Chemistry matters: A side-by-side comparison of two chemically distinct methacryloylated dECM bioresins for vat photopolymerization.

Decellularized extracellular matrix (dECM) is an excellent natural source for 3D bioprinting materials due to its inherent cell compatibility. In vat photopolymerization, the use of dECM-based bioresins is just emerging, and extensive research is needed to fully exploit their potential. In this study, two distinct methacryloyl-functionalized, photocrosslinkable dECM-based bioresins were prepared from digested porcine liver dECM through functionalization with glycidyl methacrylate (GMA) or conventional methacrylic anhydride (MA) under mild conditions for systematic comparison. Although the chemical modifications did not significantly affect the structural integrity of the dECM proteins, mammalian cells encapsulated in the respective hydrogels performed differently in long-term culture. In either case, photocrosslinking during 3D (bio)printing resulted in transparent, highly swollen, and soft hydrogels with good shape fidelity, excellent biomimetic properties and tunable mechanical properties (~ 0.2-2.5 kPa). Interestingly, at a similar degree of functionalization (DOF ~ 81.5-83.5 %), the dECM-GMA resin showed faster photocrosslinking kinetics in photorheology resulting in lower final stiffness and faster enzymatic biodegradation compared to the dECM-MA gels, yet comparable network homogeneity as assessed via Brillouin imaging. While human hepatic HepaRG cells exhibited comparable cell viability directly after 3D bioprinting within both materials, cell proliferation and spreading were clearly enhanced in the softer dECM-GMA hydrogels at a comparable degree of crosslinking. These differences were attributed to the additional hydrophilicity introduced to dECM via methacryloylation through GMA compared to MA. Due to its excellent printability and cytocompatibility, the functional porcine liver dECM-GMA biomaterial enables the advanced biofabrication of soft 3D tissue analogs using vat photopolymerization-based bioprinting.

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

Rise of tissue- and species-specific 3D bioprinting based on decellularized extracellular matrix-derived bioinks and bioresins.

Thanks to its natural complexity and functionality, decellularized extracellular matrix (dECM) serves as an excellent foundation for creating highly cell-compatible bioinks and bioresins. This enables the bioprinted cells to thrive in an environment that closely mimics their native ECM composition and offers customizable biomechanical properties. To formulate dECM bioinks and bioresins, one must first pulverize and/or solubilize the dECM into non-crosslinked fragments, which can then be chemically modified as needed. In bioprinting, the solubilized dECM-derived material is typically deposited and/or crosslinked in a layer-by-layer fashion to build 3D hydrogel structures. Since the introduction of the first liver-derived dECM-based bioinks, a wide variety of decellularized tissue have been employed in bioprinting, including kidney, heart, cartilage, and adipose tissue among others. This review aims to summarize the critical steps involved in tissue-derived dECM bioprinting, starting from the decellularization of the ECM to the standardized formulation of bioinks and bioresins, ultimately leading to the reproducible bioprinting of tissue constructs. Notably, this discussion also covers photocrosslinkable dECM bioresins, which are particularly attractive due to their ability to provide precise spatiotemporal control over the gelation in bioprinting. Both in extrusion printing and vat photopolymerization, there is a need for more standardized protocols to fully harness the unique properties of dECM-derived materials. In addition to mammalian tissues, the most recent bioprinting approaches involve the use of microbial extracellular polymeric substances in bioprinting of bacteria. This presents similar challenges as those encountered in mammalian cell printing and represents a fascinating frontier in bioprinting technology.

In vitrovascularization of hydrogel-based tissue constructs via a combined approach of cell sheet engineering and dynamic perfusion cell culture.

