Porous collagen scaffolds enable endothelial lumen formation in vitro under both static and dynamic growth conditions.

Despite recent advances in the field of tissue engineering, the development of complex tissue-like structures in vitro is compromised by the lack of integration of a functioning vasculature. In this study, we propose a mesoscale three-dimensional (3D) in vitro vascularized connective tissue model and demonstrate its feasibility to prompt the self-assembly of endothelial cells into vessel-like structures. Moreover, we investigate the effect of perfusion on the organization of the cells. For this purpose, primary endothelial cells (HUVECs) and a cell line of human foreskin fibroblasts are cultivated in ECM-like matrices made up of freeze-dried collagen scaffolds permeated with collagen type I hydrogel. A tailored bioreactor is designed to investigate the effect of perfusion on self-organization of HUVECs. Immunofluorescent staining, two-photon microscopy, second-harmonic generation imaging, and scanning electron microscopy are applied to visualize the spatial arrangement of the cells. The analyses reveal the formation of hollow, vessel-like structures of HUVECs in hydrogel-permeated collagen scaffolds under both static and dynamic conditions. In conclusion, we demonstrate the feasibility of a 3D porous collagen scaffolding system that enables and maintains the self-organization of HUVECs into vessel-like structures independent of a dynamic flow.

A Fibrin-Based Human Multicellular Gingival 3D Model Provides Biomimicry and Enables Long-Term In Vitro Studies.

Collagen-type I gels are widely used for the fabrication of 3D in vitro gingival models. Unfortunately, their long-term stability is low, which limits the variety of in vitro applications. To overcome this problem and achieve better hydrolytic stability of 3D gingival models, fibrin-based hydrogel blends with increased long-term stability in vitro are investigated. Two different fibrin-based hydrogels are tested: fibrin 2.5% (w/v) and fibrin 1% (w/v)/gelatin 5% (w/v). Appropriate numbers of primary human gingival fibroblasts (HGFs) and OKG4/bmi1/TERT (OKG) keratinocytes are optimized to achieve a homogeneous distribution of cells under the assumed 3D conditions. Both hydrogels support the viability of HGFs and the stability of the hydrogel over 28 days. In vitro cultivation at the air-liquid interface triggers keratinization of the epithelium and increases its thickness, allowing the formation of multiple tissue-like layers. The presence of HGFs in the hydrogel further enhances epithelial differentiation. In conclusion, a fibrin-based 3D gingival model mimics the histology of native gingiva in vitro and ensures its long-term stability in comparison with the previously reported collagen paralogs. These results open new perspectives for extending the period within which specific biological or pathological conditions of artificial gingival tissue can be evaluated.

Engineering Mesoscopic 3D Tumor Models with a Self-Organizing Vascularized Matrix.

Advanced in vitro systems such as multicellular spheroids and lab-on-a-chip devices have been developed, but often fall short in reproducing the tissue scale and self-organization of human diseases. A bioprinted artificial tumor model is introduced with endothelial and stromal cells self-organizing into perfusable and functional vascular structures. This model uses 3D hydrogel matrices to embed multicellular tumor spheroids, allowing them to grow to mesoscopic scales and to interact with endothelial cells. It is shown that angiogenic multicellular tumor spheroids promote the growth of a vascular network, which in turn further enhances the growth of cocultivated tumor spheroids. The self-developed vascular structure infiltrates the tumor spheroids, forms functional connections with the bioprinted endothelium, and can be perfused by erythrocytes and polystyrene microspheres. Moreover, cancer cells migrate spontaneously from the tumor spheroid through the self-assembled vascular network into the fluid flow. Additionally, tumor type specific characteristics of desmoplasia, angiogenesis, and metastatic propensity are preserved between patient-derived samples and tumors derived from this same material growing in the bioreactors. Overall, this modular approach opens up new avenues for studying tumor pathophysiology and cellular interactions in vitro, providing a platform for advanced drug testing while reducing the need for in vivo experimentation.

Wall shear stress during impingement at the building platform can exceed nozzle wall shear stress in microvalve-based bioprinting.

