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A facile and flexible approach for the integration of biomimetically branched microvasculature within bulk hydrogels is presented. For this, sacrificial scaffolds of thermoresponsive poly(2-cyclopropyl-2-oxazoline) (PcycloPrOx) are created using melt electrowriting (MEW) in an optimized and predictable way and subsequently placed into a customized bioreactor system, which is then filled with a hydrogel precursor solution. The aqueous environment above the lower critical solution temperature (LCST) of PcycloPrOx at 25 °C swells the polymer without dissolving it, resulting in fusion of filaments that are deposited onto each other (print-and-fuse approach). Accordingly, an adequate printing pathway design results in generating physiological-like branchings and channel volumes that approximate Murray's law in the geometrical ratio between parent and daughter vessels. After gel formation, a temperature decrease below the LCST produces interconnected microchannels with distinct inlet and outlet regions. Initial placement of the sacrificial scaffolds in the bioreactors in a pre-defined manner directly yields perfusable structures via leakage-free fluid connections in a reproducible one-step procedure. Using this approach, rapid formation of a tight and biologically functional endothelial layer, as assessed not only through fluorescent dye diffusion, but also by tumor necrosis factor alpha (TNF-α) stimulation, is obtained within three days.

3D bioprinting often involves application of highly concentrated polymeric bioinks to enable fabrication of stable cell-hydrogel constructs, although poor cell survival, compromised stem cell differentiation, and an inhomogeneous distribution of newly produced extracellular matrix (ECM) is frequently observed. Therefore, this study presents a bioink platform utilizing a new versatile dual-stage crosslinking approach based on thiolated hyaluronic acid (HA-SH), which not only provides stand-alone 3D printability, but also facilitates effective chondrogenic differentiation of mesenchymal stromal cells (MSCs). A range of HA-SH with different molecular weights is synthesized and crosslinked with acrylated (PEG-diacryl) and allylated (PEG-diallyl) polyethylene glycol in a two-step reaction scheme. The initial Michael addition is used to achieve ink printability, followed by UV-mediated thiol-ene reaction to stabilize the printed bioink for long-term cell culture. Bioinks with high molecular weight HA-SH (> 200 kDa) require comparably low polymer content to facilitate bioprinting. This leads to superior quality of cartilaginous constructs which possess a coherent ECM and a strongly increased stiffness of long-term cultured constructs. The dual-stage system may serve as an example to design platforms utilizing two independent crosslinking reactions at one functional group, which allows to adjust printability as well as material and biological properties of bioinks. This article is protected by copyright. All rights reserved.

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.

Pancreatic cancer is a devastating malignancy with minimal treatment options. Standard-of-care therapy, including surgery and chemotherapy, is unsatisfactory, and therapies harnessing the immune system have been unsuccessful in clinical trials. Resistance to therapy and disease progression are mediated by the tumor microenvironment, which contains excessive amounts of extracellular matrix and stromal cells, acting as a barrier to drug delivery. There is a lack of pre-clinical pancreatic cancer models that reconstruct the extracellular, cellular, and biomechanical elements of tumor tissues to assess responses toward immunotherapy. To address this limitation and explore the effects of immunotherapy in combination with chemotherapy, we developed a multicellular 3D cancer model using a star-shaped poly(ethylene glycol)-heparin hydrogel matrix. Human pancreatic cancer cells, cancer-associated fibroblasts, and myeloid cells were grown encapsulated in hydrogels to mimic key components of tumor tissues, and cell responses toward treatment were assessed. Combining the CD11b agonist ADH-503 with anti-PD-1 immunotherapy and chemotherapy led to a significant reduction in tumor cell viability, proliferation, metabolic activity, immunomodulation, and secretion of immunosuppressive and tumor growth-promoting cytokines. This article is protected by copyright. All rights reserved.

With the continuous growth of extrusion bioprinting techniques, ink formulations based on rheology modifiers are becoming increasingly popular, as they enable 3D printing of non-printable biologically-favored materials. However, benchmarking and characterization of such systems are inherently complicated due to the variety of rheology modifiers and differences in mechanisms of inducing printability. This study tries to explain induced printability in formulations by incorporating machine learning algorithms that describe the underlying basis for decision-making in classifying a printable formulation. For this purpose, a library of rheological data and printability scores for 180 different formulations of hyaluronic acid solutions with varying molecular weights and concentrations and three rheology modifiers were produced. A feature screening methodology was applied to collect and separate the impactful features, which consisted of physically interpretable and easily measurable properties of formulations. In the final step, all relevant features influencing the model’s output were analyzed by advanced yet explainable statistical methods. The outcome provides a guideline for designing new formulations based on data-driven correlations from multiple systems.