The bioengineering of artificial tissue constructs requires special attention to their fast vascularization to provide cells with sufficient nutrients and oxygen. We addressed the challenge ofin vitrovascularization by employing a combined approach of cell sheet engineering, 3D printing, and cellular self-organization in dynamic maturation culture. A confluent cell sheet of human umbilical vein endothelial cells (HUVECs) was detached from a thermoresponsive cell culture substrate and transferred onto a 3D-printed, perfusable tubular scaffold using a custom-made cell sheet rolling device. Under indirect co-culture conditions with human dermal fibroblasts (HDFs), the cell sheet-covered vessel mimic embedded in a collagen gel together with additional singularized HUVECs started sprouting into the surrounding gel, while the suspended cells around the tube self-organized and formed a dense lumen-containing 3D vascular network throughout the gel. The HDFs cultured below the HUVEC-containing cell culture insert provided angiogenic support to the HUVECs via molecular crosstalk without competing for space with the HUVECs or inducing rapid collagen matrix remodeling. The resulting vascular network remained viable under these conditions throughout the 3 week cell culture period. This static indirect co-culture setup was further transferred to dynamic flow conditions, where the medium perfusion was enabled via two independently addressable perfusion circuits equipped with two different cell culture chambers, one hosting the HDFs and the other hosting the HUVEC-laden collagen gel. Using this system, we successfully connected the collagen-embedded HUVEC culture to a dynamic medium flow, and within 1 week of the dynamic cell culture, we detected angiogenic sprouting and dense microvascular network formation via HUVEC self-organization in the hydrogel. Our approach of combining a 3D-printed and cell sheet-covered vascular precursor that retained its sprouting capacity together with the self-assembling HUVECs in a dynamic perfusion culture resulted in a vascular-like 3D network, which is a critical step toward the long-term vascularization of bioengineeredin vitrotissue constructs.

Flow-induced glycocalyx formation and cell alignment of HUVECs compared to iPSC-derived ECs for tissue engineering applications.

The relevance of cellular in vitro models highly depends on their ability to mimic the physiological environment of the respective tissue or cell niche. Static culture conditions are often unsuitable, especially for endothelial models, since they completely neglect the physiological surface shear stress and corresponding reactions of endothelial cells (ECs) such as alignment in the direction of flow. Furthermore, formation and maturation of the glycocalyx, the essential polysaccharide layer covering all endothelial surfaces and regulating diverse processes, is highly dependent on applied fluid flow. This fragile but utterly important macromolecular layer is hard to analyze, its importance is often underestimated and accordingly neglected in many endothelial models. Therefore, we exposed human umbilical vein ECs (HUVECs) and human induced pluripotent stem cell-derived ECs (iPSC-ECs) as two relevant EC models in a side-by-side comparison to static and physiological dynamic (6.6 dyn cm-2) culture conditions. Both cell types demonstrated an elongation and alignment along the flow direction, some distinct changes in glycocalyx composition on the surface regarding the main glycosaminoglycan components heparan sulfate, chondroitin sulfate or hyaluronic acid as well as an increased and thereby improved glycocalyx thickness and functionality when cultured under homogeneous fluid flow. Thus, we were able to demonstrate the maturity of the employed iPSC-EC model regarding its ability to sense fluid flow along with the general importance of physiological shear stress for glycocalyx formation. Additionally, we investigated EC monolayer integrity with and without application of surface shear stress, revealing a comparable existence of tight junctions for all conditions and a reorganization of the cytoskeleton upon dynamic culture leading to an increased formation of focal adhesions. We then fabricated cell sheets of EC monolayers after static and dynamic culture via non-enzymatic detachment using thermoresponsive polymer coatings as culture substrates. In a first proof-of-concept we were able to transfer an aligned iPSC-EC sheet to a 3D-printed scaffold thereby making a step in the direction of vascular modelling. We envision these results to be a valuable contribution to improvements of in vitro endothelial models and vascular engineering in the future.

3D-Cultured Vascular-Like Networks Enable Validation of Vascular Disruption Properties of Drugs In Vitro.

