Alpha-minimal Essential Medium Research Articles

Transcriptome-wide analysis of the differences between MCF7 cells cultured in DMEM or αMEM.

MCF7 cells have been used as an experimental model for breast cancer for decades. Typically, a culture medium is designed to supply cells with the nutrients essential for their continuous proliferation. Each medium has a specific nutritional composition. Therefore, cells cultured in different media may exhibit differences in their metabolism. However, only a few studies have investigated the effects of media on cells. In this study, we compared the effects of Dulbecco's modified Eagle medium (DMEM) and minimum essential medium alpha modification (αMEM) on MCF7 cells. The two media differentially affected the morphology, cell cycle, and proliferation of MCF7 cells, but had no effect on cell death. Replacement of DMEM with αMEM led to a decrease in ATP production and an increase in reactive oxygen species production, but did not affect the cell viability. RNA-sequencing and bioinformatic analyses revealed 721 significantly upregulated and 1247 downregulated genes in cells cultured in αMEM for 48 h compared with that in cells cultured in DMEM. The enriched gene ontology terms were related to mitosis and cell proliferation. Kyoto encyclopedia of genes and genomes analysis revealed cell cycle and DNA replication as the top two significant pathways. MCF7 cells were hypoxic when cultured in αMEM. These results show that the culture medium considerably affects cultured cells. Thus, the stability of the culture system in a study is very important to obtain reliable results.

A Simple High Yield Technique for Isolation of Wharton's Jelly-derived Mesenchymal Stem Cell.

The isolation of Mesenchymal Stem Cells (MSCs) from various tissues is possible, with the umbilical cord emerging as a competitive alternative to bone marrow. In order to fulfill the demands of cell therapy, it is essential to generate stem cells on a clinical scale while minimizing time, cost, and contamination. Here is a simple and effective protocol for isolating MSC from Wharton's Jelly (WJ-MSC) using the explant method with various supplements. Utilizing the explant method, small fragments of Wharton's jelly from the human umbilical cord were cultured in a flask. The multipotency of the isolated cells, were confirmed by their differentiation ability to osteocyte and adipocyte. Additionally, the immunophenotyping of WJ-MSCs showed positive expression of CD73, CD90, and CD105, while remaining negative for hematopoietic markers CD34 and CD45, meeting the criteria for WJ-MSC identification. Following that, to evaluate cells' proliferative capacity, various supplements, including basic Fibroblast Growth Factor (bFGF), Non-Essential amino acids (NEA), and L-Glutamine (L-Gln) were added to either alpha-Minimal Essential Medium (α-MEM) or Dulbecco's Modified Eagle's Medium-F12 (DMEM-F12), as the basic culture media. WJ-MSCs isolated by the explant method were removed from the tissue after seven days and transferred to the culture medium. These cells differentiated into adipocyte and osteocyte lineages, expressing CD73, CD90, and CD105 positively and CD34 and CD45 negatively. The results revealed that addition of bFGF to α-MEM or DMEMF12 media significantly increased the proliferation of MSCs when compared to the control group. However, there were no significant differences observed when NEA or LGln were added. Although bFGF considerably enhances cell proliferation, our study demonstrates that MSCs can grow and expand when properly prepared Wharton's jelly tissues of the human umbilical cord.

Stem cells derived from human exfoliated deciduous teeth-based media in a rat root resorption model

ObjectiveRoot resorption may occur during orthodontic treatment. Herein, we investigated the effect of a culture supernatant of stem cells derived from human exfoliated deciduous teeth on root resorption. DesignTwelve 8-week-old male Sprague–Dawley rats were used, and their maxillary first molars were pulled with excessive orthodontic force to induce root resorption. On days 1 and 7 after traction initiation, stem cells derived from human exfoliated deciduous teeth and alpha minimum essential medium (control group) were administered. After 14 days, the maxillary bone was evaluated for tooth movement. The expression of osteoprotegerin, receptor activator of nuclear factor κB ligand, tumor necrosis factor α, interleukin 1β, interleukin 6, and interleukin 17 was evaluated on the compression side and tension side. ResultsNo significant difference in tooth movement was observed between the two groups. Root resorption decreased in the group administered the culture supernatant compared with in the control. Immunohistochemical staining revealed increased osteoprotegerin expression and decreased receptor activators for nuclear factor κB ligand, tumor necrosis factor α, interleukin 1β, interleukin 6, and interleukin 17 on the compression side and tension side. ConclusionsAdministration of stem cells derived from human exfoliated deciduous teeth affected the expression of osteoprotegerin, receptor activator of nuclear factor κB ligand, tumor necrosis factor α, interleukin 1β, interleukin 6 and interleukin 17; hence, these stem cells may inhibit root resorption by regulating their expression.