Emerging biomanufacturing technologies have revolutionized the field of tissue engineering by offering unprecedented possibilities. Over the past few years, new opportunities arose by combining traditional and novel fabrication techniques, shaping the hybrid designs in biofabrication. One of the potential application fields is skin tissue engineering, in which a combination of traditional principles of wound dressing with advanced biofabrication methods could yield more efficient therapies. In this study, a hybrid design of fiber-reinforced scaffolds combined with gel casting is developed and the efficiency for in vivo wound healing applications is assessed. For this purpose, 3D fiber meshes produced by melt electrowriting are selectively filled with photocrosslinkable gelatin hydrogel matrices loaded with different growth factor carrier microspheres. Additionally, the influence of the inclusion of inorganic bioactive glass particles within the composite fibrous mesh is evaluated. Qualitative evaluation of secondary wound healing criteria and histological analysis shows that hybrid scaffolds containing growth factors and bioactive glass enhances the healing process significantly, compared to the designs merely providing a fiber-reinforced bioactive hydrogel matrix as the wound dressing. This study aims to explore a new application area for melt electrowriting as a powerful tool in fabricating hybrid therapeutic designs for skin tissue engineering.

Bioprinting has emerged as a valuable three-dimensional (3D) biomanufacturing method to fabricate complex hierarchical cell-containing constructs. Spanning from basic research to clinical translation, sterile starting materials are crucial. In this study, we present pharmacopeia compendial sterilization methods for the commonly used bioink component alginate. Autoclaving (sterilization in saturated steam) and sterile filtration followed by lyophilization as well as the pharmacopeia noncompendial method, ultraviolet (UV)-irradiation for disinfection, were assessed. The impact of the sterilization methods and their effects on physicochemical and rheological properties, bioprinting outcome, and sterilization efficiency of alginate were detailed. Only sterile filtration followed by lyophilization as the sterilization method retained alginate's physicochemical properties and bioprinting behavior while resulting in a sterile outcome. This set of methods provides a blueprint for the analysis of sterilization effects on the rheological and physicochemical pattern of bioink components and is easily adjustable for other polymers used in the field of biofabrication in the future.

Fabrication of microchannels using 3D printing of sugars as fugitive material is explored in different fields, including microfluidics. However, establishing reproducible methods for the controlled production of sugar structures with sub-100 μm dimensions remains a challenge. This study pioneers the processing of sugars by melt electrowriting (MEW) enabling the fabrication of structures with so far unprecedented resolution from Isomalt. Based on a systematic variation of process parameters, fibers with diameters down to 20 μm can be fabricated. The flexibility in the adjustment of fiber diameter by on-demand alteration of MEW parameters enables generating constructs with perfusable channels within polydimethylsiloxane molds. These channels have a diameter that can be adjusted from 30 to 200 μm in a single design. Taken together, the experiments show that MEW strongly benefits from the thermal and physical stability of Isomalt, providing a robust platform for the fabrication of small-diameter embedded microchannel systems.

Modern techniques for expanding stem cells play a substantial role in tissue engineering: the raw material that facilitates regeneration of damaged tissues and treats diseases. The environmental conditions and bioprocessing methods are the primary determinants of the rate of cultured stem cell proliferation. Bioceramic scaffolds made of calcium phosphate are effective substrates for optimal cell proliferation. The present study investigates the effects of two bioceramic scaffolds on proliferating cells in culture media. One scaffold was made of hydroxyapatite and the other was a mixture of hydroxyapatite and ferromagnetic material (Fe3O4 nanoparticles). Disk-shaped (10 mm × 2 mm) samples of the two scaffolds were prepared. Primary human fibroblast proliferation was 1.8- and 2.5-fold faster, respectively, when cultured in the presence of hydroxyapatite or ferrous nanoparticle/hydroxyapatite mixtures. Optical microscopy images revealed that the increased proliferation was due to enhanced cell-cell contact. The presence of magnetic Fe3O4 nanoparticles in the ceramic scaffolds significantly increased cell proliferation compared to hydroxyapatite scaffolds and tissue culture polystyrene.

