Marius Köpf

Cellulose Nanofibril Hydrogel Tubes as Sacrificial Templates for Freestanding Tubular Cell Constructs

The merging of defined nanoscale building blocks with advanced additive manufacturing techniques is of eminent importance for the preparation of multiscale and highly functional materials with de novo designed hierarchical architectures. Here, we demonstrate that hydrogels of cellulose nanofibrils (CNF) can be processed into complex shapes, and used as a sacrificial template to prepare freestanding cell constructs. We showcase our approach for the fabrication of hollow fibers using a controlled extrusion through a circular die into a coagulation bath. The dimensions of the hollow fibers are tunable, and the final tubes combine the nanofibrillar porosity of the CNF hydrogel with a submillimeter wall thickness and centimeter-scale length provided by the additive manufacturing technique. We demonstrate that covalent and supramolecular cross-linking of the CNFs can be used to tailor the mechanical properties of the hydrogel tubes within 1 order of magnitude and in an attractive range for the mechanosensation of cells. The resulting tubes are highly biocompatible and allow for the growth of mouse fibroblasts into confluent cell layers in their inner lumen. A detailed screening of several cellulases enables degradation of the scaffolding, temporary CNF hydrogel tube in a quick and highly cell-friendly way, and allows the isolation of coherent cell tubes. We foresee that the growing capabilities of hydrogel printing techniques in combination with the attractive features of CNFs-sustainable, globally abundant, biocompatible and enzymatically degradable-will allow making plant-based biomaterials with hierarchical structures and on-demand degradation useful, for instance, to engineer complex tissue structures to replace animal models, and for implants.

A tailored three-dimensionally printable agarose-collagen blend allows encapsulation, spreading, and attachment of human umbilical artery smooth muscle cells.

In recent years, novel biofabrication technologies have enabled the rapid manufacture of hydrogel-cell suspensions into tissue-imitating constructs. The development of novel materials for biofabrication still remains a challenge due to a gap between contradicting requirements such as three-dimensional printability and optimal cytocompatibility. We hypothesise that blending of different hydrogels could lead to a novel material with favourable biological and printing properties. In our work, we combined agarose and type I collagen in order to develop a hydrogel blend capable of long-term cell encapsulation of human umbilical artery smooth muscle cells (HUASMCs) and 3D drop-on-demand printing. Different blends were prepared with 0.25%, 0.5%, 0.75%, and 1.5% agarose and 0.2% type I collagen. The cell morphology of HUASMCs and the printing accuracy were assessed for each agarose-collagen combination, keeping the content of collagen constant. The hydrogel blend which displayed sufficient cell spreading and printing accuracy (0.5% agarose, 0.2% type I collagen, AGR0.5COLL0.2) was then characterised based on swelling and degradation over 21 days and mechanical stiffness. The cellular response regarding cell attachment of HUASMCs embedded in the hydrogel blend was further studied using SEM, TEM, and TPLSM. Printing trials were fabricated in a drop-on-demand printing process. The swelling and degradation evaluation showed an average of 20% mass loss and less than 10% swelling. AGR0.5COLL0.2 exhibited significant increase in stiffness compared to pure agarose and type I collagen. In addition, columns of AGR0.5COLL0.2 three centimeters in height were successfully printed submerged in cooled perfluorocarbon, proving the intrinsic printability of the hydrogel blend. Ultimately, a promising novel hydrogel blend showing cell spreading and attachment as well as suitability for bioprinting was identified and could, for example, serve in the manufacture of in vitro 3D models to capture more complex features of disease and drug discovery.

