Dulbecco's Modified Eagle Medium Research Articles

Non-permeable Cryoprotectants' Influence on Fibroblast Slow Freezing in Six-banded Armadillo

BACKGROUND: There is a crucial need to develop appropriate cryopreservation solutions so that somatic resource biobanks of wildlife can be established. OBJECTIVE: Here, we propose a cryopreservation protocol to optimize the preservation of skin-derived fibroblasts from six-banded armadillos (Euphractus sexcinctus Linnaeus, 1758) by comparing different concentrations of fetal bovine serum (FBS) in the absence or presence of sucrose as non-permeable cryoprotectants. MATERIALS AND METHODS: Cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium (DMEM) with 10% dimethyl sulfoxide (DMSO), varying concentrations of FBS (10, 20 and 40%) without or with 0.2 M sucrose, totaling six comparison groups. Cells not subjected to cryopreservation were used as a control. Cells were evaluated for morphological characteristics, viability, metabolism, apoptosis levels, proliferative activity and mitochondrial membrane potential (ΔΨ m). RESULTS: Cells maintained similar fusiform morphology and demonstrated high viability (> 90%) before and after cryopreservation in all groups. Cryopreserved cells with 10 and 40% of FBS without sucrose showed lower metabolism, but, when sucrose was added, this parameter was maintained as in the control group. This effect was not observed in the 20% FBS groups in the absence or presence of sucrose, with viability similar to that of the non-cryopreserved group. The addition of sucrose maintained apoptosis levels, while the 20 and 40% FBS without sucrose groups showed alterations in viable, early apoptosis and necrosis stages. Nevertheless, all cryopreserved groups showed lower proliferative activity with a higher population doubling time (16.2-19.9 h) than the non-cryopreserved group (15.2 h). Finally, the 20% FBS groups, in the absence or presence of sucrose, maintained the ΔΨm. CONCLUSION: We demonstrated that 20% FBS with sucrose was the most suitable cryopreservation solution for six-banded armadillo skin-derived fibroblast lines, promoting high cell survival after thawing.

Bone marrow-derived mesenchymal stem cells reduce CCl4-induced kidney injury and fibrosis in male Wistar rats

Aim This study explores the possible therapeutic role of rats and mice bone marrow-derived mesenchymal stem cells (BM-MSCs) on renal damage and toxicity brought on by carbon tetrachloride (CCl4) in Wistar rats. Methods Following an intraperitoneal injection of CCl4 (0.5 mL/kg b.w. twice weekly) for eight weeks, male Wistar rats were intravenously treated with rats and mice BM-MSCs (1 × 106 cells in 0.2 mL Dulbecco’s Modified Eagle Medium (DMEM)/rat/week) a week for four weeks. Kidney functions were evaluated and kidney samples were examined using hematoxylin and eosin (H&E), Masson’s trichrome (MT) staining techniques, and electron microscopy analysis. Kidney cyclooxygenase-2 (COX-2), protein 53 (p53), and tumor necrosis factor-α (TNF-α) were detected by immunohistochemical staining techniques. Additionally, bioindicators of oxidative stress and antioxidant defense systems were identified in kidney tissue. Results In CCl4-injected rats, serum creatinine, urea, and uric acid levels significantly increased, as did renal lipid peroxidation (LPO), while superoxide dismutase, glutathione peroxidase (GPx), glutathione (GSH) transferase, and GSH levels significantly dropped in the kidneys. Histologically, the kidneys displayed a wide range of structural abnormalities, such as glomerular shrinkage, tubular dilations, inflammatory leukocytic infiltration, fibroblast proliferation, and elevated collagen content. Inflammatory cytokines like COX-2 and TNF-α as well as the pro-apoptotic mediator p53 were considerably upregulated. Treatment of BM-MSCs from mice and rats with CCl4-injected rats considerably reduced the previously noted abnormalities. Conclusions By boosting antioxidant defense and reducing apoptosis and inflammation, BM-MSCs from mice and rats were able to enhance kidney function and histological integrity in rats that had received CCl4 injections.

ChIP-seq and structural analyses delineating the regulatory mechanism of master regulator EsrB in Edwardsiella piscicida.