It is well known that in microvalve-based bioprinting, the cells are subjected to wall shear stress, which can negatively affect their viability rate. We hypothesized that the wall shear stress during impingement at the building platform, hitherto not considered in microvalve-based bioprinting, can be even more critical for the processed cells than the wall shear stress inside the nozzle. To test our hypothesis, we used fluid mechanics numerical simulation based on finite volume method. In addition, viability of two functionally different cell types, HaCaT cell line and primary human umbilical vein endothelial cells (HUVECs), embedded in the cellladen hydrogel was assessed after bioprinting. Simulation results revealed that at low upstream pressure the kinetic energy was not sufficient to overcome the interfacial force for droplet formation and detachment. Oppositely, at relatively mid upstream pressure, a droplet and a ligament were formed, whereas at higher upstream pressure, a jet was formed between nozzle and platform. In the case of jet formation, the shear stress during impingement can exceed the wall shear stress in the nozzle. The amplitude of impingement shear stress depended on nozzle-to- platform distance. This was confirmed by evaluating cell viability which revealed an increase of up to 10% when increasing the nozzle-to-platform distance from 0.3 to 3 mm. In conclusion, the impingement-related shear stress can exceed the wall shear stress in the nozzle in microvalve-based bioprinting. However, this critical issue can be successfully addressed by adapting the distance between the nozzle and the building platform. Altogether, our results highlight impingement-related shear stress as another essential parameter to consider in devising bioprinting strategies.

3D geometry orchestrates the transcriptional landscape of metastatic neuroblastoma cells in a multicellular in vitro bone model.

A key challenge for the discovery of novel molecular targets and therapeutics against pediatric bone metastatic disease is the lack of bona fide in vitro cell models. Here, we show that a beta-tricalcium phosphate (ß-TCP) multicellular 3D in vitro bone microtissue model reconstitutes key phenotypic and transcriptional patterns of native metastatic tumor cells while promoting their stemness and proinvasive features. Comparing planar with interconnected channeled scaffolds, we identified geometry as a dominant orchestrator of proangiogenic traits in neuroblastoma tumor cells. On the other hand, the ß-TCP-determined gene signature was DNA replication related. Jointly, the geometry and chemical impact of ß-TCP revealed a prometastatic landscape of the engineered tumor microenvironment. The proposed 3D multicellular in vitro model of pediatric bone metastatic disease may advance further analysis of the molecular, genetic and metabolic bases of the disease and allow more efficient preclinical target validations.

Bioprinting-associated pulsatile hydrostatic pressure elicits a mild proinflammatory response in epi- and endothelial cells.

During nozzle-based bioprinting, like inkjet and microextrusion, cells are subjected to hydrostatic pressure for up to several minutes. The modality of the bioprinting-related hydrostatic pressure is either constant or pulsatile depending on the technique. We hypothesized that the difference in the modality of hydrostatic pressure affects the biological response of the processed cells differently. To test this, we used a custom-made setup to apply either controlled constant or pulsatile hydrostatic pressure on endothelial and epithelial cells. Neither bioprinting procedure visibly altered the distribution of selected cytoskeletal filaments, cell-substrate adhesions, and cell-cell contacts in either cell type. In addition, pulsatile hydrostatic pressure led to an immediate increase of intracellular ATP in both cell types. However, the bioprinting-associated hydrostatic pressure triggered a pro-inflammatory response in only the endothelial cells, with an increase of interleukin 8 (IL-8) and a decrease of thrombomodulin (THBD) transcripts. These findings demonstrate that the settings adopted during nozzle-based bioprinting cause hydrostatic pressure that can trigger a pro-inflammatory response in different barrier-forming cell types. This response is cell-type and pressure-modality dependent. The immediate interaction of the printed cells with native tissue and the immune system in vivo might potentially trigger a cascade of events. Our findings, therefore, are of major relevance in particular for novel intra-operative, multicellular bioprinting approaches.

Bioprinting-Associated Shear Stress and Hydrostatic Pressure Affect the Angiogenic Potential of Human Umbilical Vein Endothelial Cells.