Selegiline induced differentiation of rat bone marrow mesenchymal stem cells to dopaminergic neurons in vitro

Today, the use of mesenchymal stem cells (MSCs) for treating human diseases has attracted wide attention. The aim of this study is the expression of dopaminergic genes such as Nestin, patched Tumor Suppressor (PTCH), Sonic Hedgehog (SHH), Tyrosine Hydroxylase (TH) and Nuclear receptor-related factor 1 (NURR1) in MSCs after induction with selegiline. Rat bone marrow mesenchymal stem cells (rBMSCs) were extracted from femur and tibia bones and incubated with alpha Minimum Essential Medium (α-MEM) and 10% Fetal bovine serum (FBS). The stemness of cells at passage 4 was determined by the positive response to CD71 and CD90 markers and their differentiation into adipocytes and osteoblasts. The expression of SHH, PTCH, TH, NURR1 and Nestin genes in the cells after induction by 10-8 M selegiline for 48 hours was investigated by Reverse transcription polymerase chain reaction (RT-PCR) and Real Time-PCR methods. Isolated rBMSCs expressed CD71 and CD90 markers in culture conditions and could differentiate into adipocytes and osteoblasts. Induced cells showed neuronal morphology, positive response to Nestin and TH immunostaining. There was a significant increase of dopaminergic genes TH and NURR1 compared to the untreated cells. The results showed that selegiline with a dose of 10-8 M for 48 hours can lead to dopaminergic differentiation in rBMSCs.

Impact of Ovarian Factor Mediums on the Apoptotic Gene Expression and Embryo Quality Derived From Vitrified Immature Human Oocytes.

Condition mediums have a potential role in oocyte development. In this study, we evaluated the effects of different mediums on the developmental potential of vitrified immature human oocyte after IVM and parthenogenesis by ionomycin. Immature oocytes were collected from 184 women after vitrification/thawing and maturation, in three types of IVM mediums separately. Finally, 151 IVM MΙΙ oocytes were obtained and randomly divided into six groups and underwent the following intervention. Fresh and vitrified-thawing MΙΙ oocytes were activated after IVM in three conditioned mediums by ionomycin. Mediums included 1) Minimum Essential Medium Alpha (α-MEM) (as control medium), 2) α-MEM supplemented with supernatants of Mesenchyme bone marrow (B.M), 3) α-MEM with ovarian growth factors (O.F). Then, scoring of parthenote embryos was undertaken in accordance with pertinent morphological properties. Moreover, the expression of Bax and Bcl2 were determined in the parthenote embryos. Percentage of the degenerated oocyte, 2-4 cells, 4-8 cells, and 16 cells, was different in the experimental groups. Also, cytoplasmic maturation and blastocyst formation rates were significantly different (p < 0.05) between the control and the other mediums. The highest mRNA expression levels of Bcl2 and Bax genes in parthenotes were observed in the fIVM O.F and vIVM α-MEM mediums, respectively. vIVM, α-MEM and fIVM O.F showed the lowest expression of Bcl2 and Bax genes, respectively. Our findings indicate that the O.F. medium had more potent effects on oocyte growth and cytoplasmic maturation up to the blastocyst stage with the highest expression level of the BCL2 gene and the lowest relative amount of the BAX gene in this medium. The results of the present study have been verified only for parthenogenetically activated embryos, and any positive effect of the environment on the egg/embryo fertilized with sperm requires more extensive studies.

Effect of endometrial cell-conditioned medium and platelet-rich plasma on the developmental competence of mouse preantral follicles: An in vitro study.

