Bioprinting of Synthetic Cell-like Lipid Vesicles to Augment the Functionality of Tissues after Manufacturing.

Bioprinting is an automated bioassembly method that enables the formation of human tissue-like constructs to restore or replace damaged tissues. Regardless of the employed bioprinting method, cells undergo mechanical stress that can impact their survival and function postprinting. In this study, we investigate the use of a synthetic cell-like unit, giant unilamellar vesicles (GUVs), as adjuvants of the cellular function of human cells postprinting, or in future as the complete replacement of human cells. We analyzed the impact of two nozzle-based bioprinting methods (drop-on-demand and extrusion bioprinting) on the structure, stability, and function of GUVs. We showed that over 65% of the GUVs remain intact when printing at 0.5 bar, demonstrating the potential of using GUVs as a synthetic cell source. We further increased the stability of GUVs in a cell culture medium by introducing polyethylene glycol (PEG) into the GUV lipid membrane. The presence of PEG, however, diminished the structural properties of GUVs postprinting, and reduced the interaction of GUVs with human cells. Although the design of PEG-GUVs can still be modified in future studies for better cell-GUV interactions, we demonstrated that GUVs are functional postprinting. Chlorin e6-PEG-GUVs loaded with a fluorescent dye were bioprinted, and they released the dye postprinting only upon illumination. This is a new strategy to deliver carriers, such as growth factors, drugs, nutrients, or gases, inside large bioprinted specimens on a millimeter to centimeter scale. Overall, we showed that printed GUVs can augment the functionality of manufactured human tissues.

SARS-CoV-2 particles promote airway epithelial differentiation and ciliation.

Introduction: The Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which caused the coronavirus disease 2019 (COVID-19) pandemic, enters the human body via the epithelial cells of the airway tract. To trap and eject pathogens, the airway epithelium is composed of ciliated and secretory cells that produce mucus which is expelled through a process called mucociliary clearance. Methods: This study examines the early stages of contact between SARS-CoV-2 particles and the respiratory epithelium, utilizing 3D airway tri-culture models exposed to ultraviolet light-irradiated virus particles. These cultures are composed of human endothelial cells and human tracheal mesenchymal cells in a fibrin hydrogel matrix covered by mucociliated human tracheal epithelial cells. Results: We found that SARS-CoV-2 particles trigger a significant increase in ciliation on the epithelial surface instructed through a differentiation of club cells and basal stem cells. The contact with SARS-CoV-2 particles also provoked a loss of cell-cell tight junctions and impaired the barrier integrity. Further immunofluorescence analyses revealed an increase in FOXJ1 expression and PAK1/2 phosphorylation associated with particle-induced ciliation. Discussion: An understanding of epithelial responses to virus particles may be instrumental to prevent or treat respiratory infectious diseases such as COVID-19.

Hypotrochoidal scaffolds for cartilage regeneration.

The main function of articular cartilage is to provide a low friction surface and protect the underlying subchondral bone. The extracellular matrix composition of articular cartilage mainly consists of glycosaminoglycans and collagen type II. Specifically, collagen type II fibers have an arch-like organization that can be mimicked with segments of a hypotrochoidal curve. In this study, a script was developed that allowed the fabrication of scaffolds with a hypotrochoidal design. This design was investigated and compared to a regular 0-90 woodpile design. The mechanical analyses revealed that the hypotrochoidal design had a lower component Young's modulus while the toughness and strain at yield were higher compared to the woodpile design. Fatigue tests showed that the hypotrochoidal design lost more energy per cycle due to the damping effect of the unique microarchitecture. In addition, data from cell culture under dynamic stimulation demonstrated that the collagen type II deposition was improved and collagen type X reduced in the hypotrochoidal design. Finally, Alcian blue staining revealed that the areas where the stress was higher during the stimulation produced more glycosaminoglycans. Our results highlight a new and simple scaffold design based on hypotrochoidal curves that could be used for cartilage tissue engineering.

Human platelet lysate as a replacement for fetal bovine serum in human corneal stromal keratocyte and fibroblast culture.

