Minimum Essential Medium Research Articles

Poster 105: Nanowarming by Inductive Heating of Vitrified Large Articular Cartilage Specimens, In Vivo Testing with Hanford Mini Pigs

Poster 105: Nanowarming by Inductive Heating of Vitrified Large Articular Cartilage Specimens, In Vivo Testing with Hanford Mini Pigs

Potential harmful effects of viral hemorrhagic septicemia virus in mammals

Most of the emerging diseases that threaten humans are caused by RNA viruses which are extremely mutable during evolution. The fish RNA virus, viral hemorrhagic septicemia virus (VHSV) can infect a broad range of aquatic animal hosts, but the transmissibility of VHSV to mammals has not been thoroughly investigated. Therefore, our study aimed to investigate the potential adverse effects of VHSV in mammals. Briefly, the survival of VHSV was determined using only minimum essential media (MEM-2) and mammalian SNU-1411 and hepa-1c1c7s cells at 15°C and 37°C. Mice (Mus musculus, 27.3 ± 1.9 g) were intravenously injected with VHSV (2.37E+05 TCID50 · mice.–1) in triplicate. Clinical signs and survival rates were examined at 14 days post-challenge, and infection was confirmed in the surviving mice. The 50% tissue culture infective dose (TCID50) and polymerase chain reaction analysis were used to determine viral titers and the infection rate, respectively. The titer of VHSV suspended in MEM-2 at 15°C was reduced by only one log after 8 days, whereas the virus maintained at 37°C was inactivated 8 days post-inoculation (dpi). There were no recognizable cytopathic effects in either SNU-1411 or hepa-1c1c7s cells inoculated with VHSV at 15°C and 37°CVHSV in those cell lines at 37°C was rapidly decreased and eventually inactivated at 12 dpi, whereas virus at 15°C remained at low concentrations without replication. In vivo experiment showed that there were no signs of disease, mortality, or infection in VHSV-infected mice. The results of this study indicate that it is highly unlikely that VHSV can infect mammals including humans.

Gallium-Strontium Phosphate Conversion Coatings for Promoting Infection Prevention and Biocompatibility of Magnesium for Orthopedic Applications.

Device-associated infections remain a clinical challenge. The common strategies to prevent bacterial infection are either toxic to healthy mammalian cells and tissue or involve high doses of antibiotics that can prompt long-term negative consequences. An antibiotic-free coating strategy to suppress bacterial growth is presented herein, which concurrently promotes bone cell growth and moderates the dissolution kinetics of resorbable magnesium (Mg) biomaterials. Pure Mg as a model biodegradable material was coated with gallium-doped strontium-phosphate through a chemical conversion process. Gallium was distributed in a gradual manner throughout the strontium-phosphate coating, with a compact structure and a gallium-rich surface. It was demonstrated that the coating protected the underlying Mg parts from significant degradation in minimal essential media at physiological conditions over 9 days. In terms of bacteria culture, the liberated gallium ions from the coatings upon Mg specimens, even though in minute quantities, inhibited the growth of Gram-positiveStaphylococcus aureus, Gram-negative Escherichia coli, andPseudomonas aeruginosa ─ key pathogens causing infection and early failure of the surgical implantations in orthopedics and trauma. More importantly, the gallium dopants displayed minimal interferences with the strontium-phosphate-based coating which boosted osteoblasts and undermined osteoclasts in in vitro co-cultures. This work provides a new strategy to prevent bacterial infection and control the degradation behavior of Mg-based orthopedic implants, while preserving osteogenic features of the devices.

Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply.

Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B12) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine–folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle’s minimum essential medium with 1 g/L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine–folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest (p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism.

Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system.

Although DNA methylation epigenetically regulates development, data on global DNA methylation during the development of limb buds (LBs) are scarce. We aimed to investigate the global DNA methylation developmental dynamics in rat LBs cultivated in a serum supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture. Fischer rat front- and hind-LBs at the 13th and 14th gestation days (GDs) were cultivated at the air-liquid interface in Eagle’s Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively. The methylation of repetitive DNA sequences (short interspersed DNA repetitive element [SINE] rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by safranin O. On isolation, weak safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures toward day 14, except in the SF-cultivated GD13 hind-LBs, where safranin O staining was almost absent on day 3 that was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day 3. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th days was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day 3 and then decreased. In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive toward differentiation in vitro, which is of importance for regenerative medicine strategies.