Laser-based in situ embedding of metal nanoparticles into bioextruded alginate hydrogel tubes enhances human endothelial cell adhesion

Alginate is a widely used hydrogel in tissue engineering owing to its simple and non-cytotoxic gelation process, ease of use, and abundance. However, unlike hydrogels derived from mammalian sources such as collagen, alginate does not contain cell adhesion ligands. Here, we present a novel laser ablation technique for the in situ embedding of gold and iron nanoparticles into hydrogels. We hypothesized that integration of metal nanoparticles in alginate could serve as an alternative material because of its chemical biofunctionalization ability (coupling of RGD ligands) to favor cell adhesion. Cytocompatibility and biofunctionality of the gels were assessed by cell culture experiments using fibroblasts and endothelial cells. Nanoparticles with an average particle size of 3 nm (gold) and 6 nm (iron) were generated and stably maintained in alginate for up to 6 months. Using an extrusion system, several centimeter-long alginate tubes with an outer diameter of approximately 3 mm and a wall thickness of approximately 150 μm were manufactured. Confocal microscopy revealed homogeneously distributed nanoparticle agglomerates over the entire tube volume. Endothelial cells seeded on iron-loaded gels showed significantly higher viability and an increased degree of spreading, and the number of attached cells was also elevated in comparison to the control and gold-loaded alginates. We conclude that laser-based in situ integration of iron nanoparticles (⩽0.01 wt.%) in alginate is a straightforward method to generate composite materials that favor the adhesion of endothelial cells. In addition, we show that nanoparticle integration does not impair the alginate’s gelation and 3D biofabrication properties.

Mechanically Tunable Bioink for 3D Bioprinting of Human Cells

This study introduces a thermogelling bioink based on carboxylated agarose (CA) for bioprinting of mechanically defined microenvironments mimicking natural tissues. In CA system, by adjusting the degree of carboxylation, the elastic modulus of printed gels can be tuned over several orders of magnitudes (5-230 Pa) while ensuring almost no change to the shear viscosity (10-17 mPa) of the bioink solution; thus enabling the fabrication of 3D structures made of different mechanical domains under identical printing parameters and low nozzle shear stress. Human mesenchymal stem cells printed using CA as a bioink show significantly higher survival (95%) in comparison to when printed using native agarose (62%), a commonly used thermogelling hydrogel for 3D-bioprinting applications. This work paves the way toward the printing of complex tissue-like structures composed of a range of mechanically discrete microdomains that could potentially reproduce natural mechanical aspects of functional tissues.

GelMA-collagen blends enable drop-on-demand 3D printablility and promote angiogenesis

Effective vascularization is crucial for three-dimensional (3D) printed hydrogel-cell constructs to efficiently supply cells with oxygen and nutrients. Till date, several hydrogel blends have been developed that allow the in vitro formation of a capillary-like network within the gels but comparatively less effort has been made to improve the suitability of the materials for a 3D bioprinting process. Therefore, we hypothesize that tailored hydrogel blends of photo-crosslinkable gelatin and type I collagen exhibit favorable 3D drop-on-demand printing characteristics in terms of rheological and mechanical properties and that further capillary-like network formation can be induced by co-culturing human umbilical vein endothelial cells and human mesenchymal stem cells within the proposed blends. Gelatin was methacrylated (GelMA) at a high degree of functionalization, mixed with cells, type I collagen, and the photoinitiator Irgacure 2959 and then subsequently crosslinked with UV light. After 14 d of incubation, cells were immunofluorescently labeled (CD31) and displayed using two-photon laser scanning microscopy. Hydrogels were rheologically characterized and dispensable droplet volumes were measured using a custom built 3D drop-on-demand bioprinter. The cell viability remained high in controllable crosslinking conditions both in 2D and 3D. In general, higher UV light exposure and increased Irgacure concentration were associated with lower cell viabilities. Distinctive capillary-like structures were formed in 3D printable GelMA-collagen hydrogels. The characteristic crosslinking time for GelMA in the range of minutes was not altered when GelMA was blended with type I collagen. Moreover, the addition of collagen led to enhanced cell spreading, a shear thinning behavior of the hydrogel solution and increased the storage modulus of the crosslinked gel. We therefore conclude that GelMA-collagen hydrogels exhibit favorable biological as well as rheological properties which are suitable for the manufacturing of pre-vascularized tissue replacement by 3D bioprinting.