The isolation and propagation of primary human corneal stromal keratocytes (CSK) are crucial for cellular research and corneal tissue engineering. However, this delicate cell type easily transforms into stromal fibroblasts (SF) and scar inducing myofibroblasts (Myo-SF). Current protocols mainly rely on xenogeneic fetal bovine serum (FBS). Human platelet lysate (hPL) could be a viable, potentially autologous, alternative. We found high cell survival with both supplements in CSK and SF. Cell numbers and Ki67+ ratios increased with higher fractions of hPL and FBS in CSK and SF. We detected a loss in CSK marker expression (Col8A2, ALDH3A1 and LUM) with increasing fractions of FBS and hPL in CSK and SF. The expression of the Myo-SF marker SMA increased with higher amounts of FBS but decreased with incremental hPL substitution in both cell types, implying an antifibrotic effect of hPL. Immunohistochemistry confirmed the RT-PCR findings. bFGF and HGF were only found in hPL and could be responsible for suppressing the Myo-SF conversion. Considering all findings, we propose 0.5% hPL as a suitable substitution in CSK culture, as this xeno-free component efficiently preserved CSK characteristics, with non-inferiority in terms of cell viability, cell number and proliferation in comparison to the established 0.5% FBS protocol.

Digitally Fabricated and Naturally Augmented In Vitro Tissues.

Human in vitro tissues are extracorporeal 3D cultures of human cells embedded in biomaterials, commonly hydrogels, which recapitulate the heterogeneous, multiscale, and architectural environment of the human body. Contemporary strategies used in 3D tissue and organ engineering integrate the use of automated digital manufacturing methods, such as 3D printing, bioprinting, and biofabrication. Human tissues and organs, and their intra- and interphysiological interplay, are particularly intricate. For this reason, attentiveness is rising to intersect materials science, medicine, and biology with arts and informatics. This report presents advances in computational modeling of bioink polymerization and its compatibility with bioprinting, the use of digital design and fabrication in the development of fluidic culture devices, and the employment of generative algorithms for modeling the natural and biological augmentation of in vitro tissues. As a future direction, the use of serially linked in vitro tissues as human body-mimicking systems and their application in drug pharmacokinetics and metabolism, disease modeling, and diagnostics are discussed.

Prospects and Challenges of Translational Corneal Bioprinting.

Corneal transplantation remains the ultimate treatment option for advanced stromal and endothelial disorders. Corneal tissue engineering has gained increasing interest in recent years, as it can bypass many complications of conventional corneal transplantation. The human cornea is an ideal organ for tissue engineering, as it is avascular and immune-privileged. Mimicking the complex mechanical properties, the surface curvature, and stromal cytoarchitecure of the in vivo corneal tissue remains a great challenge for tissue engineering approaches. For this reason, automated biofabrication strategies, such as bioprinting, may offer additional spatial control during the manufacturing process to generate full-thickness cell-laden 3D corneal constructs. In this review, we discuss recent advances in bioprinting and biomaterials used for in vitro and ex vivo corneal tissue engineering, corneal cell-biomaterial interactions after bioprinting, and future directions of corneal bioprinting aiming at engineering a full-thickness human cornea in the lab.

Bioprinting Cell- and Spheroid-Laden Protein-Engineered Hydrogels as Tissue-on-Chip Platforms.

Human tissues, both in health and disease, are exquisitely organized into complex three-dimensional architectures that inform tissue function. In biomedical research, specifically in drug discovery and personalized medicine, novel human-based three-dimensional (3D) models are needed to provide information with higher predictive value compared to state-of-the-art two-dimensional (2D) preclinical models. However, current in vitro models remain inadequate to recapitulate the complex and heterogenous architectures that underlie biology. Therefore, it would be beneficial to develop novel models that could capture both the 3D heterogeneity of tissue (e.g., through 3D bioprinting) and integrate vascularization that is necessary for tissue viability (e.g., through culture in tissue-on-chips). In this proof-of-concept study, we use elastin-like protein (ELP) engineered hydrogels as bioinks for constructing such tissue models, which can be directly dispensed onto endothelialized on-chip platforms. We show that this bioprinting process is compatible with both single cell suspensions of neural progenitor cells (NPCs) and spheroid aggregates of breast cancer cells. After bioprinting, both cell types remain viable in incubation for up to 14 days. These results demonstrate a first step toward combining ELP engineered hydrogels with 3D bioprinting technologies and on-chip platforms comprising vascular-like channels for establishing functional tissue models.

