3D bioprinting of liver models: A systematic scoping review of methods, bioinks, and reporting quality.

Background: Effective communication is crucial for broad acceptance and applicability of alternative methods in 3R biomedical research and preclinical testing. 3D bioprinting is used to construct intricate biological structures towards functional liver models, specifically engineered for deployment as alternative models in drug screening, toxicological investigations, and tissue engineering. Despite a growing number of reviews in this emerging field, a comprehensive study, systematically assessing practices and reporting quality for bioprinted liver models is missing. Methods: In this systematic scoping review we systematically searched MEDLINE (Ovid), EMBASE (Ovid) and BioRxiv for studies published prior to June 2nd, 2022. We extracted data on methodological conduct, applied bioinks, the composition of the printed model, performed experiments and model applications. Records were screened for eligibility and data were extracted from included articles by two independent reviewers from a panel of seven domain experts specializing in bioprinting and liver biology. We used RAYYAN for the screening process and SyRF for data extraction. We used R for data analysis, and R and Graphpad PRISM for visualization. Results: Through our systematic database search we identified 1042 records, from which 63 met the eligibility criteria for inclusion in this systematic scoping review. Our findings revealed that extrusion-based printing, in conjunction with bioinks composed of natural components, emerged as the predominant printing technique in the bioprinting of liver models. Notably, the HepG2 hepatoma cell line was the most frequently employed liver cell type, despite acknowledged limitations. Furthermore, 51% of the printed models featured co-cultures with non-parenchymal cells to enhance their complexity. The included studies offered a variety of techniques for characterizing these liver models, with their primary application predominantly focused on toxicity testing. Among the frequently analyzed liver markers, albumin and urea stood out. Additionally, Cytochrome P450 (CYP) isoforms, primarily CYP3A and CYP1A, were assessed, and select studies employed nuclear receptor agonists to induce CYP activity. Conclusion: Our systematic scoping review offers an evidence-based overview and evaluation of the current state of research on bioprinted liver models, representing a promising and innovative technology for creating alternative organ models. We conducted a thorough examination of both the methodological and technical facets of model development and scrutinized the reporting quality within the realm of bioprinted liver models. This systematic scoping review can serve as a valuable template for systematically evaluating the progress of organ model development in various other domains. The transparently derived evidence presented here can provide essential support to the research community, facilitating the adaptation of technological advancements, the establishment of standards, and the enhancement of model robustness. This is particularly crucial as we work toward the long-term objective of establishing new approach methods as reliable alternatives to animal testing, with extensive and versatile applications.

Xeno-Free 3D Bioprinted Liver Model for Hepatotoxicity Assessment.

Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85-92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86-92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97-101%), akin to a Matrigel-based liver model (83-102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.

Cross-Linked Chitosan/Gelatin Beads Loaded with Chlorella vulgaris Microalgae/Zinc Oxide Nanoparticles for Adsorbing Carcinogenic Bisphenol-A Pollutant from Water.

Water polluted by phenolic compounds is a global threat to health and the environment; accordingly, we prepared a green novel sorbent biological system from a chitosan (CS), gelatin (GT), and Chlorella vulgaris freshwater microalgae (m-Alg) composite impregnated with zinc oxide nanoparticles (ZnO-NPs) for the remediation of bisphenol-A (BPA) from water. C. vulgaris was selected to be one of the constituents of the prepared composite because of its high capability in phytoremediation. The morphology and the structure of CS/GT*m-Alg/ZnO beads were characterized by SEM, FTIR, XRD, and TGA. Different monitoring experimental conditions, such as contact time, pH, BPA concentration, and sorbent dosage, were optimized. The optimum conditions for the adsorption process showed outstanding removal efficiency toward BPA at pH 4.0, contact time 40.0 min, and 40.0 mg L-1 BPA initial concentration. Langmuir, Freundlich, and Temkin isotherm models have been studied for adsorption equilibrium, and the best fit is described by the Langmuir adsorption isotherm. The adsorption kinetics has been studied using pseudo-first-order (PFO), pseudo-second-order (PSO), Elovich, and intraparticle diffusion (IPD) models. The pseudo-second-order kinetic model shows the optimum experimental fit. The monolayer adsorption capacity of the prepared CS/GT*m-Alg/ZnO for BPA was determined to be 38.24 mg g-1. The prepared CS/GT*m-Alg/ZnO beads show advantageous properties, such as their high surface area, high adsorption capacity, reusability, and cost-effectiveness.