Vascular-disrupting agents are an interesting class of anticancer compounds because of their combined mode of action in preventing new blood vessel formation and disruption of already existing vasculature in the immediate microenvironment of solid tumors. The validation of vascular disruption properties of these drugs in vitro is rarely addressed due to the lack of proper in vitro angiogenesis models comprising mature and long-lived vascular-like networks. We herein report an indirect coculture model of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) to form three-dimensional profuse vascular-like networks. HUVECs embedded and sandwiched in the collagen scaffold were cocultured with HDFs located outside the scaffold. The indirect coculture approach with the vascular endothelial growth factor (VEGF) producing HDFs triggered the formation of progressively maturing lumenized vascular-like networks of endothelial cells within less than 7 days, which have proven to be viably maintained in culture beyond day 21. Molecular weight-dependent Texas red-dextran permeability studies indicated high vascular barrier function of the generated networks. Their longevity allowed us to study the dose-dependent response upon treatment with the three known antiangiogenic and/or vascular disrupting agents brivanib, combretastatin A4 phosphate (CA4P), and 6´-sialylgalactose (SG) via semi-quantitative brightfield and qualitative confocal laser scanning microscopic (CLSM) image analysis. Compared to the reported data on in vivo efficacy of these drugs in terms of antiangiogenic and vascular disrupting effects, we observed similar trends with our 3D model, which are not reflected in conventional in vitro angiogenesis assays. High-vascular disruption under continuous treatment of the matured vascular-like network was observed at concentrations ≥3.5 ng·ml-1 for CA4P and ≥300 nM for brivanib. In contrast, SG failed to induce any significant vascular disruption in vitro. This advanced model of a 3D vascular-like network allows for testing single and combinational antiangiogenic and vascular disrupting effects with optimized dosing and may thus bridge the gap between the in vitro and in vivo experiments in validating hits from high-throughput screening. Moreover, the physiological 3D environment mimicking in vitro assay is not only highly relevant to in vivo studies linked to cancer but also to the field of tissue regeneration.

Bioprinted Multi-Cell Type Lung Model for the Study of Viral Inhibitors.

Influenza A virus (IAV) continuously causes epidemics and claims numerous lives every year. The available treatment options are insufficient and the limited pertinence of animal models for human IAV infections is hampering the development of new therapeutics. Bioprinted tissue models support studying pathogenic mechanisms and pathogen-host interactions in a human micro tissue environment. Here, we describe a human lung model, which consisted of a bioprinted base of primary human lung fibroblasts together with monocytic THP-1 cells, on top of which alveolar epithelial A549 cells were printed. Cells were embedded in a hydrogel consisting of alginate, gelatin and collagen. These constructs were kept in long-term culture for 35 days and their viability, expression of specific cell markers and general rheological parameters were analyzed. When the models were challenged with a combination of the bacterial toxins LPS and ATP, a release of the proinflammatory cytokines IL-1ß and IL-8 was observed, confirming that the model can generate an immune response. In virus inhibition assays with the bioprinted lung model, the replication of a seasonal IAV strain was restricted by treatment with an antiviral agent in a dose-dependent manner. The printed lung construct provides an alveolar model to investigate pulmonary pathogenic biology and to support development of new therapeutics not only for IAV, but also for other viruses.

Physiological Shear Stress Enhances Differentiation, Mucus-Formation and Structural 3D Organization of Intestinal Epithelial Cells In Vitro.

Gastrointestinal (GI) mucus plays a pivotal role in the tissue homoeostasis and functionality of the gut. However, due to the shortage of affordable, realistic in vitro GI models with a physiologically relevant mucus layer, studies with deeper insights into structural and compositional changes upon chemical or physical manipulation of the system are rare. To obtain an improved mucus-containing cell model, we developed easy-to-use, reusable culture chambers that facilitated the application of GI shear stresses (0.002-0.08 dyn∙cm-2) to cells on solid surfaces or membranes of cell culture inserts in bioreactor systems, thus making them readily accessible for subsequent analyses, e.g., by confocal microscopy or transepithelial electrical resistance (TEER) measurement. The human mucus-producing epithelial HT29-MTX cell-line exhibited superior reorganization into 3-dimensional villi-like structures with highly proliferative tips under dynamic culture conditions when compared to static culture (up to 180 vs. 80 µm in height). Additionally, the median mucus layer thickness was significantly increased under flow (50 ± 24 vs. 29 ± 14 µm (static)), with a simultaneous accelerated maturation of the cells into a goblet-like phenotype. We demonstrated the strong impact of culture conditions on the differentiation and reorganization of HT29-MTX cells. The results comprise valuable advances towards the improvement of existing GI and mucus models or the development of novel systems using our newly designed culture chambers.

Development of GelMA/PCL and dECM/PCL resins for 3D printing of acellular in vitro tissue scaffolds by stereolithography.

