Table of contents

After many decades of biomaterials research for peripheral nerve regeneration, a clinical product (the nerve guide), is emerging as a proven alternative for relatively short injury gaps. This review identifies aspects where 3D printing can assist in improving long-distance nerve guide regeneration strategies. These include (1) 3D printing of the customizable nerve guides, (2) fabrication of scaffolds that fill nerve guides, (3) 3D bioprinting of cells within a matrix/bioink into the nerve guide lumen and the (4) establishment of growth factor gradients along the length a nerve guide. The improving resolution of 3D printing technologies will be an important factor for peripheral nerve regeneration, as fascicular-like guiding structures provide one path to improved nerve guidance. The capability of 3D printing to manufacture complex structures from patient data based on existing medical imaging technologies is an exciting aspect that could eventually be applied to treating peripheral nerve injury. Ultimately, the goal of 3D printing in peripheral nerve regeneration is the automated fabrication, potentially customized for the patient, of structures within the nerve guide that significantly outperform the nerve autograft over large gap injuries.

Artificial blood vessels must be strong, flexible, and must not lead to blockage after implantation. It is therefore important to select an appropriate fabrication process for products to meet these requirements. This review discusses the current methods for making artificial blood vessels, focusing on fabrication principle, materials, and applications. Among these methods, 3D printing is very promising since it has the unique capability to make complicated three-dimensional structures with multiple types of materials, and can be completely digitalized. Therefore, new developments in 3D printing of artificial blood vessels are also summarized here. This review provides a reference for the fusion of multiple processes and further improvement of artificial blood vessel fabrication.

Lithography-based three-dimensional (3D) printing technologies allow high spatial resolution that exceeds that of typical extrusion-based bioprinting approaches, allowing to better mimic the complex architecture of biological tissues. Additionally, lithographic printing via digital light processing (DLP) enables fabrication of free-form lattice and patterned structures which cannot be easily produced with other 3D printing approaches. While significant progress has been dedicated to the development of cell-laden bioinks for extrusion-based bioprinting, less attention has been directed towards the development of cyto-compatible bio-resins and their application in lithography-based biofabrication, limiting the advancement of this promising technology. In this study, we developed a new bio-resin based on methacrylated poly(vinyl alcohol) (PVA-MA), gelatin-methacryloyl (Gel-MA) and a transition metal-based visible light photoinitiator. The utilization of a visible light photo-initiating system displaying high molar absorptivity allowed the bioprinting of constructs with high resolution features, in the range of 25–50 μm. Biofunctionalization of the resin with 1 wt% Gel-MA allowed long term survival (>90%) of encapsulated cells up to 21 d, and enabled attachment and spreading of endothelial cells seeded on the printed hydrogels. Cell-laden hydrogel constructs of high resolution with complex and ordered architecture were successfully bioprinted, where the encapsulated cells remained viable, homogenously distributed and functional. Bone and cartilage tissue synthesis was confirmed by encapsulated stem cells, underlining the potential of these DLP-bioprinted hydrogels for tissue engineering and biofabrication. Overall, the PVA-MA/Gel-MA bio-resin is a promising material for biofabrication and provides important cues for the further development of lithography-based bioprinting of complex, free-form living tissue analogues.

Tumour invasion into the surrounding stroma is a critical step in metastasis, and it is necessary to clarify the role of microenvironmental factors in tumour invasion. We present a microfluidic system that simulated and controlled multi-factors of the tumour microenvironment for three-dimensional (3D) assessment of tumour invasion into the stroma. The simultaneous, precise and continuous arrangement of two 3D matrices was visualised to observe the migration of cancer cell populations or single cells by transfecting cells with a fluorescent protein. A vascular endothelial layer was formed to simulate transendothelial transport of nutrients, and its endothelial barrier function was verified by the diffusion of 70 kDa fluorescein isothiocyanate (FITC)-Dextran in 3D matrices. Through high-throughput cell migration tracking observation and statistic evaluation, we clarified that cell density of the tumour directly determined its invasiveness. The results suggested that increased secretion of IL-6 among both cancer cells (MDA-MB-231) and noncancerous cells (MCF-10A or HDF-n) after co-culture contributes to cancer cell invasiveness, and this was verified by an IL-6 inhibitor assay. Finally, the drug efficacy of paclitaxel was reflected as changes in cancer cell migration ability, viability, and morphology. Together, our microfluidic devices could be a useful tool to study the mechanism of tumour invasion into the stroma and to screen anti-metastatic drugs.

Investigation of diseases of the bile duct system and identification of potential therapeutic targets are hampered by the lack of tractable in vitro systems to model cholangiocyte biology. Here, we show a step-wise method for the differentiation of murine Lgr5⁺ liver stem cells (organoids) into cholangiocyte-like cells (CLCs) using a combination of growth factors and extracellular matrix components. Organoid-derived CLCs display key properties of primary cholangiocytes, such as expressing cholangiocyte markers, forming primary cilia, transporting small molecules and responding to farnesoid X receptor agonist. Integration of organoid-derived cholangiocytes with collagen-coated polyethersulfone hollow fiber membranes yielded bioengineered bile ducts that morphologically resembled native bile ducts and possessed polarized bile acid transport activity. As such, we present a novel in vitro model for studying and therapeutically modulating cholangiocyte function.

Tissue spheroids have been proposed as building blocks in 3D biofabrication. Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational assembly has been introduced. Here we present first time report on rapid assembly of 3D tissue construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids. Chondrospheres of standard size, shape and capable to fusion have been biofabricated from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation was performed using a prototype device equipped with permanent magnets in presence of gadolinium (Gd³⁺) in culture media, which enables magnetic levitation. Mathematical modeling and computer simulations were used for prediction of magnetic field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal settings for magnetic levitation in presence of Gd³⁺. Second, we proved the ability of chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in the presence of Gd³⁺. Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-driven tissue engineering.

3D bioprinting with cell containing bioinks show great promise in the biofabrication of patient specific tissue constructs. To fulfil the multiple requirements of a bioink, a wide range of materials and bioink composition are being developed and evaluated with regard to cell viability, mechanical performance and printability. It is essential that the printability and printing fidelity is not neglected since failure in printing the targeted architecture may be catastrophic for the survival of the cells and consequently the function of the printed tissue. However, experimental evaluation of bioinks printability is time-consuming and must be kept at a minimum, especially when 3D bioprinting with cells that are valuable and costly. This paper demonstrates how experimental evaluation could be complemented with computer based simulations to evaluate newly developed bioinks. Here, a computational fluid dynamics simulation tool was used to study the influence of different printing parameters and evaluate the predictability of the printing process. Based on data from oscillation frequency measurements of the evaluated bioinks, a full stress rheology model was used, where the viscoelastic behaviour of the material was captured. Simulation of the 3D bioprinting process is a powerful tool and will help in reducing the time and cost in the development and evaluation of bioinks. Moreover, it gives the opportunity to isolate parameters such as printing speed, nozzle height, flow rate and printing path to study their influence on the printing fidelity and the viscoelastic stresses within the bioink. The ability to study these features more extensively by simulating the printing process will result in a better understanding of what influences the viability of cells in 3D bioprinted tissue constructs.

Three-dimensional bioprinting has emerged as a promising technique in tissue engineering applications through the precise deposition of cells and biomaterials in a layer-by-layer fashion. However, the limited availability of hydrogel bioinks is frequently cited as a major issue for the advancement of cell-based extrusion bioprinting technologies. It is well known that highly viscous materials maintain their structure better, but also have decreased cell viability due to the higher forces which are required for extrusion. However, little is known about the effect of the two distinct components of dynamic modulus of viscoelastic materials, storage modulus (G') and loss modulus (G''), on the printability of hydrogel-based bioinks. Additionally, 'printability' has been poorly defined in the literature, mostly consisting of gross qualitative measures which do not allow for direct comparison of bioinks. This study developed a framework for evaluating printability and investigated the effect of dynamic modulus, including storage modulus (G'), loss modulus (G''), and loss tangent (G''/G') on the printing outcome. Gelatin and alginate as model hydrogels were mixed at various concentrations to obtain hydrogel formulations with a wide range of storage and loss moduli. These formulations were then evaluated for the quantitatively defined values of extrudability, extrusion uniformity, and structural integrity. For extrudability, increasing either the loss or storage modulus increased the pressure required to extrude the bioink. A mathematical model relating the G' and G'' to the required extrusion pressure was derived based on the data. A lower loss tangent was correlated with increased structural integrity while a higher loss tangent correlated with increased extrusion uniformity. Gelatin–alginate composite hydrogels with a loss tangent in the range of 0.25–0.45 exhibited an excellent compromise between structural integrity and extrusion uniformity. In addition to the characterization of a common bioink, the methodology introduced in this paper could also be used to evaluate the printability of other bioinks in the future.

The field of how to rapidly assemble microfluidics with modular components continuously attracts researchers' attention, however, extra efforts must be devoted to solving the problems of leaking and aligning between individual modules. This paper presents a novel type of modular microfluidic device, driven by capillary force. There is no necessity for a strict seal or special alignment, and its open structures make it easy to integrate various stents and reactants. The key rationale for this method is to print different functional modules with a low-cost three-dimensional (3D) printer, then fill the channels with capillary materials and assemble them with plugs like Lego^® bricks. This rapidly reconstructed modular microfluidic device consists of a variety of common functional modules and other personalized modules, each module having a unified standard interface for easy assembly. As it can be printed by a desktop 3D printer, the manufacturing process is simple and efficient, with controllable regulation of the flow channel scale. Through diverse combinations of different modules, a variety of different functions can be achieved, without duplicating the manufacturing process. A single module can also be taken out for testing and analysis. What's more, combined with basic circuit components, it can serve as a low-cost Lego^®-like modular microfluidic circuits. As a proof of concept, the modular microfluidic device has been successfully demonstrated and used for stent degradation and cell cultures, revealing the potential use of this method in both chemical and biological research.

In this study, we developed an enzyme-based miniaturized fluorescence biosensor to detect paraoxon, one of the most well-known neurotoxic organophosphorus compounds. The biosensor was fabricated with poly(ethylene glycol) (PEG) hydrogel microarrays that entrapped acetylcholinesterase (AChE) and quantum dots (QDs) as fluorescence reporters. Metal-enhanced fluorescence (MEF) was utilized to amplify the fluorescence signal, which was achieved by decorating QDs on the surface of silica-coated silver nanoparticles (Ag@Silica). The MEF effects of Ag@Silica were optimized by tuning the thickness of the silica shells, and under the optimized conditions, the fluorescence intensity was shown to be increased 5 fold, compared with the system without MEF. PEG hydrogel microarray entrapping QD-decorated Ag@Silica and AChE was prepared via photopatterning process. The entrapped AChE hydrolyzed paraoxon to produce p-nitrophenol within the hydrogel microstructure, which subsequently quenched the fluorescence of the QDs on the surface of Ag@Silica. The MEF-assisted fluorescence detection resulted in a significant enhancement of paraoxon detection. The detection limit was approximately 1.0 × 10⁻¹⁰ M and 2.0 × 10⁻⁷ M for sensing with and without MEF, respectively. The successful integration of a hydrogel microarray system with a microfluidic system was demonstrated to be a potential application for the MEF-based micro-total-analysis-system.

Despite the recent achievements in cell-based therapies for curing type-1 diabetes (T1D), capillarization in beta (β)-cell clusters is still a major roadblock as it is essential for long-term viability and function of β-cells in vivo. In this research, we report sprouting angiogenesis in engineered pseudo islets (EPIs) made of mouse insulinoma βTC3 cells and rat heart microvascular endothelial cells (RHMVECs). Upon culturing in three-dimensional (3D) constructs under angiogenic conditions, EPIs sprouted extensive capillaries into the surrounding matrix. Ultra-morphological analysis through histological sections also revealed presence of capillarization within EPIs. EPIs cultured in 3D constructs maintained their viability and functionality over time while non-vascularized EPIs, without the presence of RHMVECs, could not retain their viability nor functionality. Here we demonstrate angiogenesis in engineered islets, where patient specific stem cell-derived human beta cells can be combined with microvascular endothelial cells in the near future for long-term graft survival in T1D patients.

The tumor microenvironment (TME) is gaining increasing attention in oncology, as it is recognized to be functionally important during tumor development and progression. Tumors are heterogeneous tissues that, in addition to tumor cells, contain tumor-associated cell types such as immune cells, fibroblasts, and endothelial cells. These other cells, together with the specific extracellular matrix (ECM), create a permissive environment for tumor growth. While the influence of tumor-infiltrating cells and mechanical properties of the ECM in tumor invasion and progression have been studied separately, their interaction within the complex TME and the epithelial -to-mesenchymal transition (EMT) is still unclear. In this work, we develop a 3D co-culture model of lung adenocarcinoma cells and macrophages in an interpenetrating network hydrogel, to investigate the influence of the macrophage phenotype and ECM stiffness in the induction of EMT. Rising ECM stiffness increases both tumor cell proliferation and invasiveness. The presence of tumor-associated macrophages and the ECM stiffness jointly contribute to an invasive phenotype, and modulate the expression of key EMT-related markers. Overall, these findings support the utility of in vitro 3D cancer models that allow one to study interactions among key components of the TME.