Assembly of thin-walled, cell-laden hydrogel conduits inflated with perfluorocarbon

Autologous vessel grafts are considered the best option for coronary artery bypass surgery. However, their restricted availability and the poor patency of allografts as well as synthetic polymer grafts, make the development of biofunctional, small-calibre blood vessel substitutes of great interest. We propose a novel free-form manufacturing technique for the extrusion of cell-laden hydrogel tubes with adjustable wall thickness. Alginate and an alginate–fibrinogen blend were coextruded with high-density perfluorocarbon, which served as a liquid-supporting core. Hydrogel conduits measuring several centimetres in length and 2 mm in diameter were fabricated. By varying the volume flow ratio of gel and perfluorocarbon the wall thickness could be adjusted precisely in a range from 0.2 to 0.3 mm. In order to adjust the operating parameters of the technique, a theoretical model of the bioextrusion process based on the gelling kinetics of alginate was developed. Experimental validation revealed a high level of accordance with this model (90–100%). Burst-pressure tests indicated high mechanical stability of the extruded conduits (>100 kPa). Fibroblasts encapsulated in the hydrogel tubes were cultured for 7 days and exhibited a high degree of cell viability (>90%) and proliferation over the first week of culture. The proposed technique holds great promise for the fully automated manufacturing of cell-laden hydrogel conduits to be used for tissue engineering purposes or as tubing in bio-artificial, medical devices such as dialysers or liver support systems, in the future.

The Effect of Addition of Calcium Phosphate Particles to Hydrogel‐Based Composite Materials on Stiffness and Differentiation of Mesenchymal Stromal Cells toward Osteogenesis

The stiffness of a hydrogel has a significant role on the mechanical stability of a scaffold. However, the stiffness of pure hydrogels can be tuned only within a limited range. Herein, it is hypothesized that the range of hydrogel stiffness can be greatly increased by the addition of calcium phosphate particles and that such composites promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Beta-tricalcium phosphate (β-TCP) particles are incorporated at concentrations of 0.5 and 5 mg mL-1 into various agarose and agarose-collagen blends. These composites are characterized with respect to stiffness, viscosity, degradation, cell morphology, viability, and osteogenesis. The osteogenic hMSCs in less stiff composites with 0.5 mg mL-1 β-TCP show the highest alkaline phosphatase expression compared to blends without β-TCP and stiffer composites with 5 mg mL-1 β-TCP. Quantitative polymerase chain reaction also shows higher expression of ALP, RUNX2, and collagen I by hMSCs in less stiff composites with 0.5 mg mL-1 β-TCP compared to blends without β-TCP and stiffer composite blends. It is concluded that by addition of calcium phosphate to specific hydrogels the stiffness can be tuned in a desired range and thus the osteogenic differentiation of embedded hMSCs can be better controlled and adjusted compared to pure hydrogels.

3D-Printing and Angiogenesis: Tailored Agarose-Type I Collagen Blends Comprise 3D Printability and Angiogenesis Potential for Tissue Engineered Substitutes

Three-dimensional (3D) bioprinting is a promising technology for manufacturing cell-laden tissue-engineered constructs. Larger tissue substitutes, however, require a vascularized network to ensure nutrition supply. Therefore, tailored bioinks combining 3D printability and cell-induced vascularization are needed. We hypothesize that tailored hydrogel blends made of agarose-type I collagen and agarose-fibrinogen are 3D printable and will allow the formation of capillary-like structures by human umbilical vein endothelial cells and human dermal fibroblasts. Samples were casted, incubated for 14 days, and analyzed by immunohistology and two-photon laser scanning microscopy. The 3D printability of the hydrogel blends was examined using a drop-on-demand printing system. The rheological behavior was also investigated. Substantial capillary network formation was observed in agarose-type I collagen hydrogel blends with concentrations of 0.2% or 0.5% collagen and 0.5% agarose. Furthermore, storage moduli of agarose-collagen blends were significantly increased compared to those of the corresponding single components (448 Pa for 0.5% agarose, 148 Pa for 0.5% collagen, and 1551 Pa for 0.5% agarose-0.5% collagen). Neither the addition of collagen nor fibrinogen significantly impaired the printing resolution. In conclusion, we present a tailored hydrogel blend that can be printed in 3D and in parallel exhibits cell-induced vascularization capability.