One of the challenges of bioprinting is to identify bioinks which support cell growth, tissue maturation, and ultimately the formation of functional grafts for use in regenerative medicine. The influence of this new biofabrication technology on biology of living cells, however, is still being evaluated. Recently we have identified a mitogenic hydrogel system based on alginate sulfate which potently supports chondrocyte phenotype, but is not printable due to its rheological properties (no yield point). To convert alginate sulfate to a printable bioink, it was combined with nanocellulose, which has been shown to possess very good printability. The alginate sulfate/nanocellulose ink showed good printing properties and the non-printed bioink material promoted cell spreading, proliferation, and collagen II synthesis by the encapsulated cells. When the bioink was printed, the biological performance of the cells was highly dependent on the nozzle geometry. Cell spreading properties were maintained with the lowest extrusion pressure and shear stress. However, extruding the alginate sulfate/nanocellulose bioink and chondrocytes significantly compromised cell proliferation, particularly when using small diameter nozzles and valves.

Bioprinting is an emerging technology in the field of tissue engineering as it allows the precise positioning of biologically relevant materials in 3D, which more resembles the native tissue in our body than current homogenous, bulk approaches. There is however a lack of materials to be used with this technology and materials such as the block copolymer Pluronic have good printing properties but do not allow long-term cell culture. Here we present an approach called nanostructuring to increase the biocompatibility of Pluronic gels at printable concentrations. By mixing acrylated with unmodified Pluronic F127 it was possible to maintain the excellent printing properties of Pluronic and to create stable gels via UV crosslinking. By subsequent elution of the unmodified Pluronic from the crosslinked network we were able to increase the cell viability of encapsulated chondrocytes at day 14 from 62% for a pure acrylated Pluronic hydrogel to 86% for a nanostructured hydrogel. The mixed Pluronic gels also showed good printability when cells where included in the bioink. The nanostructured gels were, with a compressive modulus of 1.42 kPa, mechanically weak, but we were able to increase the mechanical properties by the addition of methacrylated hyaluronic acid. Our nanostructuring approach enables Pluronic hydrogels to have the desired set of properties in all stages of the bioprinting process.

Layer-by-layer bioprinting is a logical choice for the fabrication of stratified tissues like articular cartilage. Printing of viable organ replacements, however, is dependent on bioinks with appropriate rheological and cytocompatible properties. In cartilage engineering, photocrosslinkable glycosaminoglycan-based hydrogels are chondrogenic, but alone have generally poor printing properties. By blending the thermoresponsive polymer poly(N-isopropylacrylamide) grafted hyaluronan (HA-pNIPAAM) with methacrylated hyaluronan (HAMA), high-resolution scaffolds with good viability were printed. HA-pNIPAAM provided fast gelation and immediate post-printing structural fidelity, while HAMA ensured long-term mechanical stability upon photocrosslinking. The bioink was evaluated for rheological properties, swelling behavior, printability and biocompatibility of encapsulated bovine chondrocytes. Elution of HA-pNIPAAM from the scaffold was necessary to obtain good viability. HA-pNIPAAM can therefore be used to support extrusion of a range of biopolymers which undergo tandem gelation, thereby facilitating the printing of cell-laden, stratified cartilage constructs with zonally varying composition and stiffness.

Epithelial organoids are simplified models of organs grown in vitro from embryonic and adult stem cells. They are widely used to study organ development and disease, and enable drug screening in patient-derived primary tissues. Current protocols, however, rely on animal- and tumor-derived basement membrane extract (BME) as a 3D scaffold, which limits possible applications in regenerative medicine. This prompted us to study how organoids interact with their matrix, and to develop a well-defined hydrogel that supports organoid generation and growth. It is found that soft fibrin matrices provide suitable physical support, and that naturally occurring Arg-Gly-Asp (RGD) adhesion domains on the scaffold, as well as supplementation with laminin-111, are key parameters required for robust organoid formation and expansion. The possibility to functionalize fibrin via factor XIII-mediated anchoring also allows to covalently link fluorescent nanoparticles to the matrix for 3D traction force microscopy. These measurements suggest that the morphogenesis of budding intestinal organoids results from internal pressure combined with higher cell contractility in the regions containing differentiated cells compared to the regions containing stem cells. Since the fibrin/laminin matrix supports long-term expansion of all tested murine and human epithelial organoids, this hydrogel can be widely used as a defined equivalent to BME.

A "bottom up" strategy is proposed to synthesize high aspect ratio hydroxyapatite (and brushite) platelets, and combine them with amyloid fibrils into layered hybrid nanocomposites. Their hierarchical structure, despite the differences from natural bone, confers to the nanocomposites a density and elastic modulus matching those of cancellous bone. Evidence of good adhesion and spreading of human trabecular bone-derived pre-osteoblasts cells on these nanocomposites is provided.

Chondrocyte hypertrophy is one of the key physiological processes involved in the longitudinal growth of long bones, yet regulation of hypertrophy is becoming increasingly relevant for clinical application of mesenchymal stem cells (MSCs) and screening for drugs to treat hypertrophic osteoarthritis. The extraordinary cell volume increase during hypertrophy is accompanied by an up-regulation of collagen X, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF), all which are targets of the runt-related transcription factor 2 (Runx2). Many pathways, including parathyroid hormone-related protein (PTHrP)/Indian Hedgehog, Wingless/Int (Wnt)/βcatenin, and transforming growth factor beta (TGF-β)/ Sma and Mad Related Family (Smad) pathways, can regulate hypertrophy, but factors as diverse as hypoxia, coculture, epigenetics and biomaterial composition can also potently affect Runx2 expression. Control of hypertrophic differentiation can be exploited both for cartilage repair, where a stable phenotype is desired, but also in bone regeneration, where hypertrophic cartilage could act as a template for endochondral bone formation. We hope this review will motivate the design of novel engineered microenvironments for skeletal regeneration applications.

Photo-activated materials have found widespread use in biological and medical applications and are playing an increasingly important role in the nascent field of three-dimensional (3D) bioprinting. Light can be used as a trigger to drive the formation or the degradation of chemical bonds, leading to unprecedented spatiotemporal control over a material's chemical, physical, and biological properties. With resolution and construct size ranging from nanometres to centimeters, light-mediated biofabrication allows multicellular and multimaterial approaches. It promises to be a powerful tool to mimic the complex multiscale organization of living tissues including skin, bone, cartilage, muscle, vessels, heart, and liver, among others, with increasing organotypic functionality. With this review, we comprehensively discuss photochemical reactions, photo-activated materials, and their use in state-of-the-art deposition-based (extrusion and droplet) and vat polymerization-based (one-and two-photon) bioprinting. By offering an up-to-date view on these techniques, we identify emerging trends, focusing on both the chemistry and instrument aspects, thereby allowing the readers to select the best-suited approach. Starting with photochemical reactions and photo-activated materials, we then discuss principles, applications, and limitations of each technique. With a critical eye to the most recent achievements, the reader is guided through this exciting, emerging field with special emphasis on cell-laden hydrogel constructs.

wileyonlinelibrary.comwith rapid production of patient-specifi c grafts, allowing precise control over internal and external architecture and customized mechanical properties. These techniques can be used for printing biological materials together with living cells, hence the term "bioprinting." 3D bioprinting offers researchers a unique way of depositing cell-laden biocompatible materials, the so-called bioinks, in high-resolution structures with a line thickness on the order of hundreds of microns. Due to the promise of such a technology, several commercial bioprinters have entered the market and bioinks are the subject of intense investigation. [1][2][3] Bioink formulation is often considered one of the most critical aspects of high-resolution cellular bioprinting.Cellular printing requires a bioink with two key properties, namely printability and cytocompatible crosslinking. The identifi cation of printable polymeric systems is mainly done through rheological evaluation of a material's shear thinning behavior and shear recovery. Shear thinning correlates directly with a bioink's ability to be extruded at low pressure (<3 bar), something which ensures high postprinting cell viability. [ 4 ] Shear recovery, on the other hand, relates to the ink's resistance to fl ow after printing, which ensures high fi delity of the printed structure. The presence of cells, however, greatly restricts the crosslinking options as physiologic temperature and pH need to be maintained and harsh chemicals avoided. Hydrogel bioinks can be crosslinked via covalent or physical interactions or a combination thereof. Ultraviolet light initiated crosslinking of (meth)acrylated polymers has been used most often in bioinks, but the presence of potentially toxic monomers and photoinitiators may complicate clinical translation. [5][6][7][8] Physically crosslinked gelation based on temperature, hydrophobic/hydrophilic or ionic interactions has been utilized for precrosslinking of several bioink materials including poly( N -isopropylacrylamide) conjugated hyaluronan (HA-pNIPAAm), [ 9 ] gelatin, [ 10,11 ] alginate, [ 12 ] and gellan. [ 13 ] Precrosslinking before printing or directly during deposition to stabilize the printed lines is generally followed by a fi nal crosslinking which further increases the mechanical properties and stabilizes the whole structure.For cartilage engineering applications, natural polymers from animal or plant sources including alginate, collagen, gelatin, Bioprinting is an emerging technology for the fabrication of patient-specifi c, anatomically complex tissues and organs. A novel bioink for printing cartilage grafts is developed based on two unmodifi ed FDA-compliant polysaccharides, gellan and alginate, combined with the clinical product BioCartilage (cartilage extracellular matrix particles). Cell-friendly physical gelation of the bioink occurs in the presence of cations, which are delivered by co-extrusion of a cation-loaded transient support polymer to stabilize overhanging structures. Rheological properties of...