As a crucial virulence regulator, EsrB possesses a LuxR-like superfamily domain at the C-terminal, which is conserved within the canonical NarL family regulators. Due to its critically important role in virulence and pathogenicity in fish hosts, the DNA binding ability has been believed to allow EsrB to regulate genes associated with the invasion process of host cells and basal metabolism in response to environmental stimuli. The lack of EsrB's crystal structure has been a major obstacle in understanding the molecular mechanisms of EsrB-DNA interaction which choreographs EsrB-mediated pathogenic behavior. Here, we conducted ChIP-seq and solved the crystal structure of the C-terminal of EsrB (EsrBC) at 2.20-Å resolution, which revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7' pseudopalindromic DNA motif and interacted with metabolic-related promoters with a high AT DNA motif in Dulbecco's Modified Eagle's Medium (DMEM) (mimicking in vivo environments). Our results facilitate a detailed understanding of EsrB's regulatory role in Edwardsiella piscicida pathogenesis and expand our knowledge of virulence regulators in the family Hafniaceae.

The Effect of Curcumin Administration on p53 and Caspase-3 Expression in Cervical Cancer HeLa Cell Culture

Introduction: Cervical cancer is a type of cancer with 605,000 new cases globally each year, with a mortality rate of 340,000. Curcuma longa, or curcumin, has the potential to enhance apoptosis in cervical cancer cells through the NF-kB-p53-caspase-3 pathway. Material and Methods: This in vitro experimental study employed a Completely Randomized Design (CRD) using HeLa cell cultures derived from cervical adenocarcinoma (ATCC® CCL2™). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) and treated with curcumin at concentrations of 25 µg/mL, 50 µg/mL, 100 µg/mL, 150 µg/mL, and 250 µg/mL. Cytotoxicity was assessed using the CCK-8 assay, apoptosis via flow cytometry, and p53 and caspase-3 expression using immunofluorescence. Statistical analysis was performed with IBM Statistics SPSS 25. Results: There was an increase in the percentage of apoptosis, p53 expression, and caspase-3 expression. At a dose of 100 µg/mL, total apoptosis increased significantly, reaching nearly 25% (p=0.000). P53 expression significantly increased at curcumin doses of 50 µg/mL and 100 µg/mL (p=0.021). There was a significant increase in caspase-3 expression, reaching approximately 86.2%, with a maximum effect at a dose of 100 µg/mL (p=0.002). Conclusion: Curcumin shows promising potential as an anticancer agent by reducing cell viability and enhancing apoptotic activity in HeLa cervical cancer cells.

Comparative Analysis of Cytotoxicity and Odontogenic Differentiation of Novel Bioceramic Material on Human Dental Pulp Stem Cells – An In Vitro Study

ABSTRACT Background and Objective: Bioceramic materials have revolutionized the field of endodontics by successfully transforming the outcomes of pulp therapies. Novel biomaterials are evolving by modifying the conventional mineral trioxide aggregate (MTA) to overcome their existing limitations, the major ones being prolonged setting and cytotoxic radiopacifiers. Dental white portland cement (DWPC) is a novel formulated bioceramic material introduced as an enhanced MTA alternative. This in vitro study aims to investigate the cytotoxicity and odontogenic differentiation of novel formulation DWPC with MTA Angelus, Biodentin (BD) and white portland cement (WPC) on human dental pulp stem cells (HDPSCs). Materials and Methods: HDPSCs were cultured in Dulbecco’s modified eagle’s medium (DMEM) with fetal bovine serum (FBS) and antibiotics at 37°C, 90% humidity in a 5% CO2 incubator. Experimental samples were prepared as disks. The viability of HDPSCs was measured by Mosmann’s tetrazolium toxicity (MTT) assay, and odontogenic potential was assessed using alkaline phosphatase (ALP) activity at 24 hours, 7-, 14-, and 21-day intervals. The mean and standard deviations were statistically analyzed by one-way ANOVA and post hoc Scheffe tests using IBM SPSS Software (IBM statistical package for social sciences) – Version 24.0 with a significance level set as P < 0.05. Results: All four groups tested using MTT assay showed no toxicity and possess odontogenic potential in all the experimental durations. Experimental group DWPC presented with the highest mean cell viability and ALP activity at all intervals followed by WPC, MTA, and BD (P < 0.05). Conclusion: DWPC presented good bioactivity in terms of cell viability and ALP activity. Thus, DWPC could be a promising endodontic material. However, further research is warranted to explore the clinical applicability.