Gelatin methacryloyl (GelMA) is a chemically modified extracellular matrix (ECM)-derived biopolymer that is widely used for 3D fabrication of tissue engineering scaffolds. However, its tendency for physical gelation limits its use in aqueous 3D printing resins to low concentrations, yielding a poor printing resolution in stereolithography (SLA). To obtain a GelMA-based resin that can be printed into high-resolution tissue scaffolds, we formulated resins of fish and porcine-derived GelMA in formamide using GelMA alone or mixed with star-shaped poly(ε-caprolactone) methacrylate (PCL-MA). We identified GelMA resins and GelMA/PCL-MA hybrid resins with a ratio of 70/30 wt-% to yield a suitable viscosity for SLA at 32 °C and demonstrated the resolution of the new resins in SLA by 3D printing acellular human small intestine-mimicking tissue scaffolds. The presence of PCL-MA in the hybrid resins improved the 3D printing fidelity compared to the neat GelMA resins, while GelMA provided the hybrid materials with enhanced swelling and proliferation of seeded cells. We further demonstrated the transferability of our resin formulation to native organ-derived materials by successfully replacing GelMA in the hybrid resin with solubilized, methacryloyl-functionalized decellularized liver ECM (dECM-MA) and by 3D printing multi-layer dECM/PCL-MA hydrogels.

Coassembly Generates Peptide Hydrogel with Wound Dressing Material Properties.

Multicomponent self-assembly of peptides is a powerful strategy to fabricate novel functional materials with synergetic properties that can be used for several nanobiotechnological applications. In the present study, we used a coassembly strategy to generate an injectable ultrashort bioactive peptide hydrogel formed by mixing a dipeptide hydrogelator with a macrophage attracting short chemotactic peptide ligand. Coassembly does not impede hydrogelation as shown by cryo-transmission electron microscopy (cryo-TEM), scanning electron microscopy, and rheology. Biocompatibility was shown by cytotoxicity assays and confocal microscopy. The hydrogels release the entrapped skin antibiotic ciprofloxacin, among others, in a slow and continuous manner. Such bioinspired advanced functional materials can find applications as wound dressing materials to treat chronic wound conditions like diabetic foot ulcer.

Generation of a 3D Liver Model Comprising Human Extracellular Matrix in an Alginate/Gelatin-Based Bioink by Extrusion Bioprinting for Infection and Transduction Studies.

Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional (3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.

Optimization of cell-laden bioinks for 3D bioprinting and efficient infection with influenza A virus.

Bioprinting is a new technology, which arranges cells with high spatial resolution, but its potential to create models for viral infection studies has not yet been fully realized. The present study describes the optimization of a bioink composition for extrusion printing. The bioinks were biophysically characterized by rheological and electron micrographic measurements. Hydrogels consisting of alginate, gelatin and Matrigel were used to provide a scaffold for a 3D arrangement of human alveolar A549 cells. A blend containing 20% Matrigel provided the optimal conditions for spatial distribution and viability of the printed cells. Infection of the 3D model with a seasonal influenza A strain resulted in widespread distribution of the virus and a clustered infection pattern that is also observed in the natural lung but not in two-dimensional (2D) cell culture, which demonstrates the advantage of 3D printed constructs over conventional culture conditions. The bioink supported viral replication and proinflammatory interferon release of the infected cells. We consider our strategy to be paradigmatic for the generation of humanized 3D tissue models by bioprinting to study infections and develop new antiviral strategies.

Additive Manufacturing of Vascular Grafts and Vascularized Tissue Constructs.

There is a great need for engineered vascular grafts among patients with cardiovascular diseases who are in need of bypass therapy and lack autologous healthy blood vessels. In addition, because of the severe worldwide shortage of organ donors, there is an increasing need for engineered vascularized tissue constructs as an alternative to organ transplants. Additive manufacturing (AM) offers great advantages and flexibility of fabrication of cell-laden, multimaterial, and anatomically shaped vascular grafts and vascularized tissue constructs. Various inkjet-, extrusion-, and photocrosslinking-based AM techniques have been applied to the fabrication of both self-standing vascular grafts and porous, vascularized tissue constructs. This review discusses the state-of-the-art research on the use of AM for vascular applications and the key criteria for biomaterials in the AM of both acellular and cellular constructs. We envision that new smart printing materials that can adapt to their environment and encourage rapid endothelialization and remodeling will be the key factor in the future for the successful AM of personalized and dynamic vascular tissue applications.

Three-dimensional fabrication of cell-laden biodegradable poly(ethylene glycol-co-depsipeptide) hydrogels by visible light stereolithography.

