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A novel approach, in the context of bioprinting, is the targeted printing of a defined number of cells at desired positions in predefined locations, which thereby opens up new perspectives for life science engineering. One major challenge in this application is to realize the targeted printing of cells onto a gel substrate with high cell survival rates in advanced bioinks. For this purpose, different alginate-dialdehyde—polyethylene glycol (ADA-PEG) inks with different PEG modifications and chain lengths (1–8 kDa) were characterized to evaluate their application as bioinks for drop on demand (DoD) printing. The biochemical properties of the inks, printing process, NIH/3T3 fibroblast cell distribution within a droplet and shear forces during printing were analyzed. Finally, different hydrogels were evaluated as a printing substrate. By analysing different PEG chain lengths with covalently crosslinked and non-crosslinked ADA-PEG inks, it was shown that the influence of Schiff’s bases on the viscosity of the corresponding materials is very low. Furthermore, it was shown that longer polymer chains resulted in less stable hydrogels, leading to fast degradation rates. Several bioinks highly exhibit biocompatibility, while the calculated nozzle shear stress increased from approx. 1.3 and 2.3 kPa. Moreover, we determined the number of cells for printed droplets depending on the initial cell concentration, which is crucially needed for targeted cell printing approaches.

In 3D bioprinting for cartilage regeneration, bioinks that support chondrogenic development are of key importance. Growth factors covalently bound in non-printable hydrogels have been shown to effectively promote chondrogenesis. However, studies that investigate the functionality of tethered growth factors within 3D printable bioinks are still lacking. Therefore, in this study, we established a dual-stage crosslinked hyaluronic acid-based bioink that enabled covalent tethering of transforming growth factor-beta 1 (TGF-β1). Bone marrow-derived mesenchymal stromal cells (MSCs) were cultured over three weeks in vitro, and chondrogenic differentiation of MSCs within bioink constructs with tethered TGF-β1 was markedly enhanced, as compared to constructs with non-covalently incorporated TGF-β1. This was substantiated with regard to early TGF-β1 signaling, chondrogenic gene expression, qualitative and quantitative ECM deposition and distribution, and resulting construct stiffness. Furthermore, it was successfully demonstrated, in a comparative analysis of cast and printed bioinks, that covalently tethered TGF-β1 maintained its functionality after 3D printing. Taken together, the presented ink composition enabled the generation of high-quality cartilaginous tissues without the need for continuous exogenous growth factor supply and, thus, bears great potential for future investigation towards cartilage regeneration. Furthermore, growth factor tethering within bioinks, potentially leading to superior tissue development, may also be explored for other biofabrication applications.

Hyaluronic acid (HA)-based hydrogels are very commonly applied as cell carriers for different approaches in regenerative medicine. HA itself is a well-studied biomolecule that originates from the physiological extracellular matrix (ECM) of mammalians and, due to its acidic polysaccharide structure, offers many different possibilities for suitable chemical modifications which are necessary to control, for example, network formation. Most of these chemical modifications are performed using the free acid function of the polymer and, additionally, lead to an undesirable breakdown of the biopolymer’s backbone. An alternative modification of the vicinal diol of the glucuronic acid is oxidation with sodium periodate to generate dialdehydes via a ring opening mechanism that can subsequently be further modified or crosslinked via Schiff base chemistry. Since this oxidation causes a structural destruction of the polysaccharide backbone, it was our intention to study a novel synthesis protocol frequently applied to selectively oxidize the C6 hydroxyl group of saccharides. On the basis of this TEMPO/TCC oxidation, we studied an alternative hydrogel platform based on oxidized HA crosslinked using adipic acid dihydrazide as the crosslinker.

Biofabrication, including printing technologies, has emerged as a powerful approach to the design of disease models, such as in cancer research. In breast cancer, adipose tissue has been acknowledged as an important part of the tumor microenvironment favoring tumor progression. Therefore, in this study, a 3D-printed breast cancer model for facilitating investigations into cancer cell-adipocyte interaction was developed. First, we focused on the printability of human adipose-derived stromal cell (ASC) spheroids in an extrusion-based bioprinting setup and the adipogenic differentiation within printed spheroids into adipose microtissues. The printing process was optimized in terms of spheroid viability and homogeneous spheroid distribution in a hyaluronic acid-based bioink. Adipogenic differentiation after printing was demonstrated by lipid accumulation, expression of adipogenic marker genes, and an adipogenic ECM profile. Subsequently, a breast cancer cell (MDA-MB-231) compartment was printed onto the adipose tissue constructs. After nine days of co-culture, we observed a cancer cell-induced reduction of the lipid content and a remodeling of the ECM within the adipose tissues, with increased fibronectin, collagen I and collagen VI expression. Together, our data demonstrate that 3D-printed breast cancer-adipose tissue models can recapitulate important aspects of the complex cell–cell and cell–matrix interplay within the tumor-stroma microenvironment.

Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering.

Bioprinting offers the opportunity to fabricate precise 3D tumor models to study tumor pathophysiology and progression. However, the choice of the bioink used is important. In this study, cell behavior was studied in three mechanically and biologically different hydrogels (alginate, alginate dialdehyde crosslinked with gelatin (ADA–GEL), and thiol-modified hyaluronan (HA-SH crosslinked with PEGDA)) with cells from breast cancer (MDA-MB-231 and MCF-7) and melanoma (Mel Im and MV3), by analyzing survival, growth, and the amount of metabolically active, living cells via WST-8 labeling. Material characteristics were analyzed by dynamic mechanical analysis. Cell lines revealed significantly increased cell numbers in low-percentage alginate and HA-SH from day 1 to 14, while only Mel Im also revealed an increase in ADA–GEL. MCF-7 showed a preference for 1% alginate. Melanoma cells tended to proliferate better in ADA–GEL and HA-SH than mammary carcinoma cells. In 1% alginate, breast cancer cells showed equally good proliferation compared to melanoma cell lines. A smaller area was colonized in high-percentage alginate-based hydrogels. Moreover, 3% alginate was the stiffest material, and 2.5% ADA–GEL was the softest material. The other hydrogels were in the same range in between. Therefore, cellular responses were not only stiffness-dependent. With 1% alginate and HA-SH, we identified matrices that enable proliferation of all tested tumor cell lines while maintaining expected tumor heterogeneity. By adapting hydrogels, differences could be accentuated. This opens up the possibility of understanding and analyzing tumor heterogeneity by biofabrication.

Dual setting cements composed of an in situ forming hydrogel and a reactive mineral phase combine high compressive strength of the cement with sufficient ductility and bending strength of the polymeric network. Previous studies were focused on the modification with non-degradable hydrogels based on 2-hydroxyethyl methacrylate (HEMA). Here, we describe the synthesis of suitable triblock degradable poly(ethylene glycol)-poly(lactide) (PEG-PLLA) cross-linker to improve the resorption capacity of such composites. A study with four different formulations was established. As reference, pure hydroxyapatite (HA) cements and composites with 40 wt% HEMA in the liquid cement phase were produced. Furthermore, HEMA was modified with 10 wt% of PEG-PLLA cross-linker or a test series containing only 25% cross-linker was chosen for composites with a fully degradable polymeric phase. Hence, we developed suitable systems with increased elasticity and 5–6 times higher toughness values in comparison to pure inorganic cement matrix. Furthermore, conversion rate from α-tricalcium phosphate (α-TCP) to HA was still about 90% for all composite formulations, whereas crystal size decreased. Based on this material development and advancement for a dual setting system, we managed to overcome the drawback of brittleness for pure calcium phosphate cements.

In this study, we evaluate hydrogels based on oxidized hyaluronic acid, cross-linked with adipic acid dihydrazide, for their suitability as bioinks for 3D bioprinting. Aldehyde containing hyaluronic acid (AHA) is synthesized and cross-linked via Schiff Base chemistry with bifunctional adipic acid dihydrazide (ADH) to form a mechanically stable hydrogel with good printability. Mechanical and rheological properties of the printed and casted hydrogels are tunable depending on the concentrations of AHA and ADH cross-linkers.

Abstract Purpose  In cases of severe retinal diseases, the vitreous body has to be removed and replaced by a suitable biomaterial. Currently, however, no satisfying long-term vitreous substitute is in clinical use. A novel therapeutic concept represents the combination of hyalocytes with suitable biomaterials. The goal of the present study was to evaluate the potential of bFGF and TGF-β1 as tools to control hyalocyte proliferation and the accumulation of extracellular matrix (ECM). Methods  In vitro investigation on the influence of different concentrations of bFGF and TGF-β1 on hyalocyte morphology, proliferation and ECM production. Results  Both growth factors affected hyalocyte morphology; small, round cells could be observed after bFGF supplementation, whereas the cells appeared more completely spread when cultured with TGF-β1. Hyalocyte proliferation was increased 3-fold by 10 ng/ml bFGF; 1 ng/ml TGF-β1 in contrast reduced cell proliferation to about 40% of the control. Converse effects of the growth factors could also be observed on the ECM accumulation of hyalocytes; whereas bFGF halved ECM accumulation, TGF-β1 enhanced the ECM production up to 3-fold. Precultivation of hyalocytes with bFGF for two passages had no influence on their subsequent accumulation of glycosaminoglycans (GAG). However, cells precultivated with bFGF exhibited a doubled accumulation of collagen compared to controls. Conclusions  The observed opposite effects of bFGF and TGF-β1 on hyalocyte proliferation and ECM accumulation may allow for the control of hyaloycte properties. Therefore, these two growth factors seem to be valuable tools towards the development of a cell-based vitreous substitute. [ABSTRACT FROM AUTHOR]