ObjectiveThe aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization.MethodsIn total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction.ResultsIn the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups.ConclusionOverall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

In-vitro corrosion behaviors of extruded Mg–Ga alloys in alpha minimum essential medium

The in-vitro corrosion behaviors of extruded Mg–xGa (x = 1, 3, 5) alloys in alpha minimum essential medium (α-MEM) were investigated. The bilayer corrosion film was composed of an outer layer dominated by amorphous phosphates–carbonates and an inner layer dominated by MgO–Mg(OH)2. Pure Mg and Mg–1Ga exhibited low corrosion rates and homogeneous corrosion behaviors owing to the protection from the compact corrosion films. By contrast, Mg–3Ga and Mg–5Ga exhibited high corrosion rates and inhomogeneous corrosion behaviors owing to the bulky micro-galvanic corrosion caused by Mg5Ga2. Based on these results, possible corrosion models were developed.

Osteoimmunomodulatory potential of 3D printed submicron patterns assessed in a direct co-culture model.

Modulation of the immune response following the implantation of biomaterials can have beneficial effects on bone regeneration. This involves complex interactions between the inflammatory and osteogenic cells. Therefore, the study of cell-cell interactions using direct co-culture models integrated with biomaterials is of great interest. This research aimed to study the viability, morphology, and osteogenic activity of preosteoblasts (OBs) co-cultured with pro-inflammatory macrophages (M1s) on the 3D printed (non)patterned surfaces. OBs and M1s remained alive and proliferated actively for 14days in the mixture of Dulbecco's Modified Eagle's Medium (DMEM) and alpha Minimum Essential Medium (α-MEM) (1:1), regardless of the cell ratio in the co-cultures. The spatial organization of the two types of cells changed with the time of culture from an initially uniform cell distribution to the formation of a thick layer of OBs covered by clusters of M1s. On day 7, the expression of PGE2 and TNF-α were upregulated in the co-culture relative to the mono-culture of OBs and M1s. The inflammation decreased differentiation and matrix mineralization of OBs after 28days of culture. Interestingly, the incorporation of 3D printed submicron pillars into the direct co-culture model enhanced the differentiation of preosteoblasts, as shown by relatively higher RUNX2 expression, thereby revealing the osteoimmunomodulatory potential of such surface patterns.

P-468 Organotypic culture of testicular tissue from infant boys with cryptorchidism

Abstract Study question Can organotypic culture support the survival and maturation of germ cells and niche-related cells within testicular tissue from infant boys with cryptorchidism? Summary answer The testicular structure and the number of germ cells were maintained during organotypic culture, whereas Sertoli cells and peritubular myoid cells (PTMCs) matured. What is known already Testicular tissue cryopreservation (TTC) is a strategy to safeguard the fertility of prepubertal boys who face a risk of infertility. Organotypic culture of immature testicular tissue from mice achieved production of spermatozoa. Similarly, the culture of human fetal gonads resulted in the generation of competent spermatids. However , in vitro spermatogenesis by organotypic culture of human prepubertal testicular tissue has not been achieved. It is also unknown whether germ cells as well as its niche-related cells, in testicular tissue from infant boys with cryptorchidism, can maintain and mature under in vitro conditions. Study design, size, duration Testicular tissue was cryopreserved from four infant boys with bilateral cryptorchidism undergoing orchidopexy (age range: 0.5-1.4 years), as part of a fertility preservation program. Culture media with and without retinoic acid were tested. Testicular fragments were harvested at 30 days and 60 days after culture and evaluated by histological assessment of tissue structure, germ cell development, and immunohistochemical staining for germ cell and somatic cell markers. Participants/materials, setting, methods Cryopreserved-thawed testicular tissue was cut into fragments (1-2 mm3) and placed on top of agarose gel stands and cultured at 34oC with 5% CO2 in Minimum Essential Medium-alpha supplemented with knockout serum replacement, human umbilical cord plasma, Activin A, hormones, growth factors, with or without retinoic acid. Immunohistochemical analyses were performed using germ cell markers (MAGE-A, GAGE, and VASA), Sertoli cell maturation markers (AMH, AR, SOX9), PTMC marker (alpha-SMA). Main results and the role of chance Following the 60-day culture, the lumen of the seminiferous tubules had developed. The number of germ cells per tubule remained stable during this period. However, no further germ cell maturation was observed. Germ cells showed different phenotypes of MAGEA, GAGE, and VASA expression with no significant difference in number. The number of SOX9-positive Sertoli cