Cellular Viability Research Articles

Bioactive self-assembling silk fibroin–sericin films for skin tissue engineering

The quest for an ideal wound dressing material has been a strong motivation for researchers to explore novel biomaterials for this purpose. Such explorations have led to the extensive use of silk fibroin (SF) as a suitable polymer for several applications over the years. Unfortunately, another major silk protein—sericin has not received its due attention yet in spite of having favorable biological properties. In this study, we report an approach of blending SF and silk sericin (SS) without the usage of chemical crosslinkers is made possible by the usage of formic acid which evaporates to induce β-sheets formation to form cytocompatible films. Raman spectroscopy confirms the presence of SF/SS components in blend and formation of β-sheet in films. In situ, gelation kinetics studies were conducted to understand the change in gelation properties with addition of sericin into SF. Methyl thiazolyl tetrazolium and live/dead assays were performed to study cellular attachment, viability and proliferation on SF/SS films. The antibacterial properties of SF/SS films were tested using Gram-negative and Gram-positive bacteria. The re-structured SF/SS films were stable, transparent, show good mechanical properties, antibacterial activity and cytocompatibility, therefore can serve as suitable biomaterial candidates for skin regeneration applications.

LncRNA MEG3 suppresses erastin-induced ferroptosis of chondrocytes via regulating miR-885-5p/SLC7A11 axis.

Ferroptosis is involved in osteoarthritis development; however, the roles of long noncoding RNAs (lncRNAs), including lncRNA MEG3, in the regulation of ferroptosis in osteoarthritis are still unclear. In this study, qRT‒PCR and Western blotting assays were used to detect the expression of lncRNA MEG3, miR-885-5p, SLC7A11 and GPX4; MDA and CCK-8 assays were applied to analyse cellular MDA levels and cell viability, respectively. Erastin elevated cellular MDA levels and decreased the viability of chondrocytes and the erastin-induced decline in cell viability was reversed by a ferroptosis inhibitor (ferrostatin-1). Erastin downregulated lncRNA MEG3, SLC7A11 and GPX4 and upregulated miR-885-5p. Silencing of lncRNA MEG3 increased miR-885-5p and downregulated SLC7A11 and GPX4 and further sensitized chondrocytes to erastin-induced ferroptosis. In contrast, overexpression of lncRNA MEG3 had opposite effects. Dual luciferase assays confirmed binding between lncRNA MEG3 and miR-885-5p and between miR-885-5p and the 3'UTR of SLC7A11. In the synovial fluids from patients with osteoarthritis compared with synovial fluids from normal controls, the RNA levels of lncRNA MEG3 and SLC7A11 were decreased and the miR-885-5p expression level was increased. Our findings indicated that lncRNA MEG3 overexpression alleviated ferroptosis in chondrocytes by affecting the miR-885-5p/SLC7A11 signalling pathway.

Cellular viability of fibroblasts, osteoblasts and osteoclasts in response to bone adhesive alendronate-functionalized poly(2-oxazoline)

Bone adhesive biomaterials are currently being developed to replace conventional biomaterials that do not adhere to bone. Recently, poly (2-oxazoline) polymers have been functionalized with calcium-binding alendronate groups (POx-Ale) to render these polymers bone adhesive. These polymers hold promise for application as bone fixation biomaterials, but their effects on cells present in bone tissue has not been studied yet. This study evaluated the in vitro cytocompatibility of POx-Ale for cell types that are relevant for applications in bone reconstructive surgery. Cell viability of murine pre-osteoblasts (MC3T3-E1), fibroblasts (NIH3T3) and pre-osteoclasts derived from macrophages (RAW264.7) is quantified as a function of POx-Ale polymer concentrations ranging between 5.0 and 0.3 mg mL−1. In addition, the effect of POx-Ale on cellular function is assessed by performing mineralization, gelatin zymography as well as TRAP activity anad TRAP staining assays. The results show that POx-Ale has in vitro cytotoxic effects on murine pre-osteoblasts (MC3T3-E1), fibroblasts (NIH3T3) and pre-osteoclasts (RAW264.7) when cultured in media with polymer concentrations ≥2.5 mg mL−1, and inhibits mineralization of osteoblasts and MMP activity of fibroblasts at lower polymer concentrations. However, POx-Ale did not negatively affect Tartrate-resistant acid phosphate (TRAP) activity in osteoclasts.