Self-powered water-based graphene photodetector for extremely rapid detection of SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly contagious virus, which has caused a serious public health crisis all over the world. Current coronavirus detection methods mainly rely on PCR technology, which needs high standard laboratory facilities and a long time to extract, react and analyze samples. In this report, a self-powered water-based photodetector (PD) biological detection device is designed to detect SARS-CoV-2 in the Dulbecco's Modified Eagle Medium (DMEM) solution. The graphene layer on top of liquid is covered with specific antibodies against SARS-CoV-2 spike protein. The device can detect SARS-CoV-2 exclusively with a concentration of 1 × 104 TCID50 in DMEM solution. It can push the detection limit down to 62 copies per μL with the detection time less than 100 ms. The work presents a rapid SARS-CoV-2 immunodiagnostic method without sample pretreatment or labeling.

Modified Hank's Balanced Salt Solution as a Storage Medium for Avulsed Teeth: InVitro Assessment of Periodontal Fibroblast Viability.

The optimal storage medium for an avulsed tooth should preserve the viability of periodontal fibroblasts (PDLF) to the highest degree, facilitating the re-attachment of periodontal fibers and improving the prognosis of replantation. This study compared the effect of the PDLF viability in Hank's balanced salt solution (HBSS), supplemented culture medium, that is, Dulbecco's Modified Eagle Medium (DMEM), and four modified HBSS mixtures. Periodontal tissues were obtained from extracted human teeth and processed for PDLF culture. The cells were then exposed to six experimental media: (i) HBSS, (ii) HBSS and ascorbic acid (HBSS + Vit C), (iii) HBSS and platelet-derived growth factor (HBSS + PDGF), (iv) a mixture of HBSS, PDGF, and Vit C (HBSS + PDGF + Vit C), (v) HBSS and platelet lysate (HBSS + PL), and (vi) DMEM for 3, 6, 12, and 24 h. A MTT assay was performed to determine the cell viability. Vitamin C-containing media maintained PDLF viability significantly better than HBSS + PDGF and HBSS + PL at 3, 6, 12, and 24 h (p < 0.05). The percentages of viable PDLF at 3, 6, 12, and 24 h were significantly higher than 0 h for HBSS + Vit C, HBSS + PDGF + Vit C, HBSS + PL, and DMEM (p < 0.05). All experimental media were able to maintain PDLF viability (DMEM>HBSS+Vit C; HBSS+PDGF+Vit C>HBSS+PL>HBSS+PDGF; HBSS). Although DMEM had the highest cell proliferative effect, it is impractical to be used as a transport medium due to its cost, storage, and availability. The supplementation of Vit C yielded significant cell proliferative effects; hence, HBSS + Vit C can be a better alternative as a storage medium than HBSS.

The Thermal Stability of Influenza Viruses in Milk.

Highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype (clade 2.3.4.4b) have been detected in raw milk from infected cows. Several studies have examined the time and temperature parameters to ascertain whether influenza viruses in milk can be inactivated completely under commercial pasteurization conditions, yielding conflicting results. This study aimed to investigate whether milk could help protect influenza viruses from heat treatment. After heat treatment at 49 °C for one hour, the titer reduction of the influenza A/WSN/1933 (A/H1) virus in milk was approximately 1.6 log10TCID50/mL, which was significantly lower than that (3 log10TCID50/mL) observed in the Dulbecco's Modified Eagle Medium (DMEM) control media. The influenza D/bovine/CHN/JY3002/2022 (D/Yama2019) virus in milk retained a high residual infectivity (4.68 × 103 log10TCID50/mL) after treatment at 53 °C; however, the virus in DMEM completely lost its infectivity under the same conditions. Moreover, the influenza A/chicken/CHN/Cangzhou03/2023 (A/H5) virus in DMEM could be inactivated completely using any of the three heat treatment methods: 63 °C for 30 min, 72 °C for 15 s, or 80 °C for 15 s. For the virus present in milk, only heat treatment at 80 °C for 15 s completely inactivated it. These results suggest that milk prevents influenza viruses from pasteurization inactivation.