Bioprinting-associated shear stress and hydrostatic pressure can negatively affect the functionality of dispensed cells. We hypothesized that these mechanical stimuli can potentially affect the angiogenic potential of human umbilical vein endothelial cells (HUVECs). A numerical simulation model was used to calculate the shear stress during microvalve-based droplet ejection. The impact of different levels of applied pressure and the resulting shear stress levels on the angiogenic potential of HUVECs was investigated after up to 14 days of cultivation. In vitro results showed that bioprinting-associated stress not only has short-term but also long-term effects. The short-term viability results indicate a 20% loss in post-printing cell viability in samples printed under the harshest conditions compared to those with the lowest shear stress level. Further, it was revealed that even in two-dimensional culture, HUVECs were able to form a capillary-like network organization regardless of bioprinting pressure. In three-dimensional culture experiments; however, the HUVECs printed at 3 bar were not able to form tubular structures due to their exposure to high shear stress levels. In conclusion, this study provides new insights into how the bioprinting process should be conducted to control printing-associated shear stress and hydrostatic pressure to preserve the functionality and angiogenetic potential of HUVEC.

Multiscale 3D Bioprinting by Nozzle-Free Acoustic Droplet Ejection.

Bioprinting allows the manufacture of complex cell-laden hydrogel constructs that can mature into tissue replacements in subsequent cell culture processes. The nozzles used in currently available bioprinters limit the print resolution and at dimensions below 100 µm clogging is expected. Most critically, the reduction of nozzle diameter also increases shear stress during printing. At critical shear stress, mechanical damage to printed cells triggers cell death. To overcome these limitations, a novel 3D bioprinting method based on the principle of acoustic droplet ejection (ADE) is introduced here. The absence of a nozzle in this method minimizes critical shear stress. A numerical simulation reveals that maximum shear stress during the ADE process is 2.7 times lower than with a Ø150 µm microvalve nozzle. Printing of cell clusters contained in droplets at the millimeter length scale, as well as in droplets the size of a single cell, is feasible. The precise 3D build-up of cell-laden structures is demonstrated and evidence is provided that there are no negative effects on stem cell morphology, proliferation, or differentiation capacities. This multiscale acoustic bioprinting technique thus holds promise for cell-preserving creation of complex and individualized cell-laden 3D hydrogel structures.

A 3D printed in vitro bone model for the assessment of molecular and cellular cues in metastatic neuroblastoma.

Metastasis is a complex and multifactorial process highly dependent on the interaction between disseminated tumor cells and the pre-metastatic niche. The metastatic sites detected in the bone of patients affected by neuroblastoma (NB), a malignancy of the developing sympathetic nervous system, are particularly aggressive. To improve our current knowledge of metastatic tumor cell biology and improve treatment success, appropriate in vitro and in vivo models that more closely resemble the native metastatic niche are needed. In this study, the impact of the geometry of synthetic ß-tricalcium-phosphate (ß-TCP) structures on the interaction of NB tumor cells with the stromal component has been examined. The tumor microenvironment is dynamically shaped by the stroma, which sustains the growth of NB cells inside the metastatic niche. The 3D growth conditions are a determining factor for the cell proliferation rate in ß-TCP. With respect to planar counterparts, channeled 3D ß-TCP structures stimulate more interleukin-6 and Fibronectin production and define Connexin 43 distribution inside the cells. Together, these results highlight how the biomechanical properties of the 3D microenvironment enable tumor cells to form spheroid-shaped arrangements. This, in turn, facilitates their pro-migratory and pro-invasive patterns and mimics the in vivo situation by translating realistic mechanobiological cues to the metastatic NB.

The nuclear import of the transcription factor MyoD is reduced in mesenchymal stem cells grown in a 3D micro-engineered niche.

Smart biomaterials are increasingly being used to control stem cell fate in vitro by the recapitulation of the native niche microenvironment. By integrating experimental measurements with numerical models, we show that in mesenchymal stem cells grown inside a 3D synthetic niche both nuclear transport of a myogenic factor and the passive nuclear diffusion of a smaller inert protein are reduced. Our results also suggest that cell morphology modulates nuclear proteins import through a partition of the nuclear envelope surface, which is a thin but extremely permeable annular portion in cells cultured on 2D substrates. Therefore, our results support the hypothesis that in stem cell differentiation, the nuclear import of gene-regulating transcription factors is controlled by a strain-dependent nuclear envelope permeability, probably related to the reorganization of stretch-activated nuclear pore complexes.