Eagle's Medium Research Articles

Effects of Polyunsaturated Fatty Acid/Saturated Fatty Acid Ratio and Different Amounts of Monounsaturated Fatty Acids on Adipogenesis in 3T3-L1 Cells.

(1) Background: Adipose tissue serves as a central repository for energy storage and is an endocrine organ capable of secreting various adipokines, including leptin and adiponectin. These adipokines exert profound influences on diverse physiological processes such as insulin sensitivity, appetite regulation, lipid metabolism, energy homeostasis, and body weight. Given the integral role of adipose tissue in metabolic regulation, it is imperative to investigate the effects of varying proportions and types of dietary fats on adipocyte function. In addition, our previous study showed that P/S = 5 and MUFA = 60% appeared to be beneficial in preventing white adipose tissue accumulation by decreasing plasma insulin levels and increasing hepatic lipolytic enzyme activities involved in β-oxidation. Therefore, the objective of this study was to explore the effects of a polyunsaturated fatty acid (PUFA) to saturated fatty acid (SFA) ratio of 5 and varying levels of monounsaturated fatty acids (MUFA = 30% or 60%) on lipogenesis. (2) Methods: We cultured 3T3-L1 mouse embryo fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum until confluent. Varying ratios of palmitic acid (PA), oleic acid (OA), and linoleic acid (LA) were first bound with bovine serum albumin (BSA) before being applied to 3T3-L1 adipocytes in low doses and in high doses. (3) Results: Low doses of P/S ratio = 5, MUFA = 60% (M60) fatty acids decreased the accumulation of triglycerides in mature adipocytes by decreasing the mRNA expression of adipogenic factors, such as peroxisome proliferator-activated receptors (PPARs), lipoprotein lipase (LPL), and glucose transporter-4 (GLUT-4), while increasing lipolytic enzyme (hormone-sensitive lipase, HSL) expression when compared to high doses of P/S ratio = 5, MUFA = 60% (M60), low and high doses of P/S ratio = 5, MUFA = 30% (M30). Furthermore, the treatment of M60 in low doses also decreased the secretion of leptin and increased the secretion of adiponectin in adipocytes. (4) Conclusions: The composition of P/S = 5, MUFA = 60% fatty acid in low doses appeared to result in anti-adipogenic effects on 3T3-L1 adipocytes due to the down-regulation of adipogenic effects and the transcription factor.

Anti-inflammatory effects of hesperidin on human gingival fibroblasts stimulated by lipopolysaccharide of Porphyromonas gingivalis in vitro.

Periodontal disease is an inflammatory disease caused by periodontopathogenic bacteria, the inflammatory response generated against them, and host factors. Furthermore, environmental factors can lead to disease progression. Using lipopolysaccharide (LPS)-stimulated human gingival fibroblast (HGF), this study investigated the bioactivity of HGF after exposure to hesperidin (Hesp) and the anti-inflammatory activity of Hesp against early periodontitis. HGF were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum. They were exposed to LPS for 6h, followed by Hesp (1, 10, 30, and 50µM) exposure for 4h. Cell proliferation was evaluated using reduction staining with alamerBlue™. Inflammatory cytokines [interleukin (IL)-6 and IL-8] and Toll-like receptor 4 (TLR4) levels were assessed using reverse transcription quantitative polymerase chain reaction. Hesp 50µM + LPS inhibited cell proliferation. The Hesp exposure group inhibited the expression of IL-8 and IL-6. No significant difference in TLR4 expression was observed. Hesp significantly suppressed IL-6 and IL-8 expression by inhibiting downstream signaling without inhibiting TLR4 activation.

3,3',5-Triiodothyroacetic Acid Transporters.

Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.

Effect of titanium dioxide nanoparticle (TiO2-NP) exposure in a novel Amur sturgeon Acipenser schrenckii hepatocyte cell line.

In vitro cell culture is crucial for predicting the toxicity of titanium dioxide nanoparticle (TiO2-NP). However, assessing the toxicity of TiO2-NPs in sturgeon remains difficult given the lack of sufficient cell lines. We established and characterized the first hepatocyte cell line from Acipenser schrenckii liver tissue (ASL). This ASL cell line proliferated well in Dulbecco's modified Eagle's medium at 25°C and 10% fetal bovine serum. ASL cells with a chromosome number of 244 were successfully transfected with the pEGFP-N3 plasmid. The ASL cell line's origin was verified as A. schrenckii through mitochondrial cytochrome C oxidase I and mitochondrial 16S ribosomal RNA (rRNA) sequencing. Using the ASL cell line as an in vitro model, we found that TiO2-NP exposure decreased the viability and promoted the damage of ASL cells (96-h LC50 = 331.8 μg mL-1). Increased reactive oxygen species and malondialdehyde levels in ASL cells suggested oxidative stress under TiO2-NP exposure. We also observed dysregulation of aspartate aminotransferase and alanine aminotransferase levels. By detecting calcium ions and mitochondrial membrane potential indicators, we found that the apoptotic pathway induced by endoplasmic reticulum stress played a major role at low concentrations of TiO2-NP-induced stress. Both mitochondria-mediated and endoplasmic reticulum stress promoted apoptosis under increasing TiO2-NP concentrations. In conclusion, the ASL cell line established in this study is a useful in vitro model for toxicological studies of TiO2-NP exposure in fish.

Efficient expansion culture of bovine myogenic cells with differentiation capacity using muscle extract-supplemented medium

The production of tissue-engineered skeletal muscles for cell-based therapy and cultivated meat production requires a large number of myogenic cells capable of differentiating into myotubes. A suspension culture with a stirred bioreactor is a system that is feasibly scalable, but scaling up the production of myogenic cells while maintaining differentiation capacity requires an efficient culture technique. This remains challenging in suspension culture. On the other hand, conventional meat processing generates substantial by-products, including meat trimmings containing effective substances for myogenic cell cultures. Efficiently utilizing these by-products can reduce environmental impact and disposal costs. Here, we describe an expansion culture method for bovine myogenic cells (BMCs) using Dulbecco's Modified Eagle's medium (DMEM) supplemented with muscle extracts. During the 6-day planar culture, the proliferation rate using the culture medium supplemented with muscle extracts (CMME) and 10% fetal bovine serum (FBS) was 18 ± 3.3, three times higher than that achieved using DMEM with 10% FBS. Furthermore, a combination of CMME supplemented with 10% FBS and an iMatrix-511-coated dish could maintain differentiation capacity through passaging culture. Finally, we demonstrated a suspension culture with microcarriers using CMME with 10% FBS. Through 3 passages, the cumulative proliferation rate was 191.7 ± 35.1, four times higher than that of produced DMEM with 10% FBS. Using CMME, the cells maintained differentiation capacity in comparison to that using DMEM. This approach will contribute to both the reduction of wasted by-products from conventional meat production and the development of a suspension culture system for maintaining high-quality myogenic cells.

Effectiveness and safety of biosilicate-enhanced bleaching gels on enamel with early erosion lesion.

This study evaluated the efficacy and cytotoxicity of 35% hydrogen peroxide (HP) gel incorporated with 10% (w/w) biosilicate (BioS) on sound enamel and early-stage enamel erosion lesions. Discs of enamel/dentin were selected, subjected to erosive cycles (0.3% citric acid, pH 2.6), and treated with (n = 8): HP (35% HP, positive control); HP_BioS [carboxymethyl cellulose (CMC) + HP + BioS]; BioS (CMC + BioS); CMC (negative control). The discs were adapted to artificial pulp chambers with the enamel exposed for bleaching, and the dentin facing toward the culture medium (Dulbecco's modified Eagle's medium [DMEM]). Bleaching was performed in three 30-min sessions at 7-day intervals. After bleaching, the diffusion product (DMEM extract + diffused HP) was pipetted onto MDPC-23 odontoblastic cell line and inoculated. Color parameters (ΔL, Δa, Δb), color change (ΔE00), and changes in whiteness index (ΔWID) were determined before (T0) and after the last bleaching session (T3). Cell viability (MTT, %), H2O2 diffusion (μg/mL), oxidative cell stress (OxS), and cell fluorescence (live/dead assay, in confocal microscopy) were assessed (ANOVA/Tukey; α = 0.05). No difference in ΔL, Δa, Δb, ΔE00, and ΔWID were found between HP and HP_BioS (p > 0.05). The incorporation of BioS decreased the HP diffusion into the substrates and mitigated oxidative stress in early-stage eroded enamel (p < 0.05). HP_BioS presented significantly higher cell viability compared with HP under erosion conditions. Live/dead assay indicated that BioS_HP maintained viability with larger clusters of viable cells. Incorporating BioS into HP maintained bleaching effectiveness, favored cell viability, reduced the oxidative stress, and the cytotoxicity in teeth with early-stage erosion. BioS formulation showed promising results for reducing cytotoxicity in patients seeking tooth bleaching and presenting undetectable early-stage erosion.

Rapid and simple isolation of precartilaginous stem cells: A novel approach without immunomagnetic bead sorting

ObjectiveThe present investigation was designed to devise a rapid and straightforward technique for the isolation of rat precartilaginous stem cells (PCSCs) that eschews the use of immunomagnetic bead sorting. MethodRat neonates within 24 h of birth were selected for this study. Microsurgical techniques were used to harvest the femur, tibia, and the musculature of the knee joint. The ring of LaCroix between the metaphysis of the femur and the epiphysis was excised and divided into fragments of approximately 1 mm³. Tissue sections were cultured in Dulbecco's modified Eagle's medium(DMEM)/F12 medium supplemented with 20 % fetal bovine serum and 1 % penicillin-streptomycin. Cell digestion and passaging were performed using trypsin when cells reached 70%–80 % confluence. Third-generation cells underwent immunofluorescence staining and flow cytometry to evaluate fibroblast growth factor receptor-3(FGFR-3) and proliferating cell nuclear antigen(PCNA) expression, while β-galactosidase staining was used to determine cellular senescence. ResultsWithin two days of isolation, numerous short spindle-shaped cells exhibiting distinct refractive properties were observed around the tissue fragments. These cells began to proliferate within 2–3 days and displayed ample cytoplasm. Adherent cells adopted various morphologies, including angular, triangular, and elongated spindles. By the fifth day, more than 80 % of the culture dish surface was covered with elongated cells, with some arranged in patterns reminiscent of whirlpools. Significant FGFR-3 and PCNA expression was confirmed via immunofluorescence in the third-generation cells. Additionally, flow cytometry identified that the proportion of cells positive for FGFR-3 and PCNA exceeded 98 %. Notably, the cells preserved their proliferative capacity through nine passages in vitro, with a marginal proportion showing senescence as indicated by β-galactosidase staining alone. ConclusionThe developed tissue adherence protocol was used to successfully isolate PCSCs with positive FGFR-3 and PCNA expression, rendering the immunomagnetic bead sorting superfluous. The expression of FGFR-3 and PCNA in the isolated cells persisted through the ninth passage in vitro with minimal senescence.

Leaching and cytotoxicity of bismuth oxide in ProRoot MTA - A laboratory investigation.

The present study examined the leaching and cytotoxicity of bismuth from ProRoot MTA and aimed to identify whether bismuth leaching was affected by the cement base and the immersion regime used. The leaching profile of bismuth was examined from ProRoot MTA and compared with hydroxyapatite containing 20% bismuth oxide as well as hydroxyapatite and tricalcium silicate to investigate whether bismuth release changed depending on the cement base. Bismuth leaching was determined after 30 and 180 days of ageing immersed in Dulbecco's modified Eagle's medium (DMEM) using mass spectroscopy (ICP-MS). The media were either unchanged or regularly replenished. The pH, surface microstructure and phase changes of aged materials were assessed. Wistar rat femoral bone marrow stromal cells (BMSCs) and cutaneous fibroblasts were isolated, cultured and seeded for cell counting (trypan blue live/dead) after exposure to non-aged, 30- and 180-days-aged samples in regularly replenished DMEM. Aged DMEM in contact with materials was also used to culture BMSCs to investigate the effect of material leachates on the cells. Gene expression analysis was also carried out after direct exposure of cells to non-aged materials. Differences between groups were statistically tested at a significance level of 5%. All materials exhibited alterations after immersion in DMEM and this increased with longer exposure times. The bismuth leached from ProRoot MTA as detected by ICP-MS. Aged ProRoot MTA samples exhibited a black discolouration and surface calcium carbonate deposition. ProRoot MTA influenced cell counts after direct exposure and its 180-days leachates reduced BMSC viability. After direct BMSC contact with non-aged ProRoot MTA an upregulation of metallothionein (MT1 and MT2A) expression and down-regulation of collagen-1a (Col-1a) and bone sialoprotein (BSP) expression was identified. Bismuth leaching was observed throughout 180-days observation period from all materials containing bismuth oxide. This negatively influenced cell viability and gene expression associated with bismuth exposure. This is the first study to report that metallothionein gene expression was influenced by exposure to ProRoot MTA.

Evaluation of Cardiospermum halicacabum on Bone Morphogenetic Protein-2 (BMP2) mRNA Expression in Osteoblast Cells.

Introduction Maintaining bone health is crucial for overall well-being, with osteoblasts playing a vital role in bone formation. Bone morphogenetic protein-2 (BMP2) is a key regulator, stimulating bone matrix synthesis and osteoblast differentiation. Recognizing BMP2's significance, there's growing interest in natural compounds, such as Cardiospermum halicacabum. This study explores Cardiospermum halicacabum's potential influence on BMP2 mRNA expression in osteoblast cells for insights into bone health modulation. Materials and methods This research utilized Cardiospermum halicacabum to explore its impact on MG-63 cells, a human osteoblast cell line. Osteoblast cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, and maintained at 37°C in a 5% CO2 and 95% air environment. Cell viability was evaluated by seeding osteoblast cells into 96-well plates and exposing them to different concentrations of Cardiospermum halicacabum (2.0 μg/ml and 20 μg/ml). The study observed both the promotion of osteoblast cell growth in MG-63 and morphological changes in thecells under an inverted light microscope at 10x magnification. Results were presented using one-way analysis of variance (ANOVA) conducted with IBM SPSS Statistics for Windows, Version 23 (Released 2015; IBM Corp., Armonk, New York, United States). Result The reverse transcription-polymerase chain (RT-PCR) results revealed an increased expression of BMP-2 mRNA fold change in comparison to the control group. A clear positive correlation was observed between the BMP-2 mRNA fold change and the notable increase in the concentration of Cardiospermum halicacabum. This investigation revealed a direct association of BMP-2 mRNA expressionwith the proliferation of osteoblast cells. Specifically, the BMP-2 mRNA fold change was recorded at 2.26±1.05 in Cardiospermum halicacabum at 2.0 μg/ml and 2.0 ± 0.84 at 20 μg/ml, with corresponding significances of 0.00, respectively. Conclusion Potential effects of Cardiospermum halicacabum on BMP-2 mRNA expression in osteoblast cellsand its role in bone health modulation revealed that Cardiospermum halicacabum may upregulate BMP-2 mRNA expression, suggesting its potential as a natural compound for enhancing bone formation. The observed positive correlation between Cardiospermum halicacabum concentration and BMP-2 mRNA fold change showed the significance of this botanical agent in promoting osteoblast cell proliferation. These results highlight the importance of further research to explore the applications of Cardiospermum halicacabum in managing bone disorders and improving overall bone health.

Relative Humidity-Dependent Phase Transitions in Submicron Respiratory Aerosols.

Respiratory viruses, such as influenza and severe acute respiratory syndrome coronavirus 2, represent a substantial public health burden and are largely transmitted through respiratory droplets and aerosols. Environmental factors such as relative humidity (RH) and temperature impact virus transmission rates, and a precise mechanistic understanding of the connection between these environmental factors and virus transmission would improve efforts to mitigate respiratory disease transmission. Previous studies on supermicrometer particles observed RH-dependent phase transitions and linked particle phase state to virus viability. Phase transitions in atmospheric aerosols are dependent on size in the submicrometer range, and actual respiratory particles are expelled over a large size range, including submicrometer aerosols that can transmit diseases over long distances. Here, we directly investigated the phase transitions of submicrometer model respiratory aerosols. A probe molecule, Nile red, was added to particle systems including multiple mucin/salt mixtures, a growth medium, and simulated lung fluid. For each system, the polarity-dependent fluorescence emission was measured following RH conditioning. Notably, the fluorescence measurements of mucin/NaCl and Dulbecco's modified Eagle's medium particles indicated that liquid-liquid phase separation (LLPS) also occurs in submicron particles, suggesting that LLPS can also impact the viability of viruses in submicron particles and thus affect aerosol virus transmission. Furthermore, the utility of fluorescence-based measurements to study submicrometer respiratory particle physicochemical properties in situ is demonstrated.

IMPACT OF A GEL COMPOSITION CONTAINING VARIOUS SUBSTANCES ON INDICES OF IMMUNITY AND TISSUE INFLAMMATION IN PALATINE TONSILS OF PATIENTS WITH CATARRHAL GINGIVITIS DURING CHRONIC TONSILLITIS

The study of pharmacotherapeutic (immunomodulating) action in conditions of in vitro cell cultures is currently considered appropriate and justified for determining the action of drugs at the preclinical level. It is also important to address the pathogenetic nature in using such agents, especially fro local application. This study investigated the efficacy of a multicomponent gel containing decamethoxine and hyaluronic acid in treating chronic catarrhal gingivitis among patients with chronic tonsillitis. The purpose of the work is to assess the efficacy of a gel containing decamethoxine and hyaluronic acid on the tissue of the palatine tonsils in the therapy of chronic catarrhal gingivitis in patients with chronic tonsillitis.&#x0D; Materials and methods. To investigate the effects of the main active components in the gel, we followed the recommendations of I.P. Kaidashev et al. for testing pharmacological preparations in in vitro cultures. Tissue samples from the palatine tonsils were obtained from 20 patients with chronic tonsillitis and chronic catarrhal gingivitis undergoing tonsillectomy. All procedures adhered to the guidelines set by the Laboratory of Pathophysiology and Immunology at Prof. O.S. Kolomiichenko Institute of Otolaryngology, National Academy of Sciences of Ukraine. Palatine tonsil tissue was incubated in 199 medium (Serva, Germany) containing gentamicin sulfate (Darnytsa, Ukraine) at a concentration of 120 μg/ml for 20 minutes at 4-8°C. After that, the hemorrhagic parts were removed from the whole tonsil and placed into Eagle's medium (Sigma, USA) and rinsed three to four times. Subsequently, the prepared tissue was placed in sterile vials containing enriched Eagle's medium supplemented with L-glutamine, sodium bicarbonate, 100x vitamin concentrate, 5% fetal calf serum, and 60 μg/mL gentamicin. Tonsil tissue was dissected using scissors and then washed repeatedly (5-7 times) with fresh medium to remove loosely attached cells, then, the resulting cell suspension was filtered. All procedures were conducted within a sterile laminar flow cabinet. Cell viability was assessed using light microscopy (Olympus-23, Japan) and a hemocytometer. A final cell suspension containing 2.5 million lymphocytes per milliliter of medium was prepared. Suspensions with a viability of less than 90% (blue, "dead" cells) were excluded from further experiments.&#x0D; Results. Treatment of in vitro palatine tonsil cells from patients with chronic catarrhal gingivitis and recurrent tonsillitis with the main components of the developed gel (decamethoxine and hyaluronic acid) did not induce significant changes in the concentration of α and γ-interferons. Our findings suggest that the gel composition reduces the levels of pro-inflammatory factors: interleukin-1β and immune complexes. Additionally, it appears to stimulate the production of the antimicrobial factor antistreptolysin-O by tonsil cells, potentially leading to increased antibody production against hemolytic streptococcus antigens.&#x0D; Conclusion. Based on the in vitro findings, the investigated gel composition containing decamethoxine and hyaluronic acid demonstrates potential anti-inflammatory and antimicrobial properties that stimulate the production of the antimicrobial factor antistreptolysin-O by tonsil cells, which might lead to increased antibody production against hemolytic streptococcus antigens.

Overcoming Breast Cancer Drug Resistance: A Novel Approach Using siRNA-Mediated P-glycoprotein Downregulation to Enhance Vinorelbine Efficacy.

Cancer, the second leading cause of mortality worldwide, represents a global health challenge, primarily due to drug resistance. Vinorelbine is a chemotherapeutic agent that disrupts cancer cell growth by targeting microtubules and inducing apoptosis. However, drug resistance remains a formidable obstacle. This resistance is caused by various factors including genetic mutations, drug efflux mechanisms, and DNA repair systems. Resolution of this challenge requires an innovative approach. This study investigated the potential of small interfering RNA (siRNA) to target and downregulate a vinorelbine-resistant MCF-7/ADR breast cancer cell line. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) 10% fetal bovine serum/penicillin/streptomycin. An siRNA targeting ABCB1 was designed and synthesized, and the cells were transfected with siRNA at final concentrations of 10, 20, and 30 nM. The3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess cell viability. ABCB1 mRNA expression levels were determined by real-time polymerase chain reaction (PCR). MCF-7 cells exhibited a higher sensitivity to vinorelbine than MCF-7/ADR cells. MCF-7/ADR cells exhibited resistance to vinorelbine at concentrations, 12.50 and 25.00 μM. Treatment with siRNA significantly reduced ABCB1 expression by 2.93-fold (P=0.0001). Similarly, co-treatment with siRNA and vinorelbine produced a substantial 2.89-fold decrease in ABCB1 gene expression in MCF-7 cells compared to that in MCF-7/ADR cells (P=0.0001). The results of the present study indicate that the concurrent use of siRNA and vinorelbine holds substantial promise as a therapeutic approach to overcome ABCB1-mediated multidrug resistance (MDR) in breast cancer. It is necessary to conduct comprehensive clinical trials to determine the true effectiveness of this combination therapy.

Osteogenic Differentiation and Proliferation of Apical Papilla Stem Cells Using Chitosan-Coated Nanohydroxyapatite and Bioactive Glass Nanoparticles.

The aim of this study was to evaluate the osteogenic differentiation ability and proliferation of apical papilla stem cells (SCAPs) using chitosan-coated nanohydroxyapatite and bioactive glass nanoparticles. Hydroxyapatite, chitosan-coated nanohydroxyapatite, and bioactive glass 45S5 nanoparticles were prepared and characterized using a transmission electron microscope and X-ray diffraction. SCAPs were harvested from freshly extracted impacted wisdom teeth, cultured, and characterized using flow cytometric analysis. Tested nanomaterials were mixed and samples were classified into five equal groups as follows: negative control group: SCAP with Dulbecco's modified eagle's medium, positive control group: SCAP with inductive media, first experimental group: nanohydroxyapatite with SCAP, second experimental group: chitosan-coated nanohydroxyapatite with SCAP, third experimental group: bioactive glass nanoparticles with SCAP. Osteoblastic differentiation was assessed using an alkaline phosphatase (ALP) assay. Receptor activator of nuclear factor kappa beta ligand (RANKL) expression was evaluated using specific polyclonal antibodies by fluorescence microscope. The proliferation of SCAP was assessed using cell count and viability of trypan blue in addition to an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Isolated SCAP showed a nonhematopoietic origin. Chitosan-coated nanohydroxyapatite showed the highest ALP concentration followed by nanobioactive glass, nanohydroxyapatite, and negative control. Chitosan-coated nanohydroxyapatite showed the highest H score followed by nanobioactive glass, nanohydroxyapatite, and negative control in RANKL expression. Chitosan-coated nanohydroxyapatite showed the highest viable cell count. SCAP isolation is achievable from extracted fully impacted immature third molars. All tested biomaterials have the ability to induce osteogenic differentiation and proliferation of SCAP. Composite nanoparticle materials show better osteogenic differentiation and proliferation of SCAP than single nanoparticles.

Bone marrow stem cells with or without superparamagnetic iron oxide nanoparticles as a magnetic targeting tool: Which is better in regeneration of neurolysed facial nerve? An experimental study

AimThis study was performed to evaluate neural regenerative capacities of bone marrow stem cells (BMSCs) with or without superparamagnetic iron oxide nanoparticles (SPIONs) as a magnetic targeting tool after neurolysis of the facial nerve (FN) in albino rats. MethodsThirty-eight male albino rats were selected. Two of them were euthanized for normal FN histology assessment. Thirty-six rats were injected with ethanol in the FN nerve for neurolysis induction and assessed one week post-operatively by eye blinking test. Animals were divided into three groups, each containing twelve rats: Group I (positive control) was injected with Dulbecco Modified Eagle's medium (DMEM-F12), group II was injected with BMSCs in DMEM-F12, and group III was injected with BMSCs in DMEM-F12 with poly l-lysine coated SPIONs (0.5 mmol/mL). Monitoring of SPIONs in the rat's body was carried out by MRI. A circular neodymium magnet N52 (0.57 T, 2 × 5 mm) was placed on each rat in group III just below the right ear at the site of surgery to attract SPIONs labeled BMSCs, left in place for 24 h, and then removed. From each group, six rats were euthanized at the end of the 4th and 8th week of treatment, respectively. The right FN trunks were extracted for routine histological examination using H&E stain. Immunohistochemical examination by anti-S100B was performed to characterize the thickness of the myelin sheath formed by the Schwann cells. Ultra-structural examination was performed to study changes in axons, myelin sheaths, and Schwann cells. ResultsRegeneration of nerve fibers, Schwan cells, and myelin sheaths was better in group II than in groups I and III histologically, immunohistochemically, and ultra-structurally. ConclusionBMSCs alone could ameliorate FN regeneration better than magnetic targeting treatment using BMSCs labeled with SPIONs.

Inhibition of Sodium-Glucose Cotransporter-2 during Serum Deprivation Increases Hepatic Gluconeogenesis via the AMPK/AKT/FOXO Signaling Pathway.

Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states. Huh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco's modified Eagle's medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours. SGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation. These data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.

Effects of media composition and light exposure on the electrochemical current response during scanning electrochemical microscopy live cell imaging.

Scanning Electrochemical Microscopy (SECM) has been used as a non-invasive electrochemical technique for studying cellular processes. SECM enables the quantification of cellular metabolites in real-time providing a deeper understanding of cellular responses to external stimuli. SECM imaging of living cells requires maintaining an ideal physiological environment to ensure reliable data collection on cellular reactivity. The cellular response can be directly influenced by physicochemical parameters including cell media composition, temperature and light exposure. This research demonstrates the effect of media composition on the electrochemical current signal of adenocarcinoma cervical cancer (HeLa) cells during SECM measurements using ferrocenemethanol as a redox mediator. Investigated media that are commonly used as electrolyte, are phosphate buffered saline (PBS), and Dulbecco's modified Eagle's medium (DMEM) in the absence and presence of fetal bovine serum (FBS). In addition, this research demonstrates that fluctuating light illumination impacts the stability of the cellular electrochemical current response. Our findings reveal that media composition and illumination are important parameters that must be carefully considered and monitored during SECM live cell imaging.

Phosphonoformic acid reduces hyperphosphatemia-induced vascular calcification via Pit-1.

This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC). Primary human aortic smooth muscle cells and rat aortic rings were cultured in Dulbecco's modified Eagle's medium supplemented with 0.9 mM or 2.5 mM phosphorus concentrations. Type III sodium-dependent phosphate cotransporter-1 (Pit-1) small interfering RNA and phosphonoformic acid (PFA), a Pit-1 inhibitor, were used to investigate the effects and mechanisms of Pit-1 on HPVC. Calcium content shown by Alizarin red staining, expression levels of Pit-1, and characteristic molecules for phenotypic transition of vascular smooth muscle cells were examined. Hyperphosphatemia induced the upregulation of Pit-1 expression, facilitated phenotypic transition of vascular smooth muscle cells, and led to HPVC in cellular and organ models. Treatment with Pit-1 small interfering RNA or PFA significantly inhibited Pit-1 expression, suppressed phenotypic transition, and attenuated HPVC. Our findings suggest that Pit-1 plays a pivotal role in the development of HPVC. The use of PFA as a Pit-1 inhibitor has the potential for therapeutic intervention in patients with HPVC. However, further rigorous clinical investigations are required to ensure the safety and efficacy of PFA before it can be considered for widespread implementation in clinical practice.

Recombinant Methioninase Decreased the Effective Dose of Irinotecan by 15-fold Against Colon Cancer Cells: A Strategy for Effective Low-toxicity Treatment of Colon Cancer.

Irinotecan (IRN), a topoisomerase I inhibitor and pro-drug of SN-38, is first-line treatment of colon cancer as part of FOLFIRI and FOLFOXIRI combination chemotherapy. However, IRN causes dose-limiting adverse events such as neutropenia and diarrhea. Dose reductions are sometimes required, which reduce efficacy. Recombinant methioninase (rMETase) targets the fundamental basis of cancer, methionine addiction, known as the Hoffman effect, and enhances the efficacy of numerous chemotherapy drugs. The present study determined the efficacy of rMETase when administered in combination with IRN. Cell viability was assessed by cultivating the HCT-116 human colorectal cancer cell line in 96-well plates at 1×103 cells per well in Dulbecco's modified Eagle's medium (DMEM). Subsequently, HCT-116 cells were treated with increasing concentrations of SN-38, the active form of IRN, ranging from 0.5 nM to 32 nM, and/or rMETase ranging from 0.125 to 8 U/ml. After treatment for 72 h, the half-maximal inhibitory concentration (IC50) of SN-38 alone and rMETase alone for HCT-116 cells were determined. Using the IC50 concentration of rMETase, we determined the IC50 of SN-38 in combination with rMETase. Cell viability was determined with the cell-counting Kit-8 with the WST-8 reagent.. The IC50 of rMETase alone for the HCT-116 cells was 0.55 U/ml, and the IC50 of IRN (SN-38) alone was 3.50 nM. rMETase at 0.55 U/ml lowered the IC50 of SN-38 to 0.232 nM (p<0.0001), a 15-fold reduction. rMETase and IRN are strongly synergistic, giving rise to the possibility of lowering the effective dose of IRN for the treatment of patients with colon cancer, thereby reducing its severe toxicity. This new strategy will allow more patients with cancer to be effectively treated with IRN.

Corrosion behaviour of electropolished magnesium materials

Although magnesium and its alloys are promising candidates as biodegradable implant materials, the tendency for localised corrosion mechanism in physiological environment limit their biomedical application. Electropolishing is an attractive strategy for improving the corrosion behaviour of metals, but it is still largely unexplored in magnesium materials. In this study, the characterisation of electropolished surfaces of AM50 and pure magnesium was performed, focussing on their in vitro degradation behaviour in cell medium. Corrosion rates were evaluated using potentiodynamic polarisation. The surface morphology before and after the onset of corrosion was investigated by scanning electron microscopy and confocal laser scanning microscopy. The presented electropolishing process led to improved surface performances, observable by significantly lower corrosion rates (0.08 mm·year−1 in Dulbecco's modified Eagle's medium), lower arithmetical mean height (0.05 µm), lower water contact angle (25–35°) and lower micro hardness (35–50 HV 0.1) compared to mechanically and chemically treated surfaces. MgO/Mg(OH)2 could be detected on electropolished surfaces. The localised corrosion mode could be reduced, but not entirely prevented. Electropolishing shows great potential as post-treatment of magnesium-based components, but detailed tests of the long-term corrosion behaviour are an important area of future research.

Establishment and characterization of a novel lung cell line derived from the common bottlenose dolphin.

Cetaceans are specialized marine mammals with a unique respiratory system adapted for diving behavior. Furthermore, respiratory diseases are commonly observed in these mammals. Nevertheless, much of their respiratory physiology remains unknown due to the limited supply and poor quality of their biological samples for research. In this study, we established a novel lung cell line, dLu, derived from the common bottlenose dolphin (Tursiops truncatus), which can prove useful in cetacean research, including for understanding the pathogenesis of respiratory diseases in cetaceans. The cells were cultured in a simple medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The morphology of the cells was fibroblast-like. dLu was produced by transfecting the simian virus 40 large T antigen into primary cultured cells. Although dLu exhibited approximately 80 cell divisions, it was unable to achieve complete immortalization, as the cells stopped proliferating beyond this number. dLu cells expressed toll-like receptor 3 but not toll-like receptor 4. Immunostimulation with poly(I:C) altered the gene expressions of interferon beta 1 and tumor necrosis factor alpha in dLu cells. In summary, dLu established in this study is a novel cetacean cell resource that can be easily cultured and is a useful in vitro tool in cetacean research, particularly for studying host immune responses in the lungs.