Acellular Systems Research Articles

The Development of Lung Tissue Engineering: From Biomaterials to Multicellular Systems.

The challenge of the treatment of end-stage lung disease poses an urgent clinical demand for lung tissue engineering. Over the past few years, various lung tissue-engineered constructs are developed for lung tissue regeneration and respiratory pathology study. In this review, an overview of recent achievements in the field of lung tissue engineering is proposed. The introduction of lung structure and lung injury are stated briefly at first. After that, the lung tissue-engineered constructs are categorized into three types: acellular, monocellular, and multicellular systems. The different bioengineered constructs included in each system that can be applied to the reconstruction of the trachea, airway epithelium, alveoli, and even whole lung are described in detail, followed by the highlight of relevant representative research. Finally, the challenges and future directions of biomaterials, manufacturing technologies, and cells involved in lung tissue engineering are discussed. Overall, this review can provide referable ideas for the realization of functional lung regeneration and permanent lung substitution.

343 Development of osteoclast derived exosomes for vascular calcification therapy

OBJECTIVES/GOALS: The global incidence of calcific aortic valve disease (CAVD) increased 3.5-fold since 1990. No preventative or therapeutic pharmaceutical therapies exist for CAVD. We will establish the therapeutic potential of osteoclast-derived exosomes though characterization of contents and mechanisms of action to protect against mineralization. METHODS/STUDY POPULATION: Exosomes were purified from conditioned media collected from murine myeloid precursor cells, RAW264.7 (control), and osteoclasts induced to differentiate from RAW264.7 cells (OD). Protein content of exosomes was determined using proteomic analyses. Nucleic acid contents will be identified by sequencing mRNA, miRNA, and DNA. The calcification prevention and reabsorption abilities of control and OD exosomes will be tested using human valvular interstitial cells (VIC) and smooth muscle cell calcification assays and acellular osteologic disc assays, respectively. Comparison between cellular and acellular systems will help identify mechanisms of action, and demonstrate potential therapeutic viability of OD exosomes in preventative vs resorptive treatments. RESULTS/ANTICIPATED RESULTS: OD exosomes, but not control exosomes, prevented calcification in VIC in vitro. OD exosomes contained osteoclast-specific proteins including TRAP, MMP6, cathepsin K, and bone reabsorption factors including V type proton pumps, ATPases, and integrins. These genes are also involved in resorptive activities, and were highly upregulated in OD compared to control exosomes. We anticipate miRNA signatures associated with mineral resorption will also be present. Increased knowledge of exosome cargo will illuminate their mechanism of action and allow future work to engineer increased efficacy. We also anticipate a therapeutic response when OD exosomes are applied after calcification has begun, showing exosomes promote calcium reabsorption. DISCUSSION/SIGNIFICANCE: Establishing therapeutic potential and examining mechanisms of action will pave the way for OD exosomes as a CAVD treatment. Analysis of exosome contents will determine active molecules to be enhanced in future studies. This work will lay a foundation for moving into aortic valve organoid models, which are accepted by the FDA for preclinical trials.

Protease-degradable hydrogels with multifunctional biomimetic peptides for bone tissue engineering.

Mimicking bone extracellular matrix (ECM) is paramount to develop novel biomaterials for bone tissue engineering. In this regard, the combination of integrin-binding ligands together with osteogenic peptides represents a powerful approach to recapitulate the healing microenvironment of bone. In the present work, we designed polyethylene glycol (PEG)-based hydrogels functionalized with cell instructive multifunctional biomimetic peptides (either with cyclic RGD-DWIVA or cyclic RGD-cyclic DWIVA) and cross-linked with matrix metalloproteinases (MMPs)-degradable sequences to enable dynamic enzymatic biodegradation and cell spreading and differentiation. The analysis of the intrinsic properties of the hydrogel revealed relevant mechanical properties, porosity, swelling and degradability to engineer hydrogels for bone tissue engineering. Moreover, the engineered hydrogels were able to promote human mesenchymal stem cells (MSCs) spreading and significantly improve their osteogenic differentiation. Thus, these novel hydrogels could be a promising candidate for applications in bone tissue engineering, such as acellular systems to be implanted and regenerate bone or in stem cells therapy.

Osteochondral regenerative engineering: challenges, state-of-the-art and translational perspectives.

Despite quantum leaps, the biomimetic regeneration of cartilage and osteochondral regeneration remains a major challenge, owing to the complex and hierarchical nature of compositional, structural and functional properties. In this review, an account of the prevailing challenges in biomimicking the gradients in porous microstructure, cells and extracellular matrix (ECM) orientation is presented. Further, the spatial arrangement of the cues in inducing vascularization in the subchondral bone region while maintaining the avascular nature of the adjacent cartilage layer is highlighted. With rapid advancement in biomaterials science, biofabrication tools and strategies, the state-of-the-art in osteochondral regeneration since the last decade has expansively elaborated. This includes conventional and additive manufacturing of synthetic/natural/ECM-based biomaterials, tissue-specific/mesenchymal/progenitor cells, growth factors and/or signaling biomolecules. Beyond the laboratory-based research and development, the underlying challenges in translational research are also provided in a dedicated section. A new generation of biomaterial-based acellular scaffold systems with uncompromised biocompatibility and osteochondral regenerative capability is necessary to bridge the clinical demand and commercial supply. Encompassing the basic elements of osteochondral research, this review is believed to serve as a standalone guide for early career researchers, in expanding the research horizon to improve the quality of life of osteoarthritic patients affordably.

Fish oil nano-emulsion kills macrophage: Ferroptosis triggered by catalase-catalysed superoxide eruption

Fish oil is increasingly utilised in the form of nano-emulsion as a nutrient and function fortifier. The nano-emulsions exceptionally high content of polyunsaturated fatty acids and electron donors at the oil/water interface provide an ideal site of the redox reaction. Here we report that a vigorous superoxide production in the fish oil nano-emulsion was catalysed by mammalian catalase in acellular and cellular systems. The resulting superoxide increased cytosolic reactive oxygen species (ROS) and membrane lipid peroxidation of murine macrophage, which eventually causes fatal oxidative damages. Cell death, was significantly inhibited by a catalase-specific inhibitor 3-Amino-1,2,4-triazole (3-AT), was via ferroptosis and not apoptosis. The ferroptosis was independent of free iron or glutathione peroxidase suppression. Our findings discovered a hidden health risk of the widely acclaimed fish oil emulsion, suggesting a novel cellular damage mechanism caused by dietary unsaturated fats on the alimentary tract mucosa.

P18-09 Physiological biofluids as acellular alternate model systems for rapid identification of hazardous chemicals and particulates

P18-09 Physiological biofluids as acellular alternate model systems for rapid identification of hazardous chemicals and particulates

Formation of Redox-Active Duroquinone from Vaping of Vitamin E Acetate Contributes to Oxidative Lung Injury.

In late 2019, the outbreak of e-cigarette or vaping-associated lung injuries (EVALIs) in the United States demonstrated to the public the potential health risks of vaping. While studies since the outbreak have identified vitamin E acetate (VEA), a diluent of tetrahydrocannabinol (THC) in vape cartridges, as a potential contributor to lung injuries, the molecular mechanisms through which VEA may cause damage are still unclear. Recent studies have found that the thermal degradation of e-liquids during vaping can result in the formation of products that are more toxic than the parent compounds. In this study, we assessed the role of duroquinone (DQ) in VEA vaping emissions that may act as a mechanism through which VEA vaping causes lung damage. VEA vaping emissions were collected and analyzed for their potential to generate reactive oxygen species (ROS) and induce oxidative stress-associated gene expression in human bronchial epithelial cells (BEAS-2B). Significant ROS generation by VEA vaping emissions was observed in both acellular and cellular systems. Furthermore, exposure to vaping emissions resulted in significant upregulation of NQO1 and HMOX-1 genes in BEAS-2B cells, indicating a strong potential for vaped VEA to cause oxidative damage and acute lung injury; the effects are more profound than exposure to equivalent concentrations of DQ alone. Our findings suggest that there may be synergistic interactions between thermal decomposition products of VEA, highlighting the multifaceted nature of vaping toxicity.

Development of Fluorescently Labeled, Functional Type I Collagen Molecules.

While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are examined. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ≈1.2 labels per collagen molecule preserves these characteristics. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labeled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development at low concentration, the labeled collagen is incorporated into a fine extracellular matrix (ECM) network associated with the cells within 24 h.

Induction of Gamma-Glutamyltransferase Activity and Consequent Pro-oxidant Reactions in Human Macrophages Exposed to Crocidolite Asbestos.

Asbestos is the main causative agent of malignant pleural mesothelioma. The variety known as crocidolite (blue asbestos) owns the highest pathogenic potential, due to the dimensions of its fibers as well as to its content of iron. The latter can in fact react with macrophage-derived hydrogen peroxide in the so called Fenton reaction, giving rise to highly reactive and mutagenic hydroxyl radical. On the other hand, hydroxyl radical can as well originate after thiol-dependent reduction of iron, a process capable of starting its redox cycling. Previous studies showed that glutathione (GSH) is one such thiol, and that cellular gamma-glutamyltransferase (GGT) can efficiently potentiate GSH-dependent iron redox cycling and consequent oxidative stress. As GGT is expressed in macrophages and is released upon their activation, the present study was aimed at verifying the hypothesis that GSH/GGT-dependent redox reactions may participate in the oxidative stress following the activation of macrophages induced by crocidolite asbestos. Experiments in acellular systems confirmed that GGT-mediated metabolism of GSH can potentiate crocidolite-dependent production of superoxide anion, through the production of highly reactive dipeptide thiol cysteinyl-glycine. Cultured THP-1 macrophagic cells, as well as isolated monocytes obtained from healthy donors and differentiated to macrophages in vitro, were investigated as to their expression of GGT and the effects of exposure to crocidolite. The results show that crocidolite asbestos at subtoxic concentrations (50-250 ng/1000 cells) can upregulate GGT expression, which raises the possibility that macrophage-initiated, GSH/GGT-dependent pro-oxidant reactions may participate in the pathogenesis of tissue damage and inflammation consequent to crocidolite intoxication.

Membraneless Compartmentalization Facilitates Enzymatic Cascade Reactions and Reduces Substrate Inhibition.

Living cells possess membraneless organelles formed by liquid-liquid phase separation. With the aim of better understanding the general functions of membraneless microcompartments, this paper constructs acellular multicompartment reaction systems using an aqueous multiphase system. Membraneless coacervate droplets are placed within a molecularly crowded environment, where a larger dextran (DEX) droplet is submerged in a polyethylene glycol (PEG) solution. The coacervate droplets are capable of sequestering reagents and enzymes with a long retention time, and demonstrate multistep cascading reactions through the liquid-liquid interfaces. The ability to change phase dynamics is also demonstrated through salt-mediated dissolution of coacervate droplets, which leads to the release and mixing of separately sequestered reagents and enzymes. Finally, as phase-separated materials in membraneless organelles are often substrates and substrate analogues for the enzymes sequestered or excluded in the organelles, this paper explores the interaction between DEX and dextranase, an enzyme that hydrolyzes DEX. The results reveal that dextranase suffers from substrate inhibition when partitioned directly in a DEX phase but that this inhibition can be mitigated and reactions greatly accelerated by compartmentalization of dextranase inside a coacervate droplet that is adjacent to, but phase-separated from, the DEX phase. The insight that compartmentalization of enzymes can accelerate reactions by mitigating substrate inhibition is particularly novel and is an example where artificial membraneless organelle-like systems may provide new insights into physiological cell functions.

Assessment of reactive oxygen species generated by electronic cigarettes using acellular and cellular approaches

Electronic cigarettes (e-cigs) have fast increased in popularity but the physico-chemical properties and toxicity of the generated emission remain unclear. Reactive oxygen species (ROS) are likely present in e-cig emission and can play an important role in e-cig toxicity. However, e-cig ROS generation is poorly documented. Here, we generated e-cig exposures using a recently developed versatile exposure platform and performed systematic ROS characterization on e-cig emissions using complementary acellular and cellular techniques: 1) a novel acellular Trolox-based mass spectrometry method for total ROS and hydrogen peroxide (H2O2) detection, 2) electron spin resonance (ESR) for hydroxyl radical detection in an acellular and cellular systems and 3) in vitro ROS detection in small airway epithelial cells (SAEC) using the dihydroethidium (DHE) assay. Findings confirm ROS generation in cellular and acellular systems and is highly dependent on the e-cig brand, flavor, puffing pattern and voltage. Trolox method detected a total of 1.2–8.9nmol H2O2eq./puff; H2O2 accounted for 12–68% of total ROS. SAEC cells exposed to e-cig emissions generated up to eight times more ROS compared to control. The dependency of e-cig emission profile on e-cig features and operational parameters should be taken into consideration in toxicological studies.

Artificial Antigen-Presenting Cells for Immunotherapies.

Artificial antigen-presenting cells (aAPCs) overcome many of the limitations of biologically based adoptive immunotherapy protocols. While these acellular systems can be designed with a variety of parameters, including material type, diameter, and proliferative signals for T cells, we outline methods to formulate and characterize a comprehensive polymeric microparticle aAPC platform. These aAPCs, which can be reproducibly fabricated in large quantities, efficiently stimulate antigen-specific T cell activation and proliferation by both paracrine cytokine signals and engagement of T cell surface proteins.

Gallic acid grafting modulates the oxidative potential of ferrimagnetic bioactive glass-ceramic SC-45

Magnetite-containing glass-ceramics are promising bio-materials for replacing bone tissue after tumour resection. Thanks to their ferrimagnetic properties, they generate heat when subjected to an alternated magnetic field. In virtue of this they can be employed for the hyperthermic treatment of cancer. Moreover, grafting anti-cancer drugs onto their surface produces specific anti-neoplastic activity in these biomaterials. Gallic acid (GA) exhibits antiproliferative activity which renders it a promising candidate for anticancer applications. In the present paper, the reactivity of ferrimagnetic glass-ceramic SC-45 grafted with GA (SC-45+GA) was studied in terms of ROS release, rupture of the C–H bond of the formate molecule and Fenton reactivity by EPR/spin trapping in acellular systems. The ability of these materials to cause lipid peroxidation was assessed by UV–vis/TBA assay employing linoleic acid as a model of membrane lipid. The results, compared to those obtained with SC-45, showed that GA grafting (i) significantly enhanced the Fenton reactivity and (ii) restored the former reactivity of SC-45 towards both the C–H bond and linoleic acid which had been completely suppressed by prolonged contact with water. Fe2+ centres at the surface are probably implicated. GA, acting as a pro-oxidant, reduces Fe3+ to Fe2+ by maintaining a supply of Fe2+ at the surface of SC-45+GA.

Effects of Black Raspberry Extract and Protocatechuic Acid on Carcinogen-DNA Adducts and Mutagenesis, and Oxidative Stress in Rat and Human Oral Cells.

Effects of black raspberry (BRB) extract and protocatechuic acid (PCA) on DNA adduct formation and mutagenesis induced by metabolites of dibenzo[a,l]pyrene (DBP) were investigated in rat oral fibroblasts. The DBP metabolites, (±)-anti-11,12-dihydroxy-11,12,-dihydrodibenzo[a,l]pyrene (DBP-diol) and 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) induced dose-dependent DNA adducts and mutations. DBPDE was considerably more potent, whereas the parent compound had no significant effect. Treatment with BRB extract (BRBE) and PCA resulted in reduced DBP-derived DNA adduct levels and reduced mutagenesis induced by DBP-diol, but only BRBE was similarly effective against (DBPDE). BRBE did not directly inactivate DBPDE, but rather induced a cellular response-enhanced DNA repair. When BRBE was added to cells 1 day after the DBP-diol, the BRBE greatly enhanced removal of DBP-derived DNA adducts. As oxidative stress can contribute to several stages of carcinogenesis, BRBE and PCA were investigated for their abilities to reduce oxidative stress in a human leukoplakia cell line by monitoring the redox indicator, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF) in cellular and acellular systems. BRBE effectively inhibited the oxidation, but PCA was only minimally effective against H2DCF. These results taken together provide evidence that BRBE and PCA can inhibit initiation of carcinogenesis by polycyclic aromatic hydrocarbons; and in addition, BRBE reduces oxidative stress. Cancer Prev Res; 9(8); 704-12. ©2016 AACR.

The effect of engine emissions from diesel and biodiesel fuels on oxidative damage in acellular and cellular systems

The effect of engine emissions from diesel and biodiesel fuels on oxidative damage in acellular and cellular systems

Enzymatic production of bioactive docosahexaenoic acid phenolic ester

Docosahexaenoic acid (DHA) is increasingly considered for its health benefits. However, its use as functional food ingredient is still limited by its instability. In this work, we developed an efficient and solvent-free bioprocess for the synthesis of a phenolic ester of DHA. A fed-batch process catalyzed by Candida antarctica lipase B was optimised, leading to the production of 440g/L vanillyl ester (DHA-VE). Structural characterisation of the purified product indicated acylation of the primary OH group of vanillyl alcohol. DHA-VE exhibited a high radical scavenging activity in acellular systems. In vivo experiments showed increased DHA levels in erythrocytes and brain tissues of mice fed DHA-VE-supplemented diet. Moreover, in vitro neuroprotective properties of DHA-VE were demonstrated in rat primary neurons exposed to amyloid-β oligomers. In conclusion, DHA-VE synergized the main beneficial effects of two common natural biomolecules and therefore appears a promising functional ingredient for food applications.