Fabrication of Polyaniline@ß-cyclodextrin Nanocomposite for Adsorption of Carcinogenic Phenol from Wastewater.

We synthesized a stable, eco-friendly, and low-cost polyaniline@ß-cyclodextrin (PANI@ß-CD) nanocomposite via oxidative polymerization for phenol adsorption from water waste since phenol pollution is a global danger to human and animal health and the environment. The production of the composite and synergistic alteration of PANI with ß-CD resulted in 66% reduction in particle size from 59 nm (PANI) to 20 nm (PANI@ß-CD) as well as better phenol adsorption. Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscope (SEM), and thermogravimetric analysis (TGA) were used to analyze the produced PANI@ß-CD nanocomposite. Our results show the optimum conditions for phenol adsorption: time (50 min), pH (8.0), nanosorbent dose (0.5 g), and the sorption isotherm fitted with Langmuir model; the monolayer adsorption capacity of the prepared PANI@ß-CD for phenol was determined to be 8.56 mg g-1. The average pore size, total pore volume, and surface area of PANI/ßCD nanocomposite are 15.62 nm, 0.1586 cm3/g, and 90.901 m2/g, respectively, for the pseudo second order model. Finally, modifying PANI nanoparticles with ßCD allowed reusability up to four cycles with superior adsorption performance of ∼95% using (0.01 N) HNO3.

A novel ß-cyclodextrin/alginate-combined-nickel oxide nanosorbent for adsorptive remediation of 51Cr and 56Mn radionuclides.

A Promising nanocomposite from ß-Cyclodextrin/Alginate (ß-CD/Alg) composite impregnated with nickel oxide nanoparticles (NiO) has been synthesized and characterized using diverse techniques like FT-IR, XRD, TGA, and SEM. The new nanocomposite has been investigated for the efficient remediation of 51Cr and 56Mn radionuclides from simulated contaminated radioactive water. All the controlling experimental parameters such as solution pH, contact time, initial radionuclides concentration and adsorbent mass have been investigated and optimized. The distribution coefficient values Kd (mL/g) for 51Cr and/or 56Mn radionuclides have been calculated for all factors it was found that the optimum pH values were at 5 and 6 with Kd 5300, and 4500, for 51Cr and/or 56Mn, respectively and the equilibrium was at 90 and 100 (min) with Kd values 5600 and 4800 for 51Cr and/or 56Mn, respectively.

Development of Lepidium sativum Extracts/PVA Electrospun Nanofibers as Wound Healing Dressing.