Amrubicin encapsulated PLGA NPs inhibits the PI3K/AKT signaling pathway by activating PTEN and inducing apoptosis in TMZ-resistant Glioma

Glioblastoma (GBM) remains a challenging malignancy due to its aggressive nature and the lack of efficacious therapeutic interventions. Nanotechnology-based approaches exhibit promise in GBM treatment; however, the successful translation of these strategies from preclinical models to clinical settings is hindered by inefficient nanoparticle clearance from vital organs. Addressing this concern, we investigated the therapeutic potential of amrubicin (AMR) encapsulated within poly (lactic-co-glycolic acid) nanoparticles (AMR-PLGA-NPs) in combating temozolomide (TMZ) resistant GBM. The study demonstrated that AMR-PLGA-NPs exerted a pronounced inhibitory effect on the cellular viability and migratory capacity of TMZ-resistant GBM cells. Furthermore, these nanoparticles exhibited considerable efficacy in downregulating the PI3K/AKT signaling pathway, thereby inducing apoptosis specifically in TMZ-resistant glioma cells and glioma stem-like cells through the activation of PTEN. Notably, in vivo experimentation revealed the ability of AMR-PLGA-NPs to traverse biological barriers within murine models. Collectively, these findings underscore the potential therapeutic utility of AMR-PLGA-NPs as a versatile nanoplatform for addressing the formidable challenges posed by GBM, particularly in mitigating drug resistance mechanisms. The study substantiates the stability and safety profile of AMR-PLGA-NPs, positioning them as a promising avenue for combating drug resistance in GBM therapeutics.

Abstract C100: Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis

Abstract Biological stresses that are cell-intrinsic (e.g., oncogene-induced imbalances in protein translation, lipid synthesis, mitochondrial dysfunction) or cell-extrinsic (e.g., nutrient deprivation, hypoxia, drug exposure) disrupt cellular function and viability if not mitigated. The Integrated Stress Response (ISR) consists of upstream regulatory kinases (PERK, GCN2, HRI, PKR) that respond to such stresses in part by activating the ATF4 transcription factor. ATF4 then rewires nutrient uptake, cellular metabolism, mitochondrial function, and protein chaperone networks to restore cellular homeostasis. We have shown that ATF4 is upregulated in mouse and human pancreatic ductal adenocarcinoma (PDAC), even compared to normal acinar cells where ATF4 is expressed at relatively high levels. To investigate the role of ATF4 in PDAC, we generated mice with inducible acinar-specific deletion of Atf4 using Ptf1a-CreERT to delete Atf4 following treatment of adult Atf4-floxed mice with tamoxifen. In contrast to previous studies showing germline deletion of Atf4 in mice caused premature mortality at approximately 3 weeks of age due to pancreatic insufficiency, inducible deletion of Atf4 in adult acinar cells does not cause pancreatic deficiency or animal demise. Rather, adult Ptf1a-CreERT;Atf4fl/fl (CA) mice are viable although more sensitive to cerulein-induced pancreatitis. Specifically, compared to control (WT) mice, CA mice exhibit more extensive acinar-ductal metaplasia (ADM), including more extensive expression of cytokeratin-19 (CK19), increased cell death and take longer to recover normal pancreatic architecture following in vivo cerulein challenge. Using ex vivo acinar explant assays derived from WT and CA mice, we further show that induction of ADM in WT acinar cells with TGF-a is associated with down-regulation of Atf4 and Atf4 target genes in WT acinar explants while CA explants undergo accelerated ADM. Furthermore, expression of exogenous Atf4 in acinar cells inhibited ADM. These in vivo and ex vivo findings are consistent with down-regulation of Atf4 being required for ADM. Given the tumor-promoting role of ADM in the early stages of PDAC, we next crossed the CA mice to LSL-KRasG12D mice to generate LSL-KRasG12D;Ptf1a-CreERT;Atf4fl/fl (KCA) mice to examine how loss of Atf4 affected pancreatic intraepithelial neoplasia (PanIN) and PDAC formation. Surprisingly, given the critical need to down-regulate Atf4 in ADM, we show for the first time that genetic deletion of Atf4 in pancreatic acinar cells blocks both PanIN and PDAC formation in KCA mice compared to age-matched KC mice. Rather, KCA mice developed a fatty pancreas devoid of tumor with very few CK19-positive cells forming. These results suggest that Atf4 is critical for PanIN formation and prevents PDAC. This also suggests that Atf4 plays a different role in PanIN formation than in ADM where its down-regulation was critical and indicates a dynamic role for Atf4 depending on oncogenic versus proteotoxic stress, as will be discussed. Citation Format: Leah M. Ziolkowski, Kay F. Macleod. Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C100.

SiRNA-mediated downregulation of BATF3 diminished proliferation and induced apoptosis through downregulating c-Myc expression in chronic myelogenous leukemia cells.

Despite considerable improvement in therapeutic approaches to chronic myeloid leukemia (CML) treatment, this malignancy is considered incurable due to resistance. However, investigating the molecular mechanism of CML may give rise to the development of extremely efficient targeted therapies that improve the prognosis of patients. Basic leucine zipper transcription factor ATF-like3 (BATF3), as transcription factor, is considered a key regulator of cellular activities and its function has been evaluated in tumor development and growth in several cancer types. This study aimed to evaluate the potential of the cellular impact of siRNA-mediated downregulation of BATF3 on CML cancer cells through cell proliferation, induction of apoptosis, and cell cycle distribution. The transfection of BATF3 siRNA to K562 CML cells was performed by electroporation device. To measure cellular viability and apoptosis, MTT assay and Annexin V/PI staining were carried out, respectively. Also, cell cycle assay and flow cytometry instrument were applied to assess cell cycle distribution of K562 cells. For more validation, mRNA expression of correlated genes was relatively evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The data indicated that siRNA-mediated BATF3 inactivating severely promoted the cell apoptosis. Also, the targeted therapy led to high expression of Caspase-3 gene and Bax/Bcl-2 ratio. Silenced BATF3 also induced cell cycle arrest in phase sub-G1 compared to control. Finally, a noticeable decrement was obtained in c-Myc gene expression through suppression of BATF3 in CML cells. The findings of this research illustrated the suppression of BATF3 as an effective targeted therapy strategy for CML.

Real-Time Exposure to 3D-Printing Emissions Elicits Metabolic and Pro-Inflammatory Responses in Human Airway Epithelial Cells.

Three-dimensional (3D) printer usage in household and school settings has raised health concerns regarding chemical and particle emission exposures during operation. Although the composition of 3D printer emissions varies depending on printer settings and materials, little is known about the impact that emissions from different filament types may have on respiratory health and underlying cellular mechanisms. In this study, we used an in vitro exposure chamber system to deliver emissions from two popular 3D-printing filament types, acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA), directly to human small airway epithelial cells (SAEC) cultured in an air-liquid interface during 3D printer operation. Using a scanning mobility particle sizer (SMPS) and an optical particle sizer (OPS), we monitored 3D printer particulate matter (PM) emissions in terms of their particle size distribution, concentrations, and calculated deposited doses. Elemental composition of ABS and PLA emissions was assessed using scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDX). Finally, we compared the effects of emission exposure on cell viability, inflammation, and metabolism in SAEC. Our results reveal that, although ABS filaments emitted a higher total concentration of particles and PLA filaments emitted a higher concentration of smaller particles, SAEC were exposed to similar deposited doses of particles for each filament type. Conversely, ABS and PLA emissions had distinct elemental compositions, which were likely responsible for differential effects on SAEC viability, oxidative stress, release of inflammatory mediators, and changes in cellular metabolism. Specifically, while ABS- and PLA-emitted particles both reduced cellular viability and total glutathione levels in SAEC, ABS emissions had a significantly greater effect on glutathione relative to PLA emissions. Additionally, pro-inflammatory cytokines including IL-1β, MMP-9, and RANTES were significantly increased due to ABS emissions exposure. While IL-6 and IL-8 were stimulated in both exposure scenarios, VEGF was exclusively increased due to PLA emissions exposures. Notably, ABS emissions induced metabolic perturbation on amino acids and energy metabolism, as well as redox-regulated pathways including arginine, methionine, cysteine, and vitamin B3 metabolism, whereas PLA emissions exposures caused fatty acid and carnitine dysregulation. Taken together, these results advance our mechanistic understanding of 3D-printer-emissions-induced respiratory toxicity and highlight the role that filament emission properties may play in mediating different respiratory outcomes.

Oxygen generating biomaterials at the forefront of regenerative medicine: advances in bone regeneration.

Globally, an annual count of more than two million bone transplants is conducted, with conventional treatments, including metallic implants and bone grafts, exhibiting certain limitations. In recent years, there have been significant advancements in the field of bone regeneration. Oxygen tension regulates cellular behavior, which in turn affects tissue regeneration through metabolic programming. Biomaterials with oxygen release capabilities enhance therapeutic effectiveness and reduce tissue damage from hypoxia. However, precise control over oxygen release is a significant technical challenge, despite its potential to support cellular viability and differentiation. The matrices often used to repair large-size bone defects do not supply enough oxygen to the stem cells being used in the regeneration process. Hypoxia-induced necrosis primarily occurs in the central regions of large matrices due to inadequate provision of oxygen and nutrients by the surrounding vasculature of the host tissues. Oxygen generating biomaterials (OGBs) are becoming increasingly significant in enhancing our capacity to facilitate the bone regeneration, thereby addressing the challenges posed by hypoxia or inadequate vascularization. Herein, we discussed the key role of oxygen in bone regeneration, various oxygen source materials and their mechanism of oxygen release, the fabrication techniques employed for oxygen-releasing matrices, and novel emerging approaches for oxygen delivery that hold promise for their potential application in the field of bone regeneration.

Yo-Yo Dieting Delays Male Drosophila melanogaster Aging Through Enhanced Mitochondrial Function, Relative to Sustained High-Calorie Diet Feeding.

Weight regain subsequent to weight reduction resulting from dietary interventions represents a prevalent phenomenon recognized as "Yo-yo dieting." However, the impact of prolonged Yo-yo dieting on health, especially in relation to the aging process, remains poorly understood. This study aimed to investigate the influence of Yo-yo dieting on the aging process in male Drosophila melanogaster that have been exposed to a high-calorie (HC) diet. Fruit flies were fed with either a consistent HC diet or an alternating regimen of HC and low-calorie diets every 3 days (referred to as "Yo-yo dieting") for a total of 24 days. Biochemical assays were utilized to quantify levels of oxidative stress and activities of the mitochondrial respiratory chain complexes. The frozen section staining method was employed to assess the presence of lipid droplets, reactive oxygen species, cellular viability, and mitochondrial abundance in tissues. Additionally, we examined the expression of key regulators involved in mitochondrial dynamics and biogenic signaling pathways. Yo-yo dieting resulted in an extension of the fruit flies' lifespan, concomitant with reduced body weight, decreased body protein content, and lower triglyceride levels compared to continuous a HC diet feeding. Furthermore, Yo-yo dieting ameliorated impairments in motility and intestinal barrier function. Importantly, it improved mitochondrial function and upregulated the expression of essential mitochondrial fusion proteins, namely mitofusin 1 and mitofusin 2, optic atrophy 1, and peroxisome proliferator-activated receptor-γ coactivator-1α. Therefore, the practice of Yo-yo dieting extends the lifespan of fruit flies by modulating mitochondrial dynamics and the associated biogenic signaling pathways.

FTO-mediated regulation of m6A methylation is closely related to apoptosis induced by repeated UV irradiation

BackgroundUltraviolet (UV) damage is closely related to skin photoaging and many skin diseases, including dermatic tumors. N6-methyladenosine (m6A) modification is an important epigenetic regulatory mechanism. However, the role of m6A methylation in apoptosis induced by repeated UV irradiation has not been characterized. ObjectiveTo explore m6A methylation changes and regulatory mechanisms in the repeated UV-induced skin damage process, especially apoptosis. MethodsHaCaT cells and BALB/c-Nu nude mice were exposed to repeated UVB/UVA+UVB irradiation. Colorimetry and flow cytometry were used to measure cellular viability and apoptosis. m6A-modified genes were detected via colorimetry and methylated RNA immunoprecipitation (MeRIP) sequencing. Methyltransferases and demethylases were detected via RT-PCR, western blotting and immunohistochemistry. Transfection of siRNA and plasmid was performed to knock down or overexpress the selected genes. ResultsAfter UVB irradiation, 861 m6A peaks were increased and 425 m6A peaks were decreased in HaCaT cells. The differentially modified genes were enriched in apoptosis-related pathways. The m6A demethylase FTO was decreased in both HaCaT cells and mouse skin after UV damage. Overexpressing FTO could improve cell viability, inhibit apoptosis and decrease RNA-m6A methylation, including LPCAT3-m6A, which increase LPCAT3 expression, cell viability promotion and apoptosis inhibition. ConclusionOur study identified the cell m6A methylation change lists after repeated UVB irradiation, and revealed that FTO and LPCAT3 play key roles in the m6A methylation pathogenesis of UV-induced skin cell apoptosis. FTO-m6A-LPCAT3 might serve as a novel upstream target for preventing and treating photoaging and UV-induced skin diseases.

The anti-cancer potential of active compounds extracted from Millettia griffoniana on pancreatic and colorectal cancer cells

Pancreatic and colorectal cancer are two of the most lethal cancers; this stems from poor prognosis. Current treatments may lack effectiveness and produce dangerous side effects, hence a need for alternatives. Medicinal plants like Millettia griffoniana may possess anti-cancer properties. This study aimed to isolate and identify compounds from M. griffoniana to test for potential anti-cancer activity on pancreatic and colorectal cancer. Seeds and root bark of M. griffoniana underwent solvent extraction and separation using column chromatography. Isolated compounds were screened using AlamarBlue assays and cytotoxic compounds were identified with 2D NMR. Compounds were subject to cellular viability assays using AlamarBlue and xCELLigence analysis. The Caspase Glo®-3/7 kit measured caspase activity and Real-Time PCR analysis measured apoptosis-related gene expression. 7 compounds were isolated and screened. Compounds 5 and 7 were chosen; identified as durmillone and isojamaicin. Both showed varying concentration-dependent cytotoxic activity, in AlamarBlue and xCELLigence assays, for both cell lines. Caspases 3 and 7 were up-regulated and both compounds up-regulated BAX and down-regulated BCL-2 and p53 in both cell lines. Durmillone and isojamaicin displayed cytotoxic activity on pancreatic and colorectal cancer. Apoptotic activity induced by the compounds was verified by the up-regulation of caspase activity and BAX and down-regulation of BCL-2 and p53. Further studies need to be done towards understanding the mechanisms by which these compounds bring about their cytotoxic activity.

Arsenic-induced prostate cancer: an enigma.

Arsenic exhibits varying degrees of toxicity depending on its many chemical forms. The carcinogenic properties of arsenic have already been established. However, the precise processes underlying the development of diseases following acute or chronic exposure to arsenic remain poorly known. Most of the existing investigation has focused on studying the occurrence of cancer following significant exposure to elevated levels of arsenic. Nevertheless, multiple investigations have documented diverse health consequences from prolonged exposure to low levels of arsenic. Inorganic arsenic commonly causes lung, bladder, and skin cancer. Some investigations have shown an association between arsenic in drinking water and prostate cancer, but few investigations have focused on exploring this connection. There is currently a lack of relevantanimal models demonstrating a clear link between inorganic arsenic exposure and the development of prostate cancer. Nevertheless, studies using cellular model systems have demonstrated that arsenic can potentially promote the malignant transformation of human prostate epithelial cells in vitro. The administration of elevated levels of arsenic has been demonstrated to elicit cell death in instances of acute experimental exposure. Conversely, in cases of chronic exposure, arsenic prompts cellular proliferation and sustains cellular viability, thereby circumventing the constraints imposed by telomere shortening and apoptosis. Furthermore, cells consistently exposed to the stimulus exhibit an augmented ability to invade surrounding tissues and an enhanced potential to form tumors. This review aims to portray mechanistic insights into arsenic-induced prostate cancer.

Novel polysaccharides-bile acid-cyclodextrin gel systems and effects on cellular viability and bioenergetic parameters.

Aim: The novel hydrogel systems made from sodium alginate, pectin, beta-cyclodextrin and deoxycholic acid (DCA) were proposed as potential drug-delivery matrices. Materials & methods: To ensure biocompatibility, rheological parameters were examined and hydrogels' effects on bioenergetic parameters and cellular viability on murine hepatic, and muscle and pancreatic beta cells. Results & conclusion: All hydrogels show non-Newtonian, shear thinning behavior. Cells displayed various oxygen-dependent viability patterns, with the bile acid overall adversely affecting their biological activities. All cells performed best under normoxia, with pancreatic beta cells displaying the most profound oxygen-dependentviability behavior. The cells tolerated the addition of a moderate concentration of beta-cyclodextrin to the polymer matrix.

Lidocaine Inhibits Rat Prostate Cancer Cell Invasiveness and Voltage-Gated Sodium Channel Expression in Plasma Membrane.

There is increasing evidence, mostly from breast cancer, that use of local anaesthetics during surgery can inhibit disease recurrence by suppressing the motility of the cancer cells dependent on inherent voltage-gated sodium channels (VGSCs). Here, the possibility that lidocaine could affect cellular behaviours associated with metastasis was tested using the Dunning cell model of rat prostate cancer. Mostly, the strongly metastatic (VGSC-expressing) Mat-LyLu cells were used under both normoxic and hypoxic conditions. The weakly metastatic AT-2 cells served for comparison in some experiments. Lidocaine (1-500μM) had no effect on cell viability or growth but suppressed Matrigel invasion dose dependently in both normoxia and hypoxia. Used as a control, tetrodotoxin produced similar effects. Exposure to hypoxia increased Nav1.7 mRNA expression but VGSCα protein level in plasma membrane was reduced. Lidocaine under both normoxia and hypoxia had no effect on Nav1.7 mRNA expression. VGSCα protein expression was suppressed by lidocaine under normoxia but no effect was seen in hypoxia. It is concluded that lidocaine can suppress prostate cancer invasiveness without effecting cellular growth or viability. Extended to the clinic, the results would suggest that use of lidocaine, and possibly other local anaesthetics, during surgery can suppress any tendency for post-operative progression of prostate cancer.

Instantaneous extracellular solution exchange for concurrent evaluation of membrane permeability of single cells.

The osmotic stress imposed on microorganisms by hypotonic conditions is perceived to regulate water and solute flux via cell membranes, which are crucial for survival. Some cells that fail to perceive osmotic stress die because this results in the rupture of the cell membrane. The flux through the membrane is characterized by the membrane permeability, which is measured using a stopped-flow apparatus in response to a millisecond-order osmolarity change. However, the obtained data are an ensemble average of each cell response. Additionally, the measurement of permeability, considering cellular viability, contributes to a more accurate evaluation of osmoadaptation. Here, we present a novel on-chip instantaneous extracellular solution exchange method using an air-liquid interface. The presented method provides a concurrent evaluation at the single-cell level in response to a millisecond-order osmotic shock, considering cellular viability by solution exchange. This method utilizes a liquid bridge with a locally formed droplet on the surface of a micropillar fabricated inside a microchannel. We evaluated a solution exchange time of 3.6 ms and applied this method to Synechocystis PCC 6803 under two different osmolarity conditions. The live/dead ratio of 1 M to 0.5 M osmotic down shock condition was 78.8/21.2% while that of 1 M to 0.25 M osmotic down shock condition was 40.0/60.0%. We evaluated the water permeability of two groups: cells that were still live before and after osmotic shock (hereafter named cell type 1), and cells that were live before but were dead 10 minutes after osmotic shock (hereafter named cell type 2). The results indicated that the water permeability of cell type 2 was higher than that of cell type 1. The results obtained using the presented methods confirmed that the effect of osmotic stress can be accurately evaluated using single-cell analysis.

The protective effects of alpha-pinene on high glucose-induced oxidative stress and inflammation in HepG2 cells.

Hyperglycemia, a prevalent metabolic condition observed in diabetes, leads to oxidative damage, inflammatory responses, and other consequences. Natural compounds alleviate the adverse impacts of diabetes. We aimed to explore the effects of alpha-pinene (AP) as a monoterpene on oxidative damage and inflammation caused by high glucose (HG) in the human hepatocellular liver carcinoma (HepG2) cell line. The HepG2 cells were subjected to non or HG concentration (50 mM) and treated with or without AP (8, 16, and 32 μg/ml) for 48 hr. The effect of treatments on cellular viability, malondialdehyde (MDA), glutathione (GSH), and activity of anti-oxidant enzymes, including glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), was determined. The gene expression levels of nuclear factor-κβ (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and dipeptidyl peptidase-4 (DPP-4) were estimated using quantitative real-time polymerase chain reaction (qRT-PCR). HG exposure significantly increased cell death, MDA formation, and depletion of GSH content and GPx, CAT, and SOD activity (P<0.05). We have also seen a significant induction in NF-κB, TNF-α, IL-6, and DPP-4 gene expression in hepatocytes under HG conditions (P<0.05). Interestingly, co-treatment with AP in a dose-dependent manner improved cell death and altered levels of MDA and GSH, and activity of GPx and CAT (P<0.05). AP could also modulate the gene expression of NF-κB and inflammatory biomarkers dose-dependently (P<0.05). Our findings suggested the protective effect of AP on hepatocytes under HG conditions through attenuating oxidative stress markers and suppression of inflammatory pathways.

Co-culture of STRO1 + human gingival mesenchymal stem cells and human umbilical vein endothelial cells in 3D spheroids: enhanced in vitro osteogenic and angiogenic capacities.

Stem cell spheroid is a promising graft substitute for bone tissue engineering. Spheroids obtained by 3D culture of STRO1+ Gingival Mesenchymal Stem Cells (sGMSCs) (sGMSC spheroids, GS) seldom express angiogenic factors, limiting their angiogenic differentiation in vivo. This study introduced a novel stem cell spheroid with osteogenic and angiogenic potential through 3D co-culture of sGMSCs and Human Umbilical Vein Endothelial Cells (HUVECs) (sGMSC/HUVEC spheroids, GHS). GHS with varying seeding ratios of sGMSCs to HUVECs (GHR) were developed. Cell fusion within the GHS system was observed via immunofluorescence. Calcein-AM/PI staining and chemiluminescence assay indicated cellular viability within the GHS. Furthermore, osteogenic and angiogenic markers, including ALP, OCN, RUNX2, CD31, and VEGFA, were quantified and compared with the control group comprising solely of sGMSCs (GS). Incorporating HUVECs into GHS extended cell viability and stability, initiated the expression of angiogenic factors CD31 and VEGFA, and upregulated the expression of osteogenic factors ALP, OCN, and RUNX2, especially when GHS with a GHR of 1:1. Taken together, GHS, derived from the 3D co-culture of sGMSCs and HUVECs, enhanced osteogenic and angiogenic capacities in vitro, extending the application of cell therapy in bone tissue engineering.

Cellular responses to 3D printed dental resins produced using a manufacturer recommended printer versus a third party printer.

The aim of this study was to evaluate the influence of different 3D dental resins, using a manufacturer recommended printer and a third-party printer, on cellular responses of human gingival cells. Three NextDent resins (Denture 3D+, C&B MFH and Crowntec) were used to produce specimens on printers NextDent 5100 (groups ND, NC and NT, respectively) and Phrozen Sonic Mini 4K (groups PD, PC and PT, respectively). Human gingival fibroblasts were cultured and biocompatibility was evaluated on days 1, 3 and 7. IL-6 and IL-8 concentrations were evaluated at 3 days using ELISA. Surface roughness was evaluated by a contact profilometer. SEM and fluorescence micrographs were analyzed at days 1 and 7. Statistical analyses were performed using SPSS and mean differences were tested using ANOVA and post-hoc Tukey tests (P < .05). There was an increase in cellular viability after 7 days in groups PC and PT, when compared to group PD. ND group resulted in higher concentration of IL-6 when compared to PT group. SEM and fluorescence micrographs showed less adhesion and thinner morphology of fibroblasts from group PD. No significant differences were found regarding surface roughness. The use of different printers or resins did not seem to influence surface roughness. NextDent 5100 and Phrozen Sonic Mini 4K produced resins with similar cellular responses in human gingival fibroblasts. However, Denture 3D+ resin resulted in significantly lower biocompatibility, when compared to C&B MFH and Crowntec resins. Further testing is required to support its long-term use, required for complete dentures.

Segurança clínica à administração intravenosa de células mesenquimais alógenas em cães saudáveis

ABSTRACT Despite the significance of adipose-derived stem cells in complementary and regenerative medicine, studies regarding clinical safety for allogeneic intravenous administration in healthy dogs remain insufficiently conducted. This study aimed to assess cellular viability through laboratory and microbiological evidence, along with genetic markers, in addition to evaluating clinic-laboratory safety for allogeneic intravenous administration of adipose-derived stem cells in healthy dogs. To achieve this, two cell batches and eight dogs were included in the study. The results revealed positive genetic markers for CD29, CD44, CD105, SOX2, and OCT3.4. Both batches exhibited positive cell differentiation into adipocytes, chondrocytes, and osteoblasts. Microbiological evidence showed negative results, and cell viability after thawing indicated 92% and 88.5% viable cells after 30 minutes, and 86% and 83.5% after 24 hours, respectively, for both batches. Hematological cell counts and serum biochemical enzyme levels, before and after intravenous treatment, did not exhibit statistical differences between the time points (p&gt;0.05). Median values remained within the reference range for the species during and after 30 days of treatment. Based on the cellular viability results, observed patterns, and the absence of hematological side effects, it can be concluded that intravenous therapy with allogeneic adipose-derived stem cells is clinically safe.

Improving in vitro screening compounds anti-Trypanosoma cruzi by GFP-expressing parasites.

Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.