In 3D bioprinting, bioinks with high concentrations of polymeric materials are frequently used to enable fabrication of 3D cell-hydrogel constructs with sufficient stability. However, this is often associated with restricted cell bioactivity and an inhomogeneous distribution of newly produced extracellular matrix (ECM). Therefore, this study investigates bioink compositions based on hyaluronic acid (HA), an attractive material for cartilage regeneration, which allow for reduction of polymer content. Thiolated HA and allyl-modified poly(glycidol) in varying concentrations are UV-crosslinked. To adapt bioinks to poly(ε-caprolactone) (PCL)-supported 3D bioprinting, the gels are further supplemented with 1 wt% unmodified high molecular weight HA (hmHA) and chondrogenic differentiation of incorporated human mesenchymal stromal cells is assessed. Strikingly, addition of hmHA to gels with a low polymer content (3 wt%) results in distinct increase of construct quality with a homogeneous ECM distribution throughout the constructs, independent of the printing process. Improved ECM distribution in those constructs is associated with increased construct stiffness after chondrogenic differentiation, as compared to higher concentrated constructs (10 wt%), which only show pericellular matrix deposition. The study contributes to effective bioink development, demonstrating dual function of a supplement enabling PCL-supported bioprinting and at the same time improving biological properties of the resulting constructs.

Three-dimensional (3D) bioprinting enables the controlled fabrication of complex constructs for tissue engineering applications and has been actively explored in recent years. However, its progress has been limited by the existing difficulties in the development of bioinks with suitable biocompatibility and mechanical properties and at the same time adaptability to the process. Herein, we describe the engineering of a nanocomposite agarose bioink with tailored properties using 2D nanosilicate additives. The suitability of agarose for 3D bioprinting has been debated due to its bioinert nature and compatibility with extrusion-based bioprinting. Nanosilicates were used to tailor the flow behavior of agarose solutions, and detailed rheological characterization of different bioink formulations was performed to elucidate the effect of nanosilicates on the flow behavior and gelation of agarose bioinks. The proper selection of nanosilicate concentration resulted in extrusion 3D printed structures with high shape fidelity and structural integrity. Moreover, the influence of addition of nanosilicates on the bioactivity of agarose was studied, and nanocomposite bioinks showed significant improvement in metabolic activity of encapsulated cells. The bioactivity of the nanocomposite bioinks was also evaluated through a cell spreading assay; the charged surfaces of nanosilicates resulted in full spreading and elongation of fibroblasts, and the extent of change in morphology of cells was found to be directly correlated with the nanosilicate concentration. Our findings suggested that engineered agarose-nanosilicate bioinks can be exploited as a new generation of hydrogel bioinks for extrusion 3D bioprinting with tunable flow properties and bioactivity.

Development of the biofabrication technologies is mainly hindered by the lack of bioinks which satisfy the criteria for both bioprinting and tissue engineering. Hydrogels as bioinks, are the main cell carrier in the process of bioprinting. However, the adaptation of physio-chemical properties of hydrogel bioinks to the specific requirements of 3D bioprinting process demands selection criteria which includes the biological activity of bioinks as well as mechanical and structural stability throughout the process. Natural hydrogels usually do not comply with the process of bioprinting in terms of crosslinking conditions, mechanical stability, and flow properties. To overcome the major drawbacks of natural hydrogel in adaptation with 3D bioprinting process, synergistic tuning of bioactivity and flow properties by synthesis of nanocomposite hydrogels was introduced. Agarose as a naturally derived polysaccharide has been mostly used as the support structure in 3D bioprinting. Although agarose hydrogels show high viscosity and shape fidelity after extrusion, they lack the necessary cell binding sites in their backbone and thus have limited bioactivity. Additionally, the thermal crosslinking mechanism of gelation in agarose is not compatible with extrusion 3D bioprinting. Laponite nanosilicates showed to be an effective additive being able to induce noteworthy effects on flow behavior and bioactivity of hydrogel matrix. The promising effects of incorporation of Laponite in different hydrogel matrices suggests its potential in improvement of flow behavior and bioactivity of agarose hydrogels to meet the criteria for extrusion 3D bioprinting. We hypothesized that inclusion of Laponite nanosilicates within agarose hydrogels will improve the bioactivity of the hydrogel, while the flow behavior of agarose can be altered at the same time to adapt with the extrusion 3D bioprinting.