A STUDY ON PHYTOCHEMICAL SCREENING ANTIOXIDENT AND IN VITRO-ANTICANCER ACTIVITY OF Aplotaxis auriculata ROOTS EXTRACT

The current examination was done to depict the bioactive compound constituents in plant concentrate of Aplotaxis auriculate using UV range - 210-290, FTIR, absorbance was recorded in the zone of some place in the scope of 4000 and 400 cmˉ¹. The zenith 3370 (Alcoholic and Phenolic social affairs), 2947 (Alkanes), 1656 (Alkene), 1453 (Alkane) and 668 cmˉ¹ (Aromatic) exhibits the individual get-togethers, and in vitro cell fortification against illness activities of Aplotaxis auriculata methanol remove. Cell support activity was done through DPPH, Hydrogen peroxide scrounging activity and diminishing power look at. Cell support and unfriendly to sickness practices were seen that A. auriculata extended with growing obsession. The human chest torment cell line (Michigan Cancer Foundation-7 (MCF 7)) was gotten from National Center for Cell Science (NCCS), Pune and made in Eagles Minimum Essential Medium (EMEM) containing 10% fetal dairy creatures like serum (FBS). The cells were kept up in Minimal Essential Media (MEM) improved with 10% Fetal dairy animals like serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified atmosphere of 5% CO2 at 37°C. Support social requesting were passaged following a long time after week, and the way of life medium was changed on different occasions reliably. Over piece of all authentic canny meds are standard things that perceive a principal occupation in calm improvement in medication endeavors.

Viability of Toxoplasma gondii tachyzoites in different conditions for parasite transportation.

Background and Aim:Toxoplasma gondii tachyzoite is the infective stage that causes acute infection, leading to severe toxoplasmosis. The tachyzoite stage has been extensively used for several inoculation purposes, including antigen production, immunological studies, nutrition mechanisms, and in vitro drug trials. The use of fresh tachyzoites is required for inoculation in either in vitro or in vivo studies. However, there is a lack of information on preserving live tachyzoites during transportation from laboratories to inoculation sites. Therefore, this study aimed to validate suitable preservative conditions for maintaining live parasites by determining the survival and viability of T. gondii tachyzoites on the basis of different media, temperatures, and incubation times.Materials and Methods:The free live T. gondii tachyzoites were evaluated on their viability when maintained in different media without 5% Carbon dioxide (CO2). The purified tachyzoites of the RH and PLK strains were individually suspended in normal saline (NS), phosphate-buffered saline (PBS), minimum essential medium (MEM), and MEM with 10% fetal bovine serum (MEM-FBS) and incubated for 6 h at ice-cold (IC; 3-9°C) and room temperature (RT; 25°C). Parasite survival was measured at the 0, 1st, 2nd, 3rd, 4th, 5th, and 6th h post-incubation using the trypan blue exclusion test.Results:The viability was in the range of 85.0%–91.0% for IC using NS and 81.0%–85.1% (IC) and 75.3%–77.5% (RT) using PBS. The viability was approximately 75.0%–83.0% (IC) and 70.0%–79.0% (RT) using MEM and MEM-FBS. There was a significant difference in the viability between the seven periods on the basis of one-way repeated Analysis of variance and Friedman analyses. Parasite survival slightly reduced (20.0%–30.0%) in NS and MEM-FBS at both temperatures during incubation. Notably, PBS could not support tachyzoite viability after 3 h post-incubation.Conclusion:NS was a suitable preservative for maintaining purified T. gondii tachyzoites during transportation at IC and RT without 5% CO2 supplementation. This could be a valuable medium for parasite transportation, especially when there is a large distance between the laboratory and inoculation site.

Effect of secondary particle size of nickel oxide nanoparticles on cytotoxicity in A549 cells.

The effect of nanoparticle type, shape, as well as primary and secondary particle size on toxicity remains poorly characterized. In this study, suspensions of nickel oxide (NiO) nanoparticles with the same primary particle size (< 50 nm) but different secondary particle sizes were prepared, and their cytotoxicity was investigated. A planetary ball mill wet nanopulverizer with zirconium milling balls of decreasing sizes (φ: 0.5, 0.1, and 0.05 mm) yielded NiO nanoparticles of decreasing mean particle size (310.4 ± 6.7, 172.0 ± 2.8, and 102.0 ± 0.5 nm). Stock solutions were diluted to various concentrations in 10% heat-inactivated fetal bovine serum containing minimum essential medium, and shown to have the same primary particle size, but different secondary particle sizes. Tests with A549 cells revealed that cytotoxicity increased with increasing secondary particle size: milling ball diameter φ 0.05 mm (IC50: 148 μg/mL) < φ 0.1 mm (IC50: 83.5 μg/mL) < φ 0.5 mm (IC50: 33.4 μg/mL). Uptake experiments indicated that the intracellular amount of Ni increased with increasing secondary particle size. In summary, the present findings show that differences in secondary particle size affected the cytotoxicity of NiO suspensions, which could be ascribed at least in part to differences in the amount of NiO taken up by the cells.

From lessons on the long-term effects of the preimplantation environment on later health to a "modified ART-DOHaD" animal model.

BackgroundAt its earliest stages, mammalian embryonic development is apparently simple but vulnerable. The environment during the preimplantation period, which only lasts a couple of days, has been implicated in adult health, extending to such early stages the concept of the developmental origin of health and disease (DOHaD).MethodsIn this review, we first provide a brief history of assisted reproductive technology (ART) focusing on in vitro culture and its outcomes during subsequent development mainly in mice and humans. Further, we introduce the “MEM mouse,” a novel type 2 diabetes mouse model generated by in vitro culture of preimplantation embryos in alpha minimum essential medium (αMEM).Main findingsThe association between ART and its long‐term effects has been carefully examined for its application in human infertility treatment. The “MEM mouse” develops steatohepatitis and kidney disease with diabetes into adulthood.ConclusionThe close association between the environment of preimplantation and health in postnatal life is being clarified. The approach by which severe mouse phenotypes are successfully induced by manipulating the environment of preimplantation embryos could provide new chronic disease animal models, which we call “modified ART‐DOHaD” animal models. This will also offer insights into the mechanisms underlying their long‐term effects.

Localized corrosion of magnesium alloys potentially applicable for medical implants: fundamental aspects

A comparative analysis of the corrosion activity of bioresorbable MA8 magnesium alloy (Mg&ndash; Mn &ndash; Ce system) in a medium for cultivation of mammalian cells (minimum essential medium, MEM) and 0.83 wt. % aqueous NaCl solution was performed. The development of the corrosion process on the surface of a magnesium alloy in two media was established using local scanning electrochemical methods (scanning vibrating electrode technique and scanning ion-selective electrode technique), traditional electrochemical methods (potentiodynamic polarization, electrochemical impedance spectroscopy) and hydrogen evolution measurements. The protective properties of the corrosion film formed on the alloy in MEM, as well as during exposure to NaCl solution, were established. The composition of the layer of corrosion products formed on the magnesium alloy in MEM was studied using confocal Raman spectroscopy, X-ray diffraction analysis, scanning electron microscopy, and energy dispersive X-Ray analysis. Calcium-phosphate compounds are the main products formed on the surface of a magnesium alloy during immersion in MEM. The model of corrosion mechanism of magnesium alloy in MEM, which includes three stages of the development of surface film, is proposed. The formation on the magnesium alloy in MEM of corrosion product layer, including magnesium-substituted hydroxyapatite, stabilizes the local pH below 9.0, which along with the presence of organic acids, does not allow increasing the pH during corrosion. A diagram that allows one to determine the possible reactions occurring on the surface of a magnesium alloy in MEM and the thermodynamic probability of the formation of chemical compounds based on the values of the local pH is designed. The obtained results indicate the prospect of using bioresorbable magnesium implants in surgery.

Follicular development, morphological integrity, and oxidative stress in bovine preantral follicles cultured in vitro with ascorbic acid.

The aim of this study was to evaluate the follicular development, morphological integrity, and oxidative stress of preantral ovarian follicles from Bos taurus indicus females grown in vitro with ascorbic acid. Ovaries (n = 20) from Bos taurus indicus females were collected, fragmented, and were cultured in vitro for 6 or 12 days in minimum essential medium (MEM), or MEM supplemented with 50 or 100 ng/ml ascorbic acid, with an extracellular matrix of agarose gel, in an incubator at 38.5°C; every 2 days, 100% of the culture medium was replaced. The data were analyzed using the chi-squared test and/or Fisher's exact test. In the event of a significant effect, the proportions were compared using a 2 × 2 proportion test. The oxidative stress analysis data were submitted to analysis of variance followed by the Bonferroni test. Values were considered significant when P ≤ 0.05. The addition of 100 ng/ml of ascorbic acid to the in vitro culture medium of preantral ovarian follicles from bovine females promoted follicular development, was efficient in maintaining morphological integrity, as well as the stability of reactive oxygen species, after 6 days of in vitro culture.

Upstream process optimization and micro- and macrocarrier screening for large-scale production of the oncolytic H-1 protoparvovirus

The oncolytic virus H-1PV is a promising candidate for various cancer treatments. Therefore, production process needs to be optimized and scaled up for future market release. Currently, the virus is produced with minimum essential medium in 10-layer CellSTACK® chambers with limited scalability, requiring a minimum seeding density of 7.9E3 cells/cm2. Production also requires a 5% fetal bovine serum (FBS) supplementation and has a virus yield up to 3.1E7 plaque-forming units (PFU)/cm2. Using the animal-free cell culture medium VP-SFM™ and a new feeding strategy, we demonstrate a yield boost by a mean of 0.3 log while reducing seeding density to 5.0E3 cells/cm2 and cutting FBS supplementation by up to 40% during the production process. Additionally, FBS is completely removed at the time of harvest. Eleven commercial micro- and macrocarriers were screened regarding cell growth, bead-to-bead transfer capability, and virus yield. We present a proof-of-concept study for producing H-1PV on a large scale with the microcarrier Cytodex® 1 in suspension and a macrocarrier for a fixed-bed iCELLis® bioreactor. A carrier-based H-1PV production process combined with an optimized cell culture medium and feeding strategy can facilitate future upscaling to industrial-scale production.Key points• Virus yield increase and FBS-free harvest after switching to cell culture medium VP-SFM™.• We screened carriers for cell growth, bead-to-bead transfer capability, and H-1PV yield.• High virus yield is achieved with Cytodex® 1 and macrocarrier for iCellis® in Erlenmeyer flasks.

A new murine esophageal organoid culture method and organoid-based model of esophageal squamous cell neoplasia

SummaryOrganoids mimic the physiologic and pathologic events of organs. However, no consensus on esophageal organoid (EO) culture methods has been reached. Moreover, organoid models reproducing esophageal squamous cell carcinoma (ESCC) initiation have been unavailable. Herein, we sought to develop an esophageal minimum essential organoid culture medium (E-MEOM) for culturing murine EOs and establishing an early ESCC model. We formulated E-MEOM to grow EOs from a single cell with clonal expansion, maintenance, and passage. We found that EOs cultured in E-MEOM were equivalent to the esophageal epithelium by histological analysis and transcriptomic study. Trp53 knockout and KrasG12D expression in EOs induced the development of esophageal squamous neoplasia, an early lesion of ESCC. Here we propose the new formula for EO culture with minimum components and the organoid model recapitulating ESCC initiation, laying the foundation for ESCC research and drug discovery.

Effect of base media, FSH and anti-Müllerian hormone (AMH) alone or in combination on the growth of pig preantral follicles in vitro

To compare the efficiency of North Carolina State University medium 23 (NCSU23) and Alpha Minimum Essential Medium (α-MEM) as a base medium, and to evaluate the effects of Anti-Müllerian Hormone (AMH) alone or in combination with Follicle Stimulating Hormone (FSH) on the in vitro development and steroid production of isolated porcine preantral follicles. Porcine secondary follicles were cultured in NCSU23 or α-MEM media for 4 days. Once α-MEM was determined to be the optimal culture medium, secondary follicles were cultured in α-MEM alone or supplemented with FSH (1.5 ng/mL), AMH (50 ng/mL) or the combination of the two hormones. Follicle development was evaluated by measuring follicular growth, morphology and hormone production. There was no difference between the media NCSU23 and α-MEM in terms of follicle survival and growth (P &gt; 0.05). However, at day 2, the antrum formation rate tended to be (P &lt; 0.074) higher in α-MEM than NCSU23. At day 4 of culture, the estradiol and progesterone secretion were higher in α-MEM than NCSU23 (P &lt; 0.01), while the opposite was observed for testosterone (P &lt; 0.01). The addition of AMH and/or FSH did not affect follicular survival and growth. Nevertheless, the secretion of estradiol and progesterone induced by FSH was reduced with AMH (P &lt; 0.01). α-MEM is a more effective base medium than NCSU23 for the in vitro follicular development of pig preantral follicles and AMH reduces the steroid production induced by FSH.

Isolation of Epithelial Cells from Human Dental Follicle.

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while α-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells. The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

Experimental study on the effect of three-dimensional porous structures on the vascularization rate of artificial dermis

Experimental study on the effect of three-dimensional porous structures on the vascularization rate of artificial dermis

Preclinical evaluation of acute systemic toxicity of magnesium incorporated poly(lactic-co-glycolic acid) porous scaffolds by three-dimensional printing.

ABSTRACTBiodegradable polymer scaffolds combined with bioactive components which accelerate osteogenesis and angiogenesis have promise for use in clinical bone defect repair. The preclinical acute toxicity evaluation is an essential assay of implantable biomaterials to assess the biosafety for accelerating clinical translation. We have successfully developed magnesium (Mg) particles and beta-tricalcium phosphate (β-TCP) for incorporation into poly(lactic-co-glycolic acid) (PLGA) porous composite scaffolds (PTM) using low-temperature rapid prototyping three-dimensional-printing technology. The PTM scaffolds have been fully evaluated and found to exhibit excellent osteogenic capacity for bone defect repair. The preclinical evaluation of acute systemic toxicities is essential and important for development of porous scaffolds to facilitate their clinical translation. In this study, acute systemic toxicity of the PTM scaffolds was evaluated in mice by intraperitoneal injection of the extract solutions of the scaffolds. PTM composite scaffolds with different Mg and β-TCP content (denoted as PT5M, PT10M, and PT15M) were extracted with different tissue culture media, including normal saline, phosphate-buffered saline , and serum-free minimum essential medium , to create the extract solutions. The evaluation was carried out following the National Standard. The acute toxicity was fully evaluated through the collection of extensive data, including serum/organs ion concentration, fluorescence staining, and in vivo median lethal dose measurement. Mg in major organs (heart, liver, and lung), and Mg ion concentrations in serum of mice, after intraperitoneal injection of the extract solutions, were measured and showed that the extract solutions of PT15M caused significant elevation of serum Mg ion concentrations, which exceeded the safety threshold and led to the death of the mice. In contrast, the extract solutions of PT5M and PT10M scaffolds did not cause the death of the injected mice. The median lethal dose of Mg ions in vivo for mice was determined for the first time in this study to be 110.66 mg/kg, and the safety level of serum magnesium toxicity in mice is 5.4 mM, while the calcium serum safety level is determined as 3.4 mM. The study was approved by the Animal Care and Use Committee of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences (approval No. SIAT-IRB-170401-YGS-LYX-A0346) on April 5, 2017. All these results showed that the Mg ion concentration of intraperitoneally-injected extract solutions was a determinant of mouse survival, and a high Mg ion concentration (more than 240 mM) was the pivotal factor contributing to the death of the mice, while changes in pH value showed a negligible effect. The comprehensive acute systemic toxicity evaluation for PTM porous composite scaffolds in this study provided a reference to guide the design and optimization of this composite scaffold and the results demonstrated the preclinical safety of the as-fabricated PTM scaffold with appropriate Mg content, strongly supporting the official registration process of the PTM scaffold as a medical device for clinical translation.

Cytotoxic Evaluation of Mala aluation of Malaysian K ysian Kelulut Hone elulut Honey on Human y on Human Gingival Fibr al Fibroblast Cell Line using M oblast Cell Line using MTT Assay

Kelulut honey or stingless bee honey is a type of honey produced by stingless bees of the Trigona species where the nest is found in living trees. Objective: The aim of this study was to evaluate the cytotoxic potential of Malaysian Kelulut honey by employing MTT assay on a human gingival fibroblast cell line. Methods: Human gingival fibroblast cell line was cultured in minimal essential medium alpha (α-MEM) with 10% foetal bovine serum and 1% penicillin-streptomycin solution in a 5% CO2 incubator at 37°C in a humidified atmosphere. The cells were seeded at a cell density of 5x103 cells/well in a 96-well culture plate for 24 hours. The cells were treated with seven different concentrations (200, 100, 50, 25, 12.5, 6.25, and 3.125mg/ml) of Malaysian Kelulut honey and incubated in a CO2 incubator. The negative control comprised cells treated with growth media alone. The cell viability was assessed using MTT assay at 24, 48, and 72 hours. The test plate was shaken using a microplate shaker and the absorbance of the solution was measured at 570nm using an ELISA reader with the Magellan software. Statistical analysis of the data was carried out using Kruskal-Wallis test and SPSS 24.0.0 for Windows. A p value &lt;0.05 was considered as statistically significant. Results: There was no cytotoxic effect of Malaysian Kelulut honey on HGF-1 based on the MTT assay at different concentrations and at different time points tested as the cell viability was above 70%. The highest percentage of cell viability at all three different durations of treatment were observed at 3.125mg/ml, whereas the lowest cell viability was observed at 200mg/ml of Kelulut honey concentration. However, statistically significant differences were seen between some of the concentrations at various time points. Conclusion: Since the cell viability of HGF-1 treated with Malaysian Kelulut honey was more than 70% at all concentrations ranging from 3.125mg/ml to 200mg/ml at three different time points (24, 48 and 72 hours), Malaysian Kelulut honey can be considered as non-cytotoxic on human gingival fibroblasts based on MTT assay under the present test conditions

Culture of preantral ovarian follicles of Bos taurus indicus with alpha-lipoic acid.

The aim of this study was to evaluate follicular development, morphological integrity, and antioxidant potential of preantral ovarian follicles from Bos taurus indicus females grown in vitro with alpha-lipoic acid. Ovaries (n = 24) of Bos taurus indicus (n = 12) females were collected during slaughter and fragmented. A randomly obtained fragment from each pair of ovaries was fixed in Bouin (non-cultivated control; D0). These fragments were intended for classical histology (morphology and evaluation of follicular growth), and a fragment from each pair of ovaries was frozen at -80°C (non-cultivated control; D0), and assigned for analysis of oxidative stress [thiobarbituric acid reactive substances (TBARS), nitroblue tetrazolium (NBT), and ferric reducing antioxidant power (FRAP)]. The remaining fragments were cultured in vitro for 6 (D6) or 12 (D12) days, containing only minimum essential medium (MEM) or MEM supplemented with alpha-lipoic acid (50, 100, or 250 ng/ml), on an extracellular matrix of agarose gel, in an oven at 38.5ºC. Every 2 days, 100% of the culture medium was replaced. Supplementation with 100 ng/ml was effective for maintaining follicular integrity after 6 days of culture (primordial: 51.28%; development: 36.88%; P < 0.0001). There was no difference (P > 0.05) between treatments compared with the non-cultivated control treatment (D0), using the NBT and TBARS assays. Therefore, supplementation of the in vitro culture medium of bovine preantral ovarian follicles with a concentration of 100 ng/ml of alpha-lipoic acid at 6 days of culture was effective for maintaining follicular integrity and, after 6 days, maintaining stable levels of reactive oxygen species.

Promoting Infection Prevention and Biocompatibility of Magnesium for Orthopaedic Applications via Gallium-Strontium Phosphate Conversion Coatings

Device-associated bacterial infections remain a clinical challenge. Present strategies to prevent bacterial infection are either toxic to healthy mammalian cells and tissue or involve high doses of antibiotics that can prompt long-term negative consequences in terms of side effects or drug resistance. An antibiotic-free coating strategy to prevent bacterial attachment and biofilm formation is presented, which promotes bone cell growth and moderates the dissolution kinetics of resorbable magnesium (Mg) biomaterials. Herein pure Mg substrates were coated with gallium doped strontium-phosphate through a chemical conversion process. Gallium was distributed in a graduated manner throughout the strontium-phosphate coating, with a compact structure and a gallium-rich surface. It was demonstrated that the coating protected the underlying Mg from significant degradation in minimal essential media at physiological conditions over 9 days. In terms of bacteria culture, the liberated gallium ions from the coatings upon Mg specimens, even though minute quantities, reduced the viability and biofilm formation of Gram-negative P. aeruginosa and E. coli – two important clinical strains causing infections. More importantly, the gallium dopants displayed minimal interference with the strontium-phosphate based coating which boosted osteoblasts and undermined osteoclasts, in in vitro co-cultures. This work provides a new biocompatible strategy to prevent bacterial infection and control the degradation behaviour of Mg-based orthopaedic implants, while preserving osteogenic features of the devices.