Protective effects of an aqueous Pericarpium Granati extract against inflammatory damage in mice

The protective effect of an aqueous Pericarpium Granati extract (APG) against carrageenan-induced inflammatory damage in mice was investigated. The results showed that APG displayed radical scavenging, reducing activity, and liposome protection activity in acellular systems. The implementation of an HPLC helped identify polyphenolic components including epicatechin (11.9 mg/g), gallic acid (3.9 mg/g), ellagic acid (2.4 mg/g), ferulic acid (1.0 mg/g), quercetin (0.7 mg/g), and kaempferol (0.5 mg/g). APG administration in the range of 0.25–1.0 g/kg showed a concentration dependent inhibition on inflammatory response in carrageenan induced mice. The anti-inflammatory effects of APG could be related to the suppression of tissue nitric oxide and tumor necrosis factor-α, and associated with the reduction of lipid peroxidation and an increase in antioxidant enzyme activities including catalase, superoxide dismutase, and glutathione peroxidase in vivo. The results showed that APG might serve as a natural source of anti-inflammatory and antioxidative compounds in vivo.

Emodin, an anthraquinone derivative, protects against gamma radiation-induced toxicity by inhibiting DNA damage and oxidative stress

Purpose: In the present study, we explored the modulatory effect of emodin (1,3,8-trihydroxy-6-methylanthraquinone, C15H10O5) against gamma radiation-induced DNA damage and oxidative stress in acellular and cellular systems, respectively.Materials and methods: For cellular systems, concanavalin A (ConA)-stimulated murine splenocytes were used. Cytotoxic effect of emodin (0–400 μM), radiation (3–12 Gy) and emodin + radiation was measured by MTT [3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. Gamma radiation (3–12 Gy)-induced production of reactive oxygen species (ROS), an increase in nitric oxide (NO) level and its inhibition by emodin were estimated by DCFDA (2ʹ,7ʹ-dichlorofluorescein diacetate) and Griess regent, respectively. Analysis of radiation-induced apoptosis was performed using flow cytometery and acridine orange/ethidium bromide staining. DNA damage was evaluated in acellular system using pBR322 plasmid relaxation assay.Results: Emodin was able to effectively scavenge radiation- induced free radicals (ROS and NO) in murine splenocytes. Radiation-induced apoptosis and cell death was also inhibited by emodin pre-treatment. It could significantly prevent radiation-induced DNA damage.Conclusions: Protection against gamma radiation-induced cell death and DNA damage by emodin could be attributed to its free radical scavenging activity. The present study is the first report of the radioprotective role of emodin in mammalian cells.

Evolving role of skin sparing mastectomy.

Skin sparing mastectomy (SSM) can facilitate immediate breast reconstruction and is associated with an excellent aesthetic result. The procedure is safe in selected cases; including invasive tumours < 5 cm, multi-centric tumours, ductal carcinoma in situ and for risk-reduction surgery. Inflammatory breast cancers and tumours with extensive involvement of the skin represent contra-indications to SSM due to an unacceptable risk of local recurrence. Prior breast irradiation or the need for post-mastectomy radiotherapy do not preclude SSM, however the aesthetic outcome may be compromised. Preservation of the nipple areola complex is safe for peripherally located node negative tumours. An intraoperative frozen section protocol for the retro-areolar tissue should be considered in these cases. The advent of acellular tissue matrix systems has enhanced the scope of implant-based immediate reconstruction following SSM. Cell-assisted fat transfer is emerging as a promising technique to optimise the aesthetic outcome.

Protective Effect of β-Caryophyllene, a Natural Bicyclic Sesquiterpene, Against Cerebral Ischemic Injury

β-Caryophyllene (trans-4,11,11-trimethyl-8-methylenebicyclo[7,2,0]undec-4-ene), found in various plants, is a natural bicyclic sesquiterpene with a low toxicity. Here, we show that a single intraperitoneal injection of β-caryophyllene (10 mg/kg) significantly reduced the cortical infarct volume by 67% when given immediately before middle cerebral artery occlusion (MCAO). Neurological deficits caused by MCAO were also significantly decreased by β-caryophyllene. β-Caryophyllene treatment of cortical cells exposed to oxygen-glucose deprivation revealed a significant protection in a dose-dependent manner. However, β-caryophyllene neither suppressed N-methyl-D-aspartate excitotoxicity in cultured cortical cells nor markedly decreased the oxidative stress measured in the cellular or acellular systems. By contrast, treatments with β-caryophyllene dose-dependently inhibited mRNA expression of inducible nitric oxide synthetase, interleukin (IL)-1β, IL-6, and cyclooxygenase 2 in C6 microglial cells, and decreased the level of nitric oxide and prostaglandin E₂ at a 100 μM concentration. All of these findings suggest that β-caryophyllene has a potent neuroprotective activity, and its neuroprotection may be partly related to the modulation of inflammatory mediators.