Research on human induced pluripotent stem cells (hiPSCs) is one of the fastest growing fields in biomedicine. Generated from patient's own somatic cells, hiPSCs can be differentiated towards all functional cell types and returned to the patient without immunological concerns. 3D printing of hiPSCs could enable the generation of functional organs for replacement therapies or realization of organ-on-chip systems for individualized medicine. Printing of living cells was demonstrated with immortalized cell lines, primary cells, and adult stem cells with different printing technologies and biomaterials. However, hiPSCs are more sensitive to handling procedures, in particular, when dissociated into single cells. Both pluripotency and directed differentiation are influenced by numerous environmental factors including culture media, biomaterials, and cell density. Notably, existing literature on the effect of applied biomaterials on pluripotency is rather ambiguous. In this study, laser bioprinting of undifferentiated hiPSCs in combination with different biomaterials was performed and the impact on cells' behavior, pluripotency, and differentiation was investigated. Our findings suggest that hiPSCs are indeed more sensitive to the applied biomaterials, but not to laser printing itself. With appropriate biomaterials, such as the hyaluronic acid based solutions applied in this study, hiPSCs can be successfully laser printed without losing their pluripotency.

A major challenge during the engineering of voluminous bone tissues is to maintain cell viability in the central regions of the construct. In vitro prevascularization of bone substitutes relying on endothelial cell bioprinting has the potential to resolve this issue and to replicate the native bone microvasculature. Laser-assisted bioprinting (LAB) commonly uses biological layers of hydrogel, called 'biopapers', to support patterns of printed cells and constitute the basic units of the construct. The self-assembly approach of tissue engineering allows the production of biomimetic cell-derived bone extracellular matrix including living cells. We hypothesized that self-assembled osseous sheets can serve as living biopapers to support the LAB of human endothelial cells and thus guide tubule-like structure formation. Human umbilical vein endothelial cells were bioprinted on the surface of the biopapers following a predefined pattern of lines. The osseous biopapers showed relevant matrix mineralization and pro-angiogenic hallmarks. Our results revealed that formation of tubule-like structures was favored when the cellular orientation within the biopaper was parallel to the printed lines. Altogether, we validated that human osseous cell sheets can be used as biopapers for LAB, allowing the production of human prevascularized cell-based osseous constructs that can be relevant for autologous bone repair applications.

4D printing is a highly innovative additive manufacturing process for fabricating smart structures with the ability to transform over time. Significantly different from regular 4D printing techniques, this study focuses on creating novel 4D hierarchical micropatterns using a unique photolithographic-stereolithographic-tandem strategy (PSTS) with smart soybean oil epoxidized acrylate (SOEA) inks for effectively regulating human bone marrow mesenchymal stem cell (hMSC) cardiomyogenic behaviors. The 4D effect refers to autonomous conversion of the surficial-patterned scaffold into a predesigned construct through an external stimulus delivered immediately after printing. Our results show that hMSCs actively grew and were highly aligned along the micropatterns, forming an uninterrupted cellular sheet. The generation of complex patterns was evident by triangular and circular outlines appearing in the scaffolds. This simple, yet efficient, technique was validated by rapid printing of scaffolds with well-defined and consistent micro-surface features. A 4D dynamic shape change transforming a 2-D design into flower-like structures was observed. The printed scaffolds possessed a shape memory effect beyond the 4D features. The advanced 4D dynamic feature may provide seamless integration with damaged tissues or organs, and a proof of concept 4D patch for cardiac regeneration was demonstrated for the first time. The 4D-fabricated cardiac patch showed significant cardiomyogenesis confirmed by immunofluorescence staining and qRT-PCR analysis, indicating its promising potential in future tissue and organ regeneration applications.

Recent advances in three-dimensional bioprinting technology have led to various attempts in fabricating human tissue-like structures. However, current bioprinting technologies have limitations for creating native tissue-like structures. To resolve these issues, we developed a new pre-set extrusion bioprinting technique that can create heterogeneous, multicellular, and multimaterial structures simultaneously. The key to this ability lies in the use of a precursor cartridge that can stably preserve a multimaterial with a pre-defined configuration that can be simply embedded in a syringe-based printer head. The multimaterial can be printed and miniaturized through a micro-nozzle without conspicuous deformation according to the pre-defined configuration of the precursor cartridge. Using this system, we fabricated heterogeneous tissue-like structures such as spinal cords, hepatic lobule, blood vessels, and capillaries. We further obtained a heterogeneous patterned model that embeds HepG2 cells with endothelial cells in a hepatic lobule-like structure. In comparison with homogeneous and heterogeneous cell printing, the heterogeneous patterned model showed a well-organized hepatic lobule structure and higher enzyme activity of CYP3A4. Therefore, this pre-set extrusion bioprinting method could be widely used in the fabrication of a variety of artificial and functional tissues or organs.