Hand-held bioprinting for de novo vascular formation applicable to dental pulp regeneration.

Aim of the study: Deep carious lesions may cause irreversible pulpitis and the current endodontic treatment typically removes the whole dental pulp tissue, which finally reduces lifespan of the teeth. Nowadays, the most frequent treatment is based on removing the infected tissue and filling the root canal with inert synthetic materials. Tissue engineering approaches are important alternatives to the current treatment, because they can potentially maintain the biological function of the tooth instead of sacrificing it.Materials and Methods: In this study, we propose a tissue engineering approach based on a hand-held in situ bioprinting strategy. Our approach enabled bioprinting of cell-loaded collagen-based bioinks with suitable rheological, structural and biological properties, which allowed for vasculogenesis in the root canal.Results: The rheological properties of the bioprintable bioink were measured by oscillatory amplitude sweep testing and were corroborated by macroscopic evaluation after in vitro culture, in which printed bioinks maintained their original form without contraction. Moreover, we showed evidence for successful vasculogenesis in bioprintable bioinks with comparable quality and quantity to control fibrin and collagen non-bioprintable hydrogels.Conclusions: We conclude that hand-held bioprinting holds potential for in situ treatment of dental diseases with successful evidence for vascular tube formation, as an asset for maintenance of the biological function of the tooth.

Smart Bioinks as de novo Building Blocks to Bioengineer Living Tissues.

In vitro tissues and 3D in vitro models have come of age [...].

Corneal bioprinting utilizing collagen-based bioinks and primary human keratocytes.

Corneal transplantation is the treatment of choice for patients with advanced corneal diseases. However, the outcome may be affected by graft rejection, high associated costs, surgical expertise, and most importantly the worldwide donor shortage. In recent years, bioprinting has emerged as an alternative method for fabricating tissue equivalents using autologous cells with architecture resembling the native tissue. In this study, we propose a freeform and cell-friendly drop-on-demand bioprinting strategy for creating corneal stromal 3D models as suitable implants. Corneal stromal keratocytes (CSK) were bioprinted in collagen-based bioinks as 3D biomimetic models and the geometrical outcome as well as the functionality of the bioprinted specimens were evaluated after in vitro culture. We showed that our bioprinting method is feasible to fabricate translucent corneal stromal equivalents with optical properties similar to native corneal stromal tissue, as proved by optical coherence tomography. Moreover, the bioprinted CSK were viable after the bioprinting process and maintained their native keratocyte phenotypes after 7 days in in vitro culture, as shown by immunocytochemistry. The proposed bioprinted human 3D corneal models can potentially be used clinically for patients with corneal stromal diseases.

Exploring Cancer Cell Behavior In Vitro in Three-Dimensional Multicellular Bioprintable Collagen-Based Hydrogels.

In vitro cancer 3D models are valuable tools to provide mechanistic insight into solid tumor growth, invasion, and drug delivery. The 3D spheroid model of solid tumors has been the most popular cancer model in use until now. However, previous studies have shown that these spheroid models lack sufficient morphological parameters, which may affect their response to chemicals. In this work, we proposed the fabrication of miniaturized 3D cancer models using collagen type I-based bioprintable bioinks. In the context of a mimicking model for advanced neuroblastoma studies, we showed that cancer cells contained in bioprintable bioinks formed Homer Wright-like rosettes, maintained their proliferative capacities and produced an equivalent Vimentin-rich matrix unlike that of non-bioprintable bioinks which made for poorer models. In addition, bioprintable bioinks were successfully bioprinted as compartmentalized 3D models in the centimeter scale, which was not feasible using non-bioprintable bioinks. In contrast to non-bioprintable hydrogels, we did not observe contraction in their bioprintable counterparts, which is an advantage for prospective 3D bioprinted models that should attain stable rheological and mechanical properties after bioprinting. By adopting this proposed system for the use of patient-derived primary tumor cells, the approach could be introduced as a first line strategy in precision medicine for testing the response of neuroblastoma cells to drugs, especially when disease progresses rapidly or patients do not respond to actual therapy regimens.