Enhancement of human native skin fibroblast proliferation in natural salso-bromo-iodic mineral water added to in vitro culture.

The favorable regenerative effects of some mineral waters on wound healing have long been empirically demonstrated. The aim of this experimental study is to investigate the effects of an Italian salso-bromo-iodic mineral water (Rivanazzano, Italy) on an in vitro human native fibroblast culture model to identify any potential regenerative actions. Human native fibroblasts were cultured under different experimental conditions: - Dulbecco's modified Eagle's medium (DMEM) reconstituted with distilled water (control); - DMEM reconstituted with filtered mineral water collected from the spring; - DMEM reconstituted with filtered mineral water collected at the balneotherapy facility; - DMEM reconstituted with filtered, heated mineral water collected at the balneotherapy facility; - DMEM partially replaced with filtered mineral water collected from the spring at different concentrations (10%, 20%, 30%, 40%, 50%); - DMEM partially replaced with filtered, heated mineral water collected at the balneotherapy facility at different concentrations (10%, 20%, 30%, 40%, 50%); - DMEM partially replaced with filtered mineral water collected at the balneotherapy facility at different concentrations (10%, 20%, 30%, 40%, 50%). Cell proliferation and viability were evaluated using spectrophotometric analysis following staining with the XTT Microculture Tetrazolium Assay. Statistical analyses were performed for each experimental condition at 24, 48 and 72 h. The best outcomes were observed in fibroblasts cultured with DMEM partially replaced with filtered mineral water collected from the spring, within the range of 20-50%. Our research results showed that Rivanazzano salso-bromo-iodic mineral water has a stimulating effect on in vitro human native fibroblast cultures. This activity was most pronounced with water collected from the spring, and it decreased with water collected at the balneotherapy facilities. These findings could form the basis for clinical applications in wound healing and balneotherapy.

Effect of chondroitin sulfate and bone marrow-derived mesenchymal stem cells on intervertebral disc degeneration treatment in rabbits

The present study evaluated the influence of bone marrow-derived stem cells (BM-SC) and chondroitin sulfate (CS) on histological changes in intervertebral disc (IVD). A randomized clinical trial was conducted, thirty-six healthy New Zealand rabbits were randomly distributed into four different groups (n=9): con-trol group (A), stem cell group (B), chondroitin sulfate group (C), and associa-tion of stem cells with chondroitin sulfate group (D). All animals underwent the experimental disc degeneration procedure. Group A received two intradiscal applications of DEM (Dulbecco’s modified eagle’s medium), one and two weeks after intervertebral disc degeneration (IVDD), while group B received two intradiscal applications of 1.2 x 106 BM-SC at the same time interval as group A. After IVDD, group C received eight subcutaneous applications of CS at a dose of 5 mg/kg, one application every seven days. In contrast, group D received the association of the techniques used in groups B and C. Sixty days after the end of the interventions, all animals were euthanized, then the crani-ocaudal thickness of the IVD was measured, and IVDD was classified in scores according to the histological grading model. All groups that received CS had thicker IVDs, and all the treated groups presented better scores on several items and a better overall score. It was possible to conclude that in the species studied, the isolated use of CS or BM-SC was statistically significant in improv-ing the histopathological aspects of IVDD, however, the combination of treat-ments did not prove to be more efficient.