Overcoming the problem of vascularization remains the main challenge in the field of tissue engineering. As three-dimensional (3D) bioprinting is the rising technique for the fabrication of large tissue constructs, small prevascularized building blocks were generated that can be incorporated throughout a printed construct, answering the need for a microvasculature within the small micron range (<10 μm). Uniform spheroids with an ideal geometry and diameter for bioprinting were formed, using a high-throughput non-adhesive agarose microwell system. Since monoculture spheroids of endothelial cells were unable to remain stable, coculture spheroids combining endothelial cells with fibroblasts and/or adipose tissue derived mesenchymal stem cells (ADSC) as supporting cells, were created. When applying the favorable coculture ratio, viable spheroids were obtained and endothelial cells spontaneously formed a capillary-like network and lumina, as shown by immunohistochemistry and transmission electron microscopy. Especially the presence of ADSC led to a higher vascularization and extracellular matrix production of the microtissue. Moreover, spheroids were able to assemble at random in suspension and in a hydrogel, creating a macrotissue. During at random assembly, cells reorganized, creating a branched capillary-network throughout the entire fused construct by inoculating with capillaries of adjacent spheroids. Combining the advantage of this natural capacity of microtissues to self-assemble and the controlled organization by bioprinting technologies, these prevascularized spheroids can be useful as building blocks for the engineering of large vascularized 3D tissues.

3D-printing has expanded our ability to produce reproducible and more complex scaffold architectures for tissue engineering applications. In order to enhance the biological response within these 3D-printed scaffolds incorporating nanostructural features and/or specific biological signaling may be an effective means to optimize tissue regeneration. Peptides amphiphiles (PAs) are a versatile supramolecular biomaterial with tailorable nanostructural and biochemical features. PAs are widely used in tissue engineering applications such as angiogenesis, neurogenesis, and bone regeneration. Thus, the addition of PAs is a potential solution that can greatly expand the utility of 3D bioprinting hydrogels in the field of regenerative medicine. In this paper, we firstly developed a 3D-printable thiolated-gelatin bioink supplemented with PAs to tailor the bioactivity and nanostructure which allows for the incorporation of cells. The bioink can be printed at 4 °C and stabilized to last a long time (>1 month) in culture at 37 °C by via a dual secondary crosslinking strategy using calcium ions and homobifunctional maleiminde-poly (ethylene glycol). Rheological properties of inks were characterized and were suitable for printing multi-layered structures. We additionally demonstrated enhanced functionality of ink formulations by utilizing a laminin-mimetic IKVAV-based PA system within a 3D-printable ink containing cholangiocytes. Viability and functional staining showed that the IKVAV PA nanofibers stimulated cholangioctyes to form functional tubular structures, which was not observed in other ink formulations.

Cell delivery and leakage during injection remains a challenge for cell-based intervertebral disc regeneration strategies. Cellular microencapsulation may offer a promising approach to overcome these limitations by providing a protective niche during intradiscal injection. Electrohydrodynamic spraying (EHDS) is a versatile one-step approach for microencapsulation of cells using a high voltage electric field. The primary objective of this work was to characterise key processing parameters such as applied voltage (0, 5, 10 or 15 kV), emitter needle gauge (21, 26 or 30 G), alginate concentration (1%, 2% or 3%) and flow rate (50, 100, 250 or 500 μl min⁻¹) to regulate the size and morphology of alginate microcapsules as well as subsequent cell viability when altering these parameters. The effect of initial cell seeding density (5, 10 and 20 × 10⁶ cells ml⁻¹) on subsequent matrix accumulation of microencapsulated articular chondrocytes was also evaluated. Results showed that increasing alginate concentration and thus viscosity increased overall microcapsule size but also affected the geometry towards ellipsoidal-shaped gels. Altering the electric field strength and needle diameter regulated microcapsule size towards a smaller diameter with increasing voltage and smaller needle diameter. Needle size did not appear to affect cell viability when operating with lower alginate concentrations (1% and 2%), although higher concentrations (3%) and thus higher viscosity hydrogels resulted in diminished viability with decreasing needle diameter. Increasing cell density resulted in decreased cell viability and a concomitant decrease in DNA content, perhaps due to competing nutrient demands as a result of more closely packed cells. However, higher cell densities resulted in increased levels of extracellular matrix accumulated. Overall, this work highlights the potential of EHDS as a controllable and versatile approach to fabricate microcapsules for injectable delivery which can be used in a variety of applications such as drug development or cell therapies.