This paper reports the synthesis of nanocomposite agarose hydrogels with improved bioactivity with the incorporation of anisotropic 2D nanosilicates (Laponite) to promote cell binding, growth and proliferation. Rheological measurements showed that the incorporation of nanosilicates slightly increased the gelation temperature (T-gel). The use of higher nanosilicate content at the constant agarose concentration improved the mechanical properties of the gels. Due to the non-swelling nature of agarose, the addition of nanosilicates did not result in any remarkable change in the swelling properties of the agarose gels, while collapsed agarose nanofibers were observed with the incorporation of nanosilicates. EDX analysis confirmed the presence of the embedded nanosilicates in the gel matrix. The existence of physical interactions between nanosilicate and agarose was demonstrated by FTIR over the shifting of Si-O stretching band to a lower frequency. The encapsulated NIH/3T3 fibroblast cells showed enhanced proliferation and spreading in the presence of nanosilicates.

Using decellularized extracellular matrix (dECM) hydrogels as bioinks has been an important step forward for bioprinting of functional tissue constructs, considering their rich microenvironment and their high degree of biomimicry. However, directly using dECM hydrogels as bioinks may not be suitable for bioprinting processes because of the loss of shape fidelity and geometrical precision of bioprinted structure due to their slow gelation kinetics. In this article, the development and direct bioprinting of dECM hydrogel bioink from bovine Achilles tendon were presented. The developed bioink is used for a microcapillary-based bioprinting process without any support structure and/or any additional cross-linker components. The reported decellularization and solubilization methods yield dECM pre-gels which turn into stable hydrogels in a short time at physiological conditions. The gelation kinetics and mechanical strength of bioinks with different concentrations and digestion times are characterized. A support structure-free 3D bioprinting of the developed bioink is shown by aspirating dECM bioinks and then in situ gelation and extrusion through a fine microcapillary nozzle. The viability assays indicate that the developed dECM bioink has no cytotoxic effect on encapsulated NIH 3T3 cells and the cells show lineage-specific morphology in the early days of culture as well.

Nanocrystalline merwinite was successfully synthesized by mechanical activation of a mixture of magnesium hydroxide, calcium oxide and silica gel powders followed by heat treatment. The phase development and various physical properties were investigated as a function of milling time using X-ray diffraction, scanning electron microscopy, simultaneous thermal analysis, particle size analysis and transmission electron microscopy. The results showed that formation of merwinite in annealed powders was enhanced by increasing the milling time. Nanocrystalline merwinite powder with an average crystallite size of about 36 nm was obtained after 30 hours of ball milling and subsequent annealing in 900 °C for 1 hour.

Multimaterial additive manufacturing or three-dimensional (3D) printing of hydrogel structures provides the opportunity to engineer geometrically dependent functionalities. However, current fabrication methods are mostly limited to one type of material or only provide one type of functionality. In this paper, we report a novel method of multimaterial deposition of hydrogel structures based on an aspiration-on-demand protocol, in which the constitutive multimaterial segments of extruded filaments were first assembled in liquid state by sequential aspiration of inks into a glass capillary, followed by in situ gel formation. We printed different patterned objects with varying chemical, electrical, mechanical, and biological properties by tuning process and material related parameters, to demonstrate the abilities of this method in producing heterogeneous and multi-functional hydrogel structures. Our results show the potential of proposed method in producing heterogeneous objects with spatially controlled functionalities while preserving structural integrity at the switching interface between different segments. We anticipate that this method would introduce new opportunities in multimaterial additive manufacturing of hydrogels for diverse applications such as biosensors, flexible electronics, tissue engineering and organ printing.

Collocations are linguistic phenomenon, which are notably proliferative in all languages. The definitions of collocations provided by linguists are numerous and varied. While some emphasize the multi-element structure of a collocation, other scholars admit only those composed pairs of words. Since one of the most significant features of great poets’ works is using Quranic collocations, the present study addresses the distinct features of these collocations in Orchard (Boostan of Sa’di as one of the greatest poet of 12-13 AD). In order to estimate the historical and semantic origin of lexical collocation, some translations of Orchard couplets embodying Quranic collocations were chosen both semantically and historically were evaluated.