Danazol and Stanozolol Induce Expression of Pro-Apoptotic Genes

Androgens (danazol and stanozolol) have been used to treat bone marrow failure (BMF) syndromes due to their multifaceted effects on erythropoiesis, telomere maintenance and immune homeostasis. In low and middle-income countries, androgens are used to treat aplastic anaemia, particularly in individuals who cannot afford immunosuppressive therapy and HSCT. However, patient responses to these treatments vary, with previous studies showing a 50-70% response to inherited bone marrow failures and a 40-50% response to acquired AA and MDS. There is uncertainty surrounding how androgens work, with only a few elucidated effects on the stimulation of erythropoiesis, stem cell telomere elongations and immune cell modulation. This study aims to understand the mechanisms of androgen action in hepatic cell lines since they are metabolised there and extrapolate the findings to HSCs. HepG2 cell lines at passage 6 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. Mobilised peripheral blood from stem cell donors was used to enrich CD34+ cells cultured in StemSpan SFEM Hematopoietic cell culture medium. The purity and percentage of CD34+ cells were assessed by flow cytometry, and &amp;gt; 90% purity was maintained. MTT assays determined the drug concentration in HepG2 and CD34+positive cells. HepG2 and CD34 cells were treated with danazol and stanozolol at different concentrations (100nM, 1uM and 10uM) for 24, 48, and 72 hours. Cell proliferation rates were monitored by staining the cells with CFSE dye, incubating them with the respective concentrations for 24, 48 and 72 hours and analysing the proliferation in a flow cytometer. Further, the drug-treated cells were stained with propidium iodide to examine the effects of drugs in the cell cycle at 24 hours post-drug treatment. RNA was extracted from treated cells, and expression of apoptotic (CASP8, CASP9, Fas, and BAX), senescent genes (TERF1, SIRT1, ARID1a), and androgen receptor gene was analysed using quantitative real-time PCR using relative quantification. The influence of androgens on genes involved in iron metabolism is being investigated, and protein data is awaited. Based on the MTT assay on CD34+ and HepG2 cells, 100nM and 1uM danazol and stanozolol were used for further experiments due to increased viability at 24 and 48 hrs. HepG2 cells showed a maximum proliferation at 24 hours at varying concentrations of danazol and stanozolol. We further observed an arrest of cells at the G1 phase post-drug treatment at 24 hrs. Both danazol and stanozolol, the peak significant androgen receptor expression was observed at 6 hours (fold change &amp;gt; 3.3; p = 0.0004). Pro-apoptotic gene CASP8 increased significantly with danazol over 24 hours (p = 0.025 and 0.0009, and fold change &amp;gt;1.5 and 1.4, respectively), but stanozolol caused a minor increase in expression levels. Danazol and stanozolol (100 nM conc.) significantly enhanced the expression level of CASP9 (p = 0.0253, 0.0009, and 0.007; fold change &amp;gt; 1.5 and 1.8 respectively), while FAS9 levels were significantly upregulated with stanozolol (1 uM) and danazol (p = 0.0001, 0.0001, and 0.004; fold change 2.3, 2.39, and 1.7). The mechanism of action of androgens appears complex. Genes involved in apoptotic processes are upregulated with no difference in the expression of senescent genes with both Danazol and Stanozolol. Danazol is known to have immunosuppressive effects on immune cells in AA, and this may be due to the induction of apoptotic processes in activated cells. Results from CD34 cell experiments and protein analysis may shed light on these pathways and their mechanisms.

Mouse liver blood flow is regulated by hepatic stellate cells in response to the sympathetic neurotransmitter norepinephrine

BackgroundThere is no clear information on the regulation of liver blood flow by the autonomic nervous system. We conducted this study to investigate whether quiescent hepatic stellate cells (qHSCs) regulate liver blood flow in response to the sympathetic neurotransmitter norepinephrine (NE). MethodsqHSCs isolated from mice were cultured in Dulbecco's modified Eagle medium without fetal bovine serum for 1 day on collagen gel. NE-induced qHSC contraction was evaluated using the quantitative single-cell contraction measurement method that we had developed previously. For the measurement of liver perfusion pressure in situ, a buffer solution was perfused from the portal vein in mice. ResultsNE-induced a reversible contraction of qHSCs. This contraction was suppressed by the nonmuscle myosin II inhibitor blebbistatin, the myosin light chain kinase inhibitor ML-9, the Rho kinase inhibitor H-1152, the calmodulin inhibitor W-7, the store-operated calcium channel inhibitor YM-58483, and the IP3 receptor inhibitor xestospongin C. In contrast, the transient receptor potential C channel inhibitor SKF96365 did not affect the NE-induced contraction. ConclusionThese results suggest that qHSCs contract in response to NE. New & noteworthyThe present study provides direct evidence for the first time that norepinephrine (NE) induces a reversible contraction of isolated single quiescent hepatic stellate cells (qHSCs) and further suggests that the NE-mediated qHSC contraction participates in the regulation of liver blood flow in vivo.