Incorporating 4D into Bioprinting: Real-Time Magnetically Directed Collagen Fiber Alignment for Generating Complex Multilayered Tissues.

In vitro multilayered tissues with mimetic architectures resembling native tissues are valuable tools for application in medical research. In this study, an advanced bioprinting strategy is presented for aligning collagen fibers contained in functional bioinks. Streptavidin-coated iron nanoparticles are embedded in printable bioinks with varying concentrations of low gelling temperature agarose and type I collagen. By applying a straightforward magnetic-based mechanism in hydrogels during bioprinting, it is possible to align collagen fibers in less concentrated hydrogel blends with a maximum agarose concentration of 0.5 w/v%. Conversely, more elevated concentrations of agarose in printable blends show random collagen fiber distribution. Interestingly, hydrogel blends with unidirectionally aligned collagen fibers show significantly higher compression moduli compared to hydrogel blends including random fibers. Considering its application in the field of cartilage tissue engineering, bioprinted constructs with alternating layers of aligned and random fibers are fabricated. After 21 days of culture, cell-loaded constructs with alternating layers of aligned and random fibers express markedly more collagen II in comparison to solely randomly oriented fiber constructs. These encouraging results translate the importance of the structure and architecture of bioinks used in bioprinting in light of their use for tissue engineering and personalized medical applications.

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 (ß-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 ß-TCP show the highest alkaline phosphatase expression compared to blends without ß-TCP and stiffer composites with 5 mg mL-1 ß-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 ß-TCP compared to blends without ß-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.

Three-Dimensional Printing and Angiogenesis: Tailored Agarose-Type I Collagen Blends Comprise Three-Dimensional 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.

Bioprinting Organotypic Hydrogels with Improved Mesenchymal Stem Cell Remodeling and Mineralization Properties for Bone Tissue Engineering.

3D-manufactured hydrogels with precise contours and biological adhesion motifs are interesting candidates in the regenerative medicine field for the culture and differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). 3D-bioprinting is a powerful technique to approach one step closer the native organization of cells. This study investigates the effect of the incorporation of collagen type I in 3D-bioprinted polysaccharide-based hydrogels to the modulation of cell morphology, osteogenic remodeling potential, and mineralization. By combining thermo-responsive agarose hydrogels with collagen type I, the mechanical stiffness and printing contours of printed constructs can be improved compared to pure collagen hydrogels which are typically used as standard materials for MSC osteogenic differentiation. The results presented here show that MSC not only survive the 3D-bioprinting process but also maintain the mesenchymal phenotype, as proved by live/dead staining and immunocytochemistry (vimentin positive, CD34 negative). Increased solids concentrations of collagen in the hydrogel blend induce changes in cell morphology, namely, by enhancing cell spreading, that ultimately contribute to enhanced and directed MSC osteogenic differentiation. 3D-bioprinted agarose-collagen hydrogels with high-collagen ratio are therefore feasible for MSC osteogenic differentiation, contrarily to low-collagen blends, as proved by two-photon microscopy, Alizarin Red staining, and real-time polymerase chain reaction.

Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity.

A microvalve-based bioprinting system for the manufacturing of high-resolution, multimaterial 3D-structures is reported. Applying a straightforward fluid-dynamics model, the shear stress at the nozzle site can precisely be controlled. Using this system, a broad study on how cell viability and proliferation potential are affected by different levels of shear stress is conducted. Complex, multimaterial 3D structures are printed with high resolution. This work pioneers the investigation of shear stress-induced cell damage in 3D bioprinting and might help to comprehend and improve the outcome of cell-printing studies in the future.

Adhesion of human mesenchymal stem cells can be controlled by electron beam-microstructured titanium alloy surfaces during osteogenic differentiation.

Several studies focusing on bone tissue engineering demonstrated that given microstructuring of an implant surface has a strong effect on its interaction with cells, and their adhesion and differentiation. In the present study, geometrically structured titanium alloy surfaces are shown to be able to guide cell adhesion during differentiation in vitro. For this reason, using an electron beam texturing technique, TiAl6V4 surfaces were selectively targeted in the micrometer range. The effect of such textured titanium alloy surfaces on cell adhesion during osteogenic differentiation was analyzed for human mesenchymal stem cells (MSC), the natural precursor cells of bone tissue. Cytotoxicity, cell viability and differentiation were analyzed. Immunofluorescence stainings demonstrated that in contrast to MSC in an expansion medium, MSC in an osteogenic induction medium produce adhesion proteins such as ß3-integrins and thereby connect in an oriented way to the generated microstructures on titanium alloy surfaces. These results are of relevance for developing tailored titanium alloy implant surfaces which exhibit an improved cell response.

The stiffness and structure of three-dimensional printed hydrogels direct the differentiation of mesenchymal stromal cells toward adipogenic and osteogenic lineages.

The mechanical and physicochemical effects of three-dimensional (3D) printable hydrogels on cell behavior are paramount features to consider before manufacturing functional tissues. We hypothesize that besides good printability and cytocompatibility of a supporting hydrogel for the manufacture of individual tissues, it is equally essential to consider beforehand the desired tissue (bone, cartilage, fat). In light of its application, the structure and stiffness of printable hydrogel matrices influence cell geometry, which in turn impacts the differentiation fate. Embedded human mesenchymal stromal cells in printable type I collagen- and chitosan-agarose blends were induced to differentiate toward osteoblasts and adipocytes. Hydrogels' printability in air versus submerged printing in perfluorocarbon was evaluated according to the height, diameter, uniformity, and stability of 3D printed vertical cylinders. Bipotent differentiation within hydrogels was assessed histologically (morphology, cellularity), by immunohistochemistry (vimentin, smooth muscle actin), two-photon microscopy (spatial distribution), and real-time polymerase chain reaction (ALP, BGLAP, OPN, RUNX2, COL 1, aP2, PPARγ-2). Agarose and agarose blends revealed the most valid printability properties by generating uniform cylinders with an average height of 4 mm. Osteogenic differentiation was preferably achieved in anisotropic soft collagen-rich substrates, whereas adipogenic differentiation mostly occurred in isotropic stiff agarose-rich matrices. The conjugation of type I collagen to agarose with varying ratios is possibly a suitable bioink for a broad range of 3D printed mesenchymal tissues.

Two-photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage-, IL-4 activated macrophage- and osteoclast-based in vitro degradation of beta-tricalcium phosphate bone substitute material.

Two-photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two-photon laser scanning microscopy. Beta-tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin-4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta-tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL-4 activated macrophages. An average of ~32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL-4 activated macrophages. For the first time by applying two-photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design.

Biofabrication under fluorocarbon: a novel freeform fabrication technique to generate high aspect ratio tissue-engineered constructs.

Bioprinting is a recent development in tissue engineering, which applies rapid prototyping techniques to generate complex living tissues. Typically, cell-containing hydrogels are dispensed layer-by-layer according to a computer-generated three-dimensional model. The lack of mechanical stability of printed hydrogels hinders the fabrication of high aspect ratio constructs. Here we present submerged bioprinting, a novel technique for freeform fabrication of hydrogels in liquid fluorocarbon. The high buoyant density of fluorocarbons supports soft hydrogels by floating. Hydrogel constructs of up to 30-mm height were generated. Using 3% (w/v) agarose as the hydrogel and disposable syringe needles as nozzles, the printer produced features down to 570-µm diameter with a lateral dispensing accuracy of 89 µm. We printed thin-walled hydrogel cylinders measuring 4.8 mm in height, with an inner diameter of ∼2.9 mm and a minimal wall thickness of ∼650 µm. The technique was successfully applied in printing a model of an arterial bifurcation. We extruded under fluorocarbon, cellularized alginate tubes with 5-mm outer diameter and 3-cm length. Cells grew vigorously and formed clonal colonies within the 7-day culture period. Submerged bioprinting thus seems particularly suited to fabricate hollow structures with a high aspect ratio like vascular grafts for cardiovascular tissue engineering as well as branching or cantilever-like structures, obviating the need for a solid support beneath the overhanging protrusions.