Lepidium sativum L. (Garden cress/Hab El Rashad) (Ls), family Brassicaceae, has considerable importance in traditional medicine worldwide because of its antioxidant and anti-inflammatory activities. Ls fruits were used in Ayurvedic medicines as a useful drug for injuries, skin, and eye diseases. The aim of this study was to examine the effectiveness of the total ethanol extract (TEE) and polysaccharide (Poly) of Ls seeds loaded on poly(vinyl alcohol) (PVA) nanofibers (NFs) as a wound healing dressing and to correlate the activity with the constituents of each. TEE and Poly were phytochemically analyzed qualitatively and quantitatively. Qualitative analysis proved the presence of phenolic acids, flavonoids, tannins, sterols, triterpenes, and mucilage. Meanwhile, quantitative determinations were carried out spectrophotometrically for total phenolic and total flavonoid contents. High-performance liquid chromatography (HPLC) for TEE identified 15 phenolic acids and flavonoid compounds, with gallic acid and catechin as the majors. Separation, purification, and identification of the major compounds were achieved through a Puriflash system, column Sephadex LH20, and spectroscopic data (1H, 13C NMR, and UV). Eight compounds (gallic acid, catechin, rutin, kaempferol-3-O-rutinoside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, quercetin, and kaempferol) were obtained. Gas-liquid chromatography (GLC) analysis for Poly identified 11 compounds, with galactose being the main. The antioxidant activity for both extracts was measured by three different methods based on different mechanisms: 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing ability of plasma (FRAP), and 3-ethylbenzothiazoline-6-sulfonic acid (ABTS). TEE has the highest effectiveness as an antioxidant agent with IC50 82.6 ± 8.35 µg/mL for DPPH and 772.47 and 758.92 µM Trolox equivalent/mg extract for FRAP and ABTS, respectively. The PVA nanofibers (NFs) for each sample were fabricated by electrospinning. The fabricated NFs were characterized by SEM and Fourier transform infrared spectroscopy (FTIR); the results revealed successful encapsulation of TEE and Poly in the prepared NFs. Moreover, the swelling index of TEE in the prepared NFs shows that it is the most appropriate for use as a wound dressing. Cytotoxicity studies indicated a high cell viability with IC50 216 µg/mL and 1750 µg/mL for TEE and Poly, respectively. Moreover, the results revealed that nanofibers possess higher cell viability compared to solutions with the same sample quantities: 9-folds for TEE and 4-folds for Poly of amount 400 µg. The in vitro wound healing test showed that the TEE nanofibers performed better than Poly nanofibers in accelerating wound healing, with 90% for TEE, more than that for the Poly extract (82%), after 48 h. These findings implied that the incorporation of TEE in PVA nanofibers was more efficient than incorporation of Poly in improving the biological activity in wound healing. In conclusion, the TEE and polysaccharides of L. sativum L seed are ideal candidates for nanofibrous wound dressings. Furthermore, the contents of phenolic acids and flavonoids in TEE, which have potential antioxidant activity, make the TEE of L. sativum more favorable for wound healing dressing.

Spontaneous Formation of 3D Breast Cancer Tissues on Electrospun Chitosan/Poly(ethylene oxide) Nanofibrous Scaffolds.

Three-dimensional (3D) tissue culture has attracted a great deal of attention as a result of the need to replace the conventional two-dimensional cell cultures with more meaningful methods, especially for understanding the sophisticated nature of native tumor microenvironments. However, most techniques for 3D tissue culture are laborious, expensive, and limited to spheroid formation. In this study, a low-cost and highly effective nanofibrous scaffold is presented for spontaneous formation of reproducible 3D breast cancer microtissues. Experimentally, aligned and non-aligned chitosan/poly(ethylene oxide) nanofibrous scaffolds were prepared at one of two chitosan concentrations (2 and 4 wt %) and various electrospinning parameters. The resulting fabricated scaffolds (C2P1 and C4P1) were structurally and morphologically characterized, as well as analyzed in silico. The obtained data suggest that the fiber diameter, surface roughness, and scaffold wettability are tunable and can be influenced based on the chitosan concentration, electrospinning conditions, and alignment mode. To test the usefulness of the fabricated scaffolds for 3D cell culture, a breast cancer cell line (MCF-7) was cultured on their surfaces and evaluated morphologically and biochemically. The obtained data showed a higher proliferation rate for cells grown on scaffolds compared to cells grown on two-dimensional adherent plates (tissue culture plate). The MTT assay revealed that the rate of cell proliferation on nanofibrous scaffolds is statistically significantly higher compared to tissue culture plate (P ≤ 0.001) after 14 days of culture. The formation of spheroids within the first few days of culture shows that the scaffolds effectively support 3D tissue culture from the outset of the experiment. Furthermore, 3D breast cancer tissues were spontaneously formed within 10 days of culture on aligned and non-aligned nanofibrous scaffolds, which suggests that the scaffolds imitate the in vivo extracellular matrix in the tumor microenvironment. Detailed mechanisms for the spontaneous formation of the 3D microtissues have been proposed. Our results suggest that scaffold surface topography significantly influences tissue formation and behavior of the cells.