Nanobubbles and Fibroblast Growth: An In Vitro Study on Cell Migration and Proliferation.

Nanobubbles are studied for their unique properties and possible applications in wound healing processes. This study investigates the effects of hydrogen (H₂), oxygen (O₂), and ozone (O₃) nanobubbles on fibroblast migration and proliferation usingin vitroscratch wound healing assays. Fibroblast cells were treated with Dulbecco's Modified Eagle Medium (DMEM) combined with nanobubble solutions, and cell density was measured at 24 and 48 hours. While no significant difference was observed at 24 hours (p=0.52), ozone nanobubbles significantly reduced cell density at 48 hours (p=0.005), indicating cytotoxic effects. Hydrogen and oxygen nanobubble treatments did not show statistically significant differences from the control. These results highlight the cytotoxic effects of ozone nanobubbles on fibroblasts, which may impact their potential application in wound healing. While the study shows the cytotoxic effects of ozone nanobubbles,in vivowound healing and antimicrobial impacts remain unexplored and warrant further study.

Sperm Cryopreservation in Canaries to Protect Endangered Songbird Species: Comparison of Different Cryoprotectants.

Sperm cryopreservation is a rather complex process that needs to be adapted to wild and domestic bird species to ensure adequate efficiency. This study aimed to determine the usability of different cryoprotectants in the cryopreservation of Gloster canary sperm. For this purpose, sperm samples were collected from 12 2-year-old male Gloster canaries three times a week using cloacal massage for 4 weeks. After individual evaluation, sperm samples from the canaries were combined. Mixed sperm were divided into two groups in the study. Overall, 8% dimethyl sulfoxide (DMSO) and ethylene glycol (EG) were used as cryoprotectants. Sperm samples were drawn into straws after adding Dulbecco's Modified Eagle Medium (DMEM) extender with high glucose ratio and two different cryoprotectants in a 1:1 ratio and frozen to -80°C with liquid nitrogen vapour and then stored in liquid nitrogen at -196°C. Frozen-thawed semen samples were evaluated regarding motility, vitality, plasma membrane integrity (hypoosmotic swelling test [HOST]), density and abnormal spermatozoa rate. The highest motility value after freezing and thawing was determined in the EG group with 31.667%±4.773%. In addition, vitality, plasma membrane integrity and normal sperm morphology were statistically significantly higher in the EG-frozen group, whereas head and tail abnormality was low (p<0.05). This study determined that a DMEM extender containing 8% EG was more advantageous than a DMEM containing DMSO regarding spermatological parameters and could be used for long-term storage of canary sperm.

Mechanical Properties of 3D Printed PLA Scaffolds for Bone Regeneration

Abstract The growing interest in biodegradable scaffolds for bone regeneration created a need to investigate new materials suitable for scaffold formation. Poly(lactic acid) (PLA) is a polymer commonly used in biomedical engineering, e.g. in tissue engineering as a biodegradable material. However, the mechanical behavior of PLA along its degradation time is still not explored well. For this reason, the mechanical properties of PLA scaffolds affected by incubation in physiological medium needs to be investigated to show the potential of PLA to be used as a material for biodegradable scaffold formation. The purpose of this research is to determine the mechanical properties of PLA scaffolds before and after incubation, and to apply constitutive material models for further behavior prediction. Two sets of PLA scaffolds were printed by the 3D printer “Prusa i3 MK3S” and sterilized by ultraviolet light and ethanol solution. The first set of specimens was incubated in DMEM (Dulbecco’s Modified Eagle Medium) for 60, 120, and 180 days maintaining 36.5 °C temperature. The mechanical properties of the scaffolds were determined after performing the compression test in the “Mecmesin MultiTest 2.5-i” testing stand with a force applied at two different speed modes. The obtained data was curve fitted with the hyperelastic material models for a model suitability study. The second set of specimens was incubated in PBS (Phosphate Buffered Saline) for 20 weeks and used in a polymer degradation study. The obtained results show that the mechanical properties of PLA scaffolds do not decrease during incubation in physiological medium for a predicted new bone tissue formation period, though hydrolysis starts at the very beginning and increases with time. PLA as a material seems to be suitable for the use in bone tissue engineering as it allows to form biocompatible and biodegradable scaffolds with high mechanical strength, required for effective tissue formation.