Stereolithography (SLA) holds great promise in fabrication of cell-laden hydrogels with biomimetic complexity for use in tissue engineering and pharmaceutics. However, the availability of biodegradable photocrosslinkable hydrogel polymers for SLA is very limited. In this study, a water-soluble methacrylated poly(ethylene glycol-co-depsipeptide) was synthesized to yield a biodegradable photocrosslinkable macromer for SLA. Structural analysis confirmed the inclusion of biodegradable peptide and ester groups and photocrosslinkable methacrylate groups into the polymer backbone. The new macromer combined with RGDS peptide was used for SLA fabrication of hydrogels in absence and presence of cells. With the increasing light exposure time in SLA, mechanical stiffness of the hydrogels increased from 3 ± 1 kPa to 38 ± 13 kPa. Total mass loss of the samples within 7 days in PBS was 13%-21% and within 24 days 35%-66%. Due to degradation, the mechanical stiffness decreased by one order magnitude within 7-day incubation in PBS. Encapsulated endothelial cells proliferated in the hydrogels during 10-day in vitro cell culturing study. The macromer was further used in SLA to fabricate bifurcating tubular structures as preliminary vessel grafts. The new biodegradable, photocrosslinkable polymer is a significant addition to the very limited material selection currently available for SLA-based fabrication of cell-laden tissue engineering constructs.

Novel additive manufactured scaffolds for tissue engineered trachea research.

CONCLUSIONS: This study demonstrates proof of concept for controlled manufacturing methods that utilize novel tailored biopolymers (3D photocuring technology) or conventional bioresorbable polymers (fused deposition modeling, FDM) for macroscopic and microscopic geometry control. The manufactured scaffolds could be suitable for tissue engineering research. OBJECTIVES: To design novel trachea scaffold prototypes for tissue engineering purposes, and to fabricate them by additive manufacturing. METHODS: A commercial 3D model and CT scans of a middle-aged man were obtained for geometrical observations and measurements of human trachea. Model trachea scaffolds with variable wall thickness, interconnected pores, and various degrees of porosity were designed. Photocurable polycaprolactone (PCL) polymer was used with 3D photocuring technology. Thermoplastic polylactide (PLA) and PCL were used with FDM. Cell cultivations were performed for biocompatibility studies. RESULTS: Scaffolds of various sizes and porosities were successfully produced. Both thermoplastic PLA and PCL and photocurable PCL could be used effectively with additive manufacturing technologies to print high-quality tubular porous biodegradable structures. Optical microscopic and SEM images showed the viability of cells. The cells were growing in multiple layers, and biocompatibility of the structures was shown.

Preparation of poly(ε-caprolactone)-based tissue engineering scaffolds by stereolithography.

A photocrosslinkable poly(ε-caprolactone) (PCL)-based resin was developed and applied using stereolithography. No additional solvents were required during the structure preparation process. Three-armed PCL oligomers of varying molecular weights were synthesized, functionalized with methacrylic anhydride, and photocrosslinked, resulting in high gel content networks. Stereolithography was used to build designed porous scaffolds using the resin containing PCL macromer, Irgacure 369 photoinitiator, inhibitor and dye. A suitable resin viscosity was obtained by heating the resin during the curing process. The scaffolds precisely matched the computer-aided designs, with no observable material shrinkage. The average porosity was 70.5 ± 0.8%, and the average pore size was 465 µm. The pore network was highly interconnected. The photocrosslinkable, biodegradable PCL resin is well suited for the solvent-free fabrication of tissue engineering scaffolds by stereolithography.

Porous biodegradable scaffold: predetermined porosity by dissolution of poly(ester-anhydride) fibers from polyester matrix.

A novel selective leaching method for the porogenization of the biodegradable scaffolds was developed. Continuous, predetermined pore structure was prepared by dissolving fast eroding poly(epsilon-caprolactone)-based poly(ester-anhydride) fibers from the photo-crosslinked poly(epsilon-caprolactone) matrix. The porogen fibers dissolved in the phosphate buffer (pH 7.4, 37 degrees C) within a week, resulting in the porosity that replicated exactly the single fiber dimensions and the overall arrangement of the fibers. The amount of the porosity, estimated with micro-CT, corresponded with the initial amount of the fibers. The potential to include bioactive agents in the porogen fibers was demonstrated with the bioactive glass.