Biofabrication processes can affect biological quality attributes of encapsulated cells within constructs. Currently, assessment of the fabricated constructs is performed offline by subjecting the constructs to destructive assays that require staining and sectioning. This drawback limits the translation of biofabrication processes to industrial practice. In this work, we investigate the dielectric response of viable cells encapsulated in bioprinted 3D hydrogel constructs to an applied alternating electric field as a label-free non-destructive monitoring approach. The relationship between β-dispersion parameters (permittivity change—Δε, Cole–Cole slope factor—α, critical polarization frequency—f_c) over the frequency spectrum and critical cellular quality attributes are investigated. Results show that alginate constructs containing a higher number of viable cells (human adipose derived stem cells—hASC and osteosarcoma cell line—MG63) were characterized by significantly higher Δε and α (both p < 0.05). When extended to bioprinting, results showed that changes in hASC proliferation and viability in response to changes in critical bioprinting parameters (extrusion pressure, temperature, processing time) significantly affected ∆ε, α, and f_c. We also demonstrated monitoring of hASC distribution after bioprinting and changes in proliferation over time across the cross-section of a bioprinted medial knee meniscus construct. The trends in ∆ε over time were in agreement with the alamarBlue assay results for the whole construct, but this measurement approach provided a localized readout on the status of encapsulated cells. The findings of this study support the use of dielectric impedance spectroscopy as a label-free and non-destructive method to characterize the critical quality attributes of bioprinted constructs.

Despite the promise of stem cell engineering and the new advances in bioprinting technologies, one of the major challenges in the manufacturing of large scale bone tissue scaffolds is the inability to perfuse nutrients throughout thick constructs. Here, we report a scalable method to create thick, perfusable bone constructs using a combination of cell-laden hydrogels and a 3D printed sacrificial polymer. Osteoblast-like Saos-2 cells were encapsulated within a gelatin methacrylate (GelMA) hydrogel and 3D printed polyvinyl alcohol pipes were used to create perfusable channels. A custom-built bioreactor was used to perfuse osteogenic media directly through the channels in order to induce mineral deposition which was subsequently quantified via micro-CT. Histological staining was used to verify mineral deposition around the perfused channels, while COMSOL modeling was used to simulate oxygen diffusion between adjacent channels. This information was used to design a scaled-up construct containing a 3D array of perfusable channels within cell-laden GelMA. Progressive matrix mineralization was observed by cells surrounding perfused channels as opposed to random mineral deposition in static constructs. Micro-CT confirmed that there was a direct relationship between channel mineralization within perfused constructs and time within the bioreactor. Furthermore, the scalable method presented in this work serves as a model on how large-scale bone tissue replacement constructs could be made using commonly available 3D printers, sacrificial materials, and hydrogels.

Three-dimensional bioprinting of biomaterials shows great potential for producing cell-encapsulated scaffolds to repair nerves after injury or disease. For this, preparation of biomaterials and bioprinting itself are critical to create scaffolds with both biological and mechanical properties appropriate for nerve regeneration, yet remain unachievable. This paper presents our study on bioprinting Schwann cell-encapsulated scaffolds using composite hydrogels of alginate, fibrin, hyaluronic acid, and/or RGD peptide, for nerve tissue engineering applications. For the preparation of composite hydrogels, suitable hydrogel combinations were identified and prepared by adjusting the concentration of fibrin based on the morphological spreading of Schwann cells. In bioprinting, the effects of various printing process parameters (including the air pressure for dispensing, dispensing head movement speed, and crosslinking conditions) on printed structures were investigated and, by regulating these parameters, mechanically-stable scaffolds with fully interconnected pores were printed. The performance of Schwann cells within the printed scaffolds were examined in terms of viability, proliferation, orientation, and ability to produce laminin. Our results show that the printed scaffolds can promote the alignment of Schwann cells inside scaffolds and thus provide haptotactic cues to direct the extension of dorsal root ganglion neurites along the printed strands, demonstrating their great potential for applications in the field of nerve tissue engineering.