Investigating wall shear stress and the static pressure in bone scaffolds: a study of porosity and fluid flow dynamics.

In bone tissue engineering, scaffolds are crucial as they provide a suitable structure for cell proliferation. Transporting Dulbecco's Modified Eagle Medium (DMEM) to the cells and regulating the scaffold's biocompatibility are both controlled by the dynamics of the fluid passing through the scaffold pores. Scaffold design selection and modeling are thus important in tissue engineering to achieve successful bone regeneration. This study aims to design and analyze three scaffold designs-Face-Centered Cubic (FCC), and two newly developed designs Octagonal Truss and a Square Pyramid with four porosity variations. The research aims to analyze the effect of design and porosity variation on pressure and wall shear stress, essential for analyzing scaffold biocompatibility in tissue engineering. Three scaffold designs with varying porosities with strut diameters ranging from 0.3 to 0.6mm were modeled to analyze the behavior using BioMed Clear Resin. The fluid dynamics within these scaffolds were then examined using Computational Fluid Dynamics (CFD) to understand how different porosity levels affect fluid flow pressure and wall shear stress. The findings revealed variations in wall shear stress and their influence on cell proliferation. The maximum value of wall shear stress (WSS) is observed in the Square Pyramid model. The analysis shows that WSS at the inlet decreases as strut diameters increase or porosity percentages rise offering valuable insights for the development of effective scaffold designs. It can be concluded from the results that the Square Pyramid design has the highest value of WSS, thus increasing the chances of cell growth. From a biological perspective, the results of this work show promise for creating better scaffolds for tissue engineering.

Regulation of hepatic transporters OATP1A2 and OATP1B1 by the action of nitric oxide (II)

Nitric oxide II (NO) is a signaling molecule that has a wide range of physiological effects, including the regulation of gastrointestinal processes. The liver actively expresses the clinically significant transporters OATP1A2 and OATP1B1, which are involved in the influx of biologically active and medicinal substances. That is why it seems relevant to determine the pathways of regulation of hepatic transporters under the influence of NO. Aim. To study the effect of NO on the relative amount and expression of the transporters OATP1A2 and OATP1B1 in vitro in HepG2 cells. Materials and methods. The study was performed on a culture of HepG2 cells, which were cultured in 6-well plates at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) with a high glucose content (4500 mg/l) containing L-glutamine (4 mM), 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (all components from Sigma-Aldrich, Germany). S-nitrosoglutathione (Sigma-Aldrich, Germany) was added to the culture medium at concentrations of 1, 10, 50, 100 and 500 µM, incubated for 24 and 72 hours. Water for injection (solvent) was added to control cells in an equivalent volume S-nitrosoglutathione). The relative amounts of OATP1A2 and OATP1B1 proteins were assessed by Western blot, and the expression of SLCO1A2 and SLCO1B1 by real-time PCR. The results of the study. In the course of this study, it was shown that the addition of S-nitrosoglutathione in the concentration range of 10-500 μM and exposure duration of 24 and 72 hours causes an increase in the intracellular level of nitric oxide metabolites, which indicates the adequacy of the use of this NO donor. At the same time, under the influence of NO, there was an increase in the relative amount of the studied transporters - OATP1A2 at an exposure period of 24 hours and S-nitrosoglutathione concentrations of 50 and 100 μM, OATP1B1-24 and 72 hours, at concentrations of 10-500 μM, a similar trend was noted for the expression genes SLCO1A2 and SLCO1B1. Conclusion. The NO donor - S-nitrosoglutathione causes an increase in the relative amount of OATP family transporters - OATP1A2 and OATP1B1, due to increased expression of the SLCO1A2 and SLCO1B1 genes, in vitro in HepG2 cells.

