Comparison of the Behavior of 3D-Printed Endothelial Cells in Different Bioinks
Biomaterials with characteristics similar to extracellular matrix and with suitable bioprinting properties are essential for vascular tissue engineering. In search for suitable biomaterials, this study investigated the three hydrogels alginate/hyaluronic acid/gelatin (Alg/HA/Gel), pre-crosslinked alginate di-aldehyde with gelatin (ADA-GEL), and gelatin methacryloyl (GelMA) with respect to their mechanical properties and to the survival, migration, and proliferation of human umbilical vein endothelial cells (HUVECs). In addition, the behavior of HUVECs was compared with their behavior in Matrigel. For this purpose, HUVECs were mixed with the inks both as single cells and as cell spheroids and printed using extrusion-based bioprinting. Good printability with shape fidelity was determined for all inks. The rheological measurements demonstrated the gelling consistency of the inks and shear-thinning behavior. Different Young’s moduli of the hydrogels were determined. However, all measured values where within the range defined in the literature, leading to migration and sprouting, as well as reconciling migration with adhesion. Cell survival and proliferation in ADA-GEL and GelMA hydrogels were demonstrated for 14 days. In the Alg/HA/Gel bioink, cell death occurred within 7 days for single cells. Sprouting and migration of the HUVEC spheroids were observed in ADA-GEL and GelMA. Similar behavior of the spheroids was seen in Matrigel. In contrast, the spheroids in the Alg/HA/Gel ink died over the time studied. It has been shown that Alg/HA/Gel does not provide a good environment for long-term survival of HUVECs. In conclusion, ADA-GEL and GelMA are promising inks for vascular tissue engineering.
1. Introduction
As cardio-vascular diseases remain one of the major causes of mortality and morbidity, especially in the context of an ever-aging population, the need for vascular replacement and, ideally, the advent of engineered blood vessels as well as vascular prostheses is increasing. Currently, the options for vascular grafts are still very limited. On the one hand, alloplastic substitutes suffer from a short patency rate, and allogeneic and xenogeneic vessel grafts are immunologically rejected and may hence lead to severe problems, including shrinkage with postoperative stenosis, so the demand for advanced engineered grafts is enormous [1]. However, even synthetic grafts still have shortcomings. Therefore, to overcome these limitations, researchers are constantly searching for new biomaterials and new compositions of materials [2]. The main goal for suitable biomaterials is to mimic the natural tissue to achieve functional and structural tissue formation [3]. Therefore, the ideal condition for cell growth and angiogenesis would be an extracellular matrix (ECM), as found in the human body [4].
To this end, sophisticated in vivo experiments are performed to analyze the angiogenesis of implanted constructs [5]. The ECM is composed of various types of proteins and glycans. It provides structural and biochemical support for cell growth and provides cells with biochemical and biomechanical stimulation so that tissue differentiation and the development of organelles are triggered [3,6]. Even though the building blocks of ECM all have very high biocompatibility, they cannot be manufactured as precisely as synthetic polymers [4]. In 3D bioprinting applications, hydrogel-based inks are used as a matrix to build scaffolds and can be originally composed of synthetic or natural polymers. Synthetic polymers are easy to handle and can tailor their properties to specific applications. However, they are often poorly biocompatible; therefore, natural-based hydrogels are favored. Hence, the inks used for 3D bioprinting applications must be printable and biocompatible and should also possess suitable mechanical and structural properties [1]. Hydrogels are hydrophilic, polymeric networks that can absorb large amounts of water and, due to their soft, rubbery consistency, provide a tissue-like environment for the encapsulated cells [7]. Hydrogels based on alginate dialdehyde (ADA) are ideally suited as materials for tissue engineering due to their biocompatibility, biodegradability, and rapid degradation rates [8].
Since alginate is biocompatible and rapidly ionically gelled, it is suitable for the encapsulation of cells and for biofabrication. The use of alginate is limited by insufficient material-cell interaction and inefficient cell adhesion. However, the incorporation of gelatin by covalent crosslinking with alginate dialdehyde (ADA) can overcome these limitations. Gelatin, which is present in ADA-GEL, is a biodegradable protein produced by acidic or basic hydrolysis of collagen [9]. Gelatin is particularly well suited for vascular tissue engineering because it possesses certain properties that facilitate angiogenesis [10]. For example, gelatin contains integrin-binding motif (RGD) sequences that enable endothelial cells to degrade the hydrogel, migrate, spread, and provide adequate cell attachment [10,11]. As gelatin is produced from collagen, it performs similar cell functions and is also important for cell proliferation and differentiation [12,13]. The potential of ADA-GEL for tissue engineering and biofabrication has already been demonstrated [14,15].
In 2021, Schmid et al. (2021) [7] reported another alginate-gelatin-based ink, which consists of the three components alginate, hyaluronic acid (HA), and gelatin (Alg/HA/Gel). While alginate is a polysaccharide derived from brown algae, HA is a natural glycosaminoglycan found in almost all connective tissues. Since HA is a natural extracellular matrix material that is naturally biocompatible and has water-binding properties, it is suitable as a biopolymer for tissue engineering. HA plays an important role in many cell activities and tissue functions in the body, such as cell migration, proliferation, differentiation, and angiogenesis [16]. The newly developed Alg/HA/Gel ink has been demonstrated to have good printability, high shape fidelity, and high tumor cell survival [7].
In addition, there are also gelatin-based inks that have been modified. For example, the biodegradation of gelatin can be adapted by functionalization with methyl acrylate (MA), resulting in gelatin methacryloyl (GelMA). Since crude gelatin forms a hydrogel, which has low mechanical strength and is liquid in cell culture, a crosslinking chemical is used to improve stiffness [17,18]. The formation of covalently crosslinked hydrogels is achieved by a photoinitiator system under mild conditions, which triggers the formation of free radicals that polymerize the various methacrylamide and methacrylate groups inside the gelatin. Light exposure of the ink in the presence of a photoinitiator increases the stability of the hydrogel by the formation of irreversible chemical crosslinks between the protein chains and results in the encapsulation of the cells, which allows them to be highly viable [10,11,19]. The encapsulation of HUVECs in GelMA as well as the use of GelMA for soft tissue engineering applications have already been successfully demonstrated in the literature [10,11]. In contrast, the encapsulation of HUVECs in the form of single cells and spheroids in the hydrogels Alg/HA/Gel and ADA-GEL for bioprinting has been described little, if at all, in the literature [20]. Therefore, in the following study, small scaffolds from the three inks described were printed incorporating HUVECs with an extrusion-based bioprinter. Extrusion-based bioprinting offers the advantage over conventional hydrogel models that hydrogels and cells can be combined at different concentrations to create macroporous structures and complex 3D architectures [7,21] and can be used for vascular models [22]. Furthermore, extrusion-based bioprinting is characterized by its simplicity, affordability, accuracy, and reproducibility [23].
Here, endothelial cells were printed both as single cells and as cell spheroids in the different hydrogels to create a 3D microenvironment. The mechanical properties, such as the stiffness and degradation behavior of the inks, were analyzed. With respect to a successful printing process, the rheological properties and the printability of the inks were further investigated. To study the behavior of endothelial cells in different inks, single cells were observed for 14 days for their metabolic activity, cell survival, and alignment. The spheroids were studied for 7 days in terms of sprouting, migration, survival, and alignment. To optimally compare the behavior of the cells in the printed constructs, both the single cells and the spheroids were additionally printed in Matrigel.
2.2.1. ADA-GEL
The ADA-GEL was prepared following the protocol of Hazur et al. (2020) [24]. In the first step, 2 mL of a 6.25 wt.% stock solution of ADA (13% degree of oxidation) (prepared from Alginate VIVAPHARM® PH 163 S2 JRS PHARMA GmbH & Co. KG, Rosenberg, Germany) was prepared. For this, 0.125 g of ADA and 2 mL of Dulbecco’s Phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) were homogeneously dissolved in a 50 mL beaker with constant stirring. Meanwhile, a second solution consisting of 6.25 wt.% gelatin (Sigma-Aldrich, St. Louis, MO, USA) in PBS containing 250 mmol/L CaCO3 (Calcium carbonate precipitated for analysis, EMSURE®, Merck KGaA, Darmstadt, Germany) was prepared. For this, 0.188 g gelatin was dissolved in 3 mL PBS on a hot plate with constant stirring. Once the gelatin dissolved completely, 75 mg of CaCO3 was added, and the solution was homogenized for an additional 10 min. Once the stock solution containing the ADA-GEL dissolved completely, 2 mL of the second prepared gelatin/CaCO3 solution was added using a micropipette and stirred for another 20–30 min at 37 °C. Meanwhile, the third and final solution was prepared. To prepare 2 mL of a 250 mmol/L D-Glucono-δ-lactone (GDL, Sigma-Aldrich, St. Louis, MO, USA) solution, 2 mL of ultrapure water containing 89 mg GDL was stirred for 1 min. The solution should be prepared just before the mixing step. An amount of 1 mL of the GDL solution was added dropwise to the ADA-GEL/CaCO3 mixture, and the entire mixture was stirred for 3 h at 37 °C. After the ink has been stirred, it can be carefully transferred with the cells into a cartridge for 3D printing.
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Schulik, J.; Salehi, S.; Boccaccini, A.R.; Schrüfer, S.; Schubert, D.W.; Arkudas, A.; Kengelbach-Weigand, A.; Horch, R.E.; Schmid, R. Comparison of the Behavior of 3D-Printed Endothelial Cells in Different Bioinks. Bioengineering 2023, 10, 751.
https://doi.org/10.3390/bioengineering10070751
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