk hydrogels are thus a highly attractive novel bioink for biofabrication.     

Hyaluronic acid vinyl esters: A toolbox toward controlling mechanical properties of hydrogels for 3D microfabrication

In this study, a thorough exploration of constitutional parameters of thiol-ene photocrosslinkable hydrogels based on hyaluronic acid vinyl ester was conducted in order to decipher their impact on material properties. These constitutional parameters originated from the process of synthesis (macromer size and degree of substitution) and from the process of formulation (photoinitiator concentration, macromer content, and thiol-to-ene ratio). Various macromers were obtained with a broad variety of degrees of substitution. Photorheology measurements were performed in order to determine the influence of the structure parameters on photoreactivity and the physical properties of hydrogels. Final crosslink densities and photoreactivities dramatically increase with increasing number of functional groups, macromer concentrations as well as with photoinitiator concentration. Swellabilities of the hydrogels were determined as complementary reference values. Mass swelling ratios as well as mass loss increased with decreasing degree of substitution as a result of increased mesh size and hydrophilicity. Finally, hyaluronic acid vinyl ester formulations were used to encapsulate fluorescent-labeled immortalized human adipose-derived mesenchymal stem cells in 3D via UV and by high-resolution two-photon polymerization. Cell-survival was successfully studied via confocal laser scanning microscopy during the course of 2 weeks.     

Control of Nanoparticle Release Kinetics from 3D Printed Hydrogel Scaffolds

The convergence of biofabrication with nanotechnology is largely unexplored but enables geometrical control of cell‐biomaterial arrangement combined with controlled drug delivery and release. As a step towards integration of these two fields of research, this study demonstrates that modulation of electrostatic nanoparticle–polymer and nanoparticle–nanoparticle interactions can be used for tuning nanoparticle release kinetics from 3D printed hydrogel scaffolds. This generic strategy can be used for spatiotemporal control of the release kinetics of nanoparticulate drug vectors in biofabricated constructs.     

Designing Gelatin Methacryloyl (GelMA)‐Based Bioinks for Visible Light Stereolithographic 3D Biofabrication

Bioinks play a key role in determining the capability of the biofabricatoin processes and the resolution of the printed constructs. Excellent biocompatibility, tunable physical properties, and ease of chemical or biological modifications of gelatin methacryloyl (GelMA) have made it an attractive choice as bioinks for biomanufacturing of various tissues or organs. However, the current preparation methods for GelMA‐based bioinks lack the ability to tailor their physical properties for desired bioprinting methods. Inherently, GelMA prepolymer solution exhibits a fast sol–gel transition at room temperature, which is a hurdle for its use in stereolithography (SLA) bioprinting. Here, synthesis parameters are optimized such as solvents, pH, and reaction time to develop GelMA bioinks which have a slow sol–gel transition at room temperature and visible light crosslinkable functions. A total of eight GelMA combinations are identified as suitable for digital light processing (DLP)‐based SLA (DLP‐SLA) bioprinting through systematic characterizations of their physical and rheological properties. Out of various types of GelMA, those synthesized in reverse osmosis (RO) purified water (referred to as RO‐GelMA) are regarded as most suitable to achieve high DLP‐SLA printing resolution. RO‐GelMA‐based bioinks are also found to be biocompatible showing high survival rates of encapsulated cells in the photocrosslinked gels. Additionally, the astrocytes and fibroblasts are observed to grow and integrate well within the bioprinted constructs. The bioink's superior physical and photocrosslinking properties offer pathways of tuning the scaffold microenvironment and highlight the applicability of developed GelMA bioinks in various tissue engineering and regenerative medicine applications.     

Development of 3D Biofabricated Cell Laden Hydrogel Vessels and a Low‐Cost Desktop Printed Perfusion Chamber for In Vitro Vessel Maturation

The vascular system represents the key supply chain for nutrients and oxygen inside the human body. Engineered solutions to produce sophisticated alternatives for autologous or artificial vascular implants to sustainably replace diseased vascular tissue still remain a key challenge in tissue engineering. In this paper, cell‐laden 3D bioplotted hydrogel vessel‐like constructs made from alginate di‐aldehyde (ADA) and gelatin (GEL) are presented. The aim is to increase the mechanical stability of fibroblast‐laden ADA‐GEL vessels, tailoring them for maturation under dynamic cell culture conditions. BaCl₂ is investigated as a crosslinker for the oxidized alginate‐gelatin system. Normal human dermal fibroblast (NHDF)‐laden vessel constructs are optimized successfully in terms of higher stiffness by increasing ADA concentration and using BaCl₂, with no toxic effects observed on NHDF. Contrarily, BaCl₂ crosslinking of ADA‐GEL accelerates cell attachment, viability, and growth from 7d to 24h compared to CaCl₂. Moreover, alignment of cells in the longitudinal direction of the hydrogel vessels when extruding the cell‐laden hydrogel crosslinked with Ba²⁺ is observed. It is possible to tune the stiffness of ADA‐GEL by utilizing Ba²⁺ as crosslinker. In addition, a customized, low‐cost 3D printed polycarbonate (PC) perfusion chamber for perfusion of vessel‐like constructs is introduced.     

A Humanized In Vitro Model of Innervated Skin for Transdermal Analgesic Testing

Sensory innervation of the skin is essential for its function, homeostasis, and wound healing mechanisms. Thus, to adequately model the cellular microenvironment and function of native skin, in vitro human skin equivalents (hSE) containing a sensory neuron population began to be researched. In this work, a fully human 3D platform of hSE innervated by induced pluripotent stem cell-derived nociceptor neurospheres (hNNs), mimicking the native mode of innervation, is established. Both the hSE and nociceptor population exhibit morphological and phenotypical characteristics resembling their native counterparts, such as epidermal and dermal layer formation and nociceptor marker exhibition, respectively. In the co-culture platform, neurites develop from the hNNs and navigate in 3D to innervate the hSE from a distance. To probe both skin and nociceptor functionality, a clinically available capsaicin patch (Qutenza) is applied directly over the hSE section and neuron reaction is analyzed. Application of the patch causes an exposure time-dependent neurite regression and degeneration. In platforms absent of hSE, axonal degeneration is further increased, highlighting the role of the skin construct as a barrier. In sum, an in vitro tool of functional innervated skin with high interest for preclinical research is established.