Phenotypic characteristics of adipocyte-like cells generated from C2C12 myoblasts cultured with chicken serum

The aim of this study was to clarify the transcriptional and metabolic characteristics of C2C12 myoblasts cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 20 % chicken serum (CHS) (C2C12–CHS cells) compared with C2C12 myoblasts cultured in DMEM containing 20 % fetal bovine serum (FBS) (C2C12-FBS cells). After 3 days of culture, C2C12–CHS cells showed a marked accumulation of lipid droplets, accompanied by increased expression levels of brown adipocyte-related genes (i.e., Bmp7, Prdm16, Ucp1, Cidea, Pgc1α, Cox7a1, Cox8, and β3-adorenoceptor). Furthermore, stimulation of β3-adorenoceptor by its selective agonist, mirabegron, increased the mRNA expression of Ucp1 and Pgc1α in C2C12–CHS cells. Wide-targeted metabolomic analysis performed by gas chromatography-tandem mass spectrometry revealed that the metabolic profile of C2C12–CHS cells was obviously different to that of C2C12-FBS cells. Additionally, the metabolomic analysis indicated that β3-adrenoceptor stimulation by mirabegron upregulated energy metabolism in C2C12–CHS cells as seen in brown adipocytes. These results suggest that C2C12–CHS cells may differentiate into brown adipocyte-like cells, accompanied by increased functional β3-adrenoceptor.

The Utilization of Central Composite Design for the Production of Hydrogel Blends for 3D Printing

Central composite design (CCD) is a statistical experimental design technique that utilizes a combination of factorial and axial points to study the effects of multiple variables on a response. This study focused on optimizing hydrogel formulations for 3D printing using CCD. Three biopolymers were selected: sodium alginate (SA), gelatin (GEL), and carboxymethyl cellulose (CMC). The maximum and minimum concentrations of each polymer were established using a Google Scholar search, and CCD was employed to generate various combinations for hydrogel preparation. The hydrogels were characterized in accordance with their swelling degree (SD) in phosphate-buffered saline (PBS) and Dulbecco’s Modified Eagle Medium (DMEM), as well as their printability in 2D and 3D assays. The formulation consisting of 7.5% SA, 7.5% GEL, and 2.5% CMC exhibited the best swelling properties and exceptional printability, surpassing all other tested formulations. This study highlights the effectiveness of design of experiment methodologies in accelerating the development of optimized hydrogel formulations for various applications in 3D printing and suggests avenues for future research to explore their performance in specific biological contexts.

Significant Impact of let-7d MicroRNA on Breast Cancer Cell Lines Post-Radiation Treatment

Introduction: Radiotherapy is a common treatment for breast cancer treatment, that induces DNA damage. These DNA damages are addressed through various repair pathways, which regulate the DNA repair systems and confer radio-resistance. Non-coding RNAs are a big proportion of genome transcripts without the potential to encode proteins. Related studies demonstrated that radiation affects the expression of non-coding RNAs. Let-7d, a tumor suppressor in numerous cancers, has some target genes that play a role in the DNA repair system. Materials and Methods: Human breast cancer cell lines MDA-MB-231 and MCF-7 were cultured in a Dulbecco's Modified Eagle Medium. The exponentially growing cells were treated with some doses of X-rays. After radiation treatment and cell harvesting, RNA was extracted, and cDNA synthesis was done. The let-7d miRNA expression changes were calculated with real-time quantitative reverse transcription polymerase chain reaction. Results: The results implied that radiation caused increased let-7d expression in breast cancer cell lines after radiation treatment. In addition, the results showed that 24 h after radiation, the expression of let-7d in the radioresistant cell line was higher than the radiosensitive one; 48 h after radiation, the expression of let-7d in the radiosensitive cell line was higher than the other one. Conclusions: The results demonstrated that radiation treatment increased let-7d miRNA expression in both radiosensitive and radio-resistant breast cancer cell lines. Therefore, let-7d might be involved in the radiosensitivity of breast cancer.