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With the aim of understanding and recapitulating cellular interactions of hepatocytes in their physiological microenvironment and to generate an artificial 3D in vitro model, a co-culture system using 3D extrusion bioprinting was developed. A bioink based on alginate and methylcellulose (algMC) was first shown to be suitable for bioprinting of hepatocytes; the addition of Matrigel to algMC enhanced proliferation and morphology of them in monophasic scaffolds. Towards a more complex system that allows studying cellular interactions, we applied core–shell bioprinting to establish tailored 3D co-culture models for hepatocytes. The bioinks were specifically functionalized with natural matrix components (based on human plasma, fibrin or Matrigel) and used to co-print fibroblasts and hepatocytes in a spatially defined, coaxial manner. Fibroblasts acted as supportive cells for co-cultured hepatocytes, stimulating the expression of certain biomarkers of hepatocytes like albumin. Furthermore, matrix functionalization positively influenced both cell types in their respective compartments by enhancing their adhesion, viability, proliferation and function. In conclusion, we established a functional co-culture model with independently tunable compartments for different cell types via core–shell bioprinting. This provides the basis for more complex in vitro models allowing co-cultivation of hepatocytes with other liver-specific cell types to closely resemble the liver microenvironment.

For the generation of multi-layered full thickness osteochondral tissue substitutes with an individual geometry based on clinical imaging data, combined extrusion-based 3D printing (3D plotting) of a bioink laden with primary chondrocytes and a mineralized biomaterial phase was introduced. A pasty calcium phosphate cement (CPC) and a bioink based on alginate-methylcellulose (algMC) – both are biocompatible and allow 3D plotting with high shape fidelity – were applied in monophasic and combinatory design to recreate osteochondral tissue layers. The capability of cells reacting to chondrogenic biochemical stimuli inside the algMC-based 3D hydrogel matrix was assessed. Towards combined osteochondral constructs, the chondrogenic fate in the presence of CPC in co-fabricated and biphasic mineralized pattern was evaluated. Majority of expanded and algMC-encapsulated cells survived the plotting process and the cultivation period, and were able to undergo redifferentiation in the provided environment to produce their respective extracellular matrix (ECM) components (i.e. sulphated glycosaminoglycans, collagen type II), examined after 3 weeks. The presence of a mineralized zone as located in the physiological calcified cartilage region suspected to interfere with chondrogenesis, was found to support chondrogenic ECM production by altering the ionic concentrations of calcium and phosphorus in in vitro culture conditions.

One of the biggest challenges in tissue engineering and regenerative medicine is to ensure oxygen supply of cells in the (temporary) absence of vasculature. With the vision to exploit photosynthetic oxygen production by microalgae, co-cultivated in close vicinity to oxygen-consuming mammalian cells, we are searching for culture conditions that are compatible for both sides. Herein, we investigated the impact of long-term illumination on mammalian cells which is essential to enable photosynthesis by microalgae: four different cell types—primary human fibroblasts, dental pulp stem cells, and osteoblasts as well as the murine beta-cell line INS-1—were continuously exposed to warm white light, red or blue light over seven days. We observed that illumination with red light has no adverse effects on viability, metabolic activity and growth of the cells whereas exposure to white light has deleterious effects that can be attributed to its blue light portion. Quantification of intracellular glutathione did not reveal a clear correlation of this effect with an enhanced production of reactive oxygen species. Finally, our data indicate that the cytotoxic effect of short-wavelength light is predominantly a direct effect of cell illumination; photo-induced changes in the cell culture media play only a minor role.

Highly viscous bioinks offer great advantages for the three-dimensional fabrication of cell-laden constructs by microextrusion printing. However, no standardised method of mixing a high viscosity biomaterial ink and a cell suspension has been established so far, leading to non-reproducible printing results. A novel method for the homogeneous and reproducible mixing of the two components using a mixing unit connecting two syringes is developed and investigated. Several static mixing units, based on established mixing designs, were adapted and their functionality was determined by analysing specific features of the resulting bioink. As a model system, we selected a highly viscous ink consisting of fresh frozen human blood plasma, alginate, and methylcellulose, and a cell suspension containing immortalized human mesenchymal stem cells. This bioink is crosslinked after fabrication. A pre-crosslinked gellan gum-based bioink providing a different extrusion behaviour was introduced to validate the conclusions drawn from the model system. For characterisation, bioink from different zones within the mixing device was analysed by measurement of its viscosity, shape fidelity after printing and visual homogeneity. When taking all three parameters into account, a comprehensive and reliable comparison of the mixing quality was possible. In comparison to the established method of manual mixing inside a beaker using a spatula, a significantly higher proportion of viable cells was detected directly after mixing and plotting for both bioinks when the mixing unit was used. A screw-like mixing unit, termed “HighVisc”, was found to result in a homogenous bioink after a low number of mixing cycles while achieving high cell viability rates.

Three-dimensional (3D) bioprinting has become a fast-developing research field in the last few years. Many different technical solutions are available, with extrusion-based printing being the most promising and versatile method. In addition, a variety of biomaterials are already available for 3D printing of live cells. The real challenge, however, remains bioprinting of macroscopic, volumetric constructs of well-defined structures since hydrogels used for cell-embedding must consist of rather soft materials. This article describes recent developments to overcome these limitations that prevent clinical applications of bioprinted human tissues. New approaches include technical solutions such as in situ cross-linking or gelation processes that now can be performed during the bioprinting process, modified bioinks that combine suitable viscosity and cytocompatible gelation mechanisms, and utilization of additional materials to provide mechanical strength to the cell-laden constructs.

Combining the volumetric fabrication of hydrogel constructs using extrusion bioprinting with highly precise drop‐on‐demand (DoD) bioprinting offers exciting opportunities in biofabrication. This technical report presents a technique in which a solenoid micro‐pipette is operated as an additional tool in an extrusion (bio)printing system to deposit small volumes of bioinks into extrusion‐printed hydrogel constructs. Using three exemplary approaches, we show that this enables the patterned placement of cells or growth factors within 3D constructs and thus influences developmental processes. Human cells within low‐viscosity bioinks, deposited into extrusion‐printed hydrogel constructs by filling inter‐strand cavities or by injection into the hydrogel strands, maintained their viability and functionality up to 28 days. As demonstrated for salivary gland cells, the properties of the hydrogel matrix can influence the fate of the injected cells: In a stiff alginate (Alg)‐based hydrogel, they formed aggregates, which is beneficial for organoid formation, and in softer hydrogels, they migrated to neighboring cell clusters. Locally injected signaling factors such as vascular endothelial growth factor (VEGF) attracted endothelial cells and fibroblasts, which migrated into previously cell‐free hydrogel areas. The combination of extrusion and DoD bioprinting opens new approaches to integrate different cell types and functionalizations in one construct, facilitating the creation of more complex and dynamic models.

3D plotting is an additive manufacturing technology enabling biofabrication, thus the integration of cells or biologically sensitive proteins or growth factors into the manufacturing process. However, most (bio-)inks developed for 3D plotting were not shown to be processed into clinically relevant geometries comprising critical overhangs and cavities, which would collapse without a sufficient support material. Herein, we have developed a support hydrogel ink based on methylcellulose (mc), which is able to act as support as long as the co-plotted main structure is not stable. Therefore, 6 w/v %, 8 w/v % and 10 w/v % mc were allowed to swell in water, resulting in viscous inks, which were characterized for their rheological and extrusion properties. The successful usage of 10 w/v % mc as support ink was proven by multichannel plotting of the support together with a plottable calcium phosphate cement (CPC) acting as main structure. CPC scaffolds displaying critical overhangs or a large central cavity could be plotted accurately with the newly developed mc support ink. The dissolution properties of mc allowed complete removal of the gel without residuals, once CPC setting was finished. Finally, we fabricated a scaphoid bone model by computed tomography data acquisition and co-extrusion of CPC and the mc support hydrogel.

In tissue engineering, additive manufacturing (AM) technologies have brought considerable progress as they allow the fabrication of three-dimensional (3D) structures with defined architecture. 3D plotting is a versatile, extrusion-based AM technology suitable for processing a wide range of biomaterials including hydrogels. In this study, composites of highly concentrated alginate and gellan gum were prepared in order to combine the excellent printing properties of alginate with the favorable gelling characteristics of gellan gum. Mixtures of 16.7 wt % alginate and 2 or 3 wt % gellan gum were found applicable for 3D plotting. Characterization of the resulting composite scaffolds revealed an increased stiffness in the wet state (15%–20% higher Young’s modulus) and significantly lower volume swelling in cell culture medium compared to pure alginate scaffolds (~10% vs. ~23%). Cytocompatibility experiments with human mesenchymal stem cells (hMSC) revealed that cell attachment was improved—the seeding efficiency was ~2.5–3.5 times higher on the composites than on pure alginate. Additionally, the composites were shown to support hMSC proliferation and early osteogenic differentiation. In conclusion, print fidelity of highly concentrated alginate-gellan gum composites was comparable to those of pure alginate; after plotting and crosslinking, the scaffolds possessed improved qualities regarding shape fidelity, mechanical strength, and initial cell attachment making them attractive for tissue engineering applications.

Soft tissue infections in open fractures or burns are major cause for high morbidity in trauma patients. Sustained, long-term and localized delivery of antimicrobial agents is needed for early eradication of these infections. Traditional (topical or systemic) antibiotic delivery methods are associated with a variety of problems, including their long-term unavailability and possible low local concentration. Novel approaches for antibiotic delivery via wound coverage/healing scaffolds are constantly being developed. Many of these approaches are associated with burst release and thus seldom maintain long-term inhibitory concentrations. Using 3D core/shell extrusion printing, scaffolds consisting of antibiotic depot (in the core composed of low concentrated biomaterial ink 3% alginate) surrounded by a denser biomaterial ink (shell) were fabricated. Denser biomaterial ink (composed of alginate and methylcellulose or alginate, methylcellulose and Laponite) retained scaffold shape and modulated antibiotic release kinetics. Release of antibiotics was observed over seven days, indicating sustained release characteristics and maintenance of potency. Inclusion of Laponite in shell, significantly reduced burst release of antibiotics. Additionally, the effect of shell thickness on release kinetics was demonstrated. Amalgamation of such a modular delivery system with other biofabrication methods could potentially open new strategies to simultaneously treat soft tissue infections and aid wound regeneration.

In this study, the bone-regenerative potential of bioactive factors derived from adipose tissue, platelet-rich plasma (PRP) and conditioned medium from hypoxia-treated human telomerase immortalized bone-marrow-derived mesenchymal stem cells (hTERT-MSC) was investigated in vitro with the aim to develop cost-effective and efficient bone substitutes for optimized regeneration of bone defects. Adipose tissue was harvested from human donors undergoing reconstructive surgery, and adipose tissue extract (ATE) was prepared. Platelet lysates (PL) were produced by repeated freeze-thaw cycles of PRP, and hypoxia-conditioned medium (HCM) was obtained by culturing human telomerase immortalized bone-marrow-derived mesenchymal stromal cells for 5 days with 1% O2. Besides analysis by cytokine and angiogenesis arrays, ELISA was performed. Angiogenic potential was investigated in cocultures of bone-marrow-derived (BM)-MSC and human umbilical vein endothelial cells. Multiple angiogenic proteins and cytokines were detected in all growth factor mixtures. HCM and ATE contained high amounts of angiogenin and CCL2/MCP-1, whereas PL contained high amounts of IGFBP-1. Culturing cells with HCM and ATE significantly increased specific ALP activity of BM-MSC as well as tubule length and junctions of endothelial networks, indicating osteogenic and angiogenic stimulation. To achieve a synergism between chemoattractive potential and osteogenic and angiogenic differentiation capacity, a combination of different growth factors appears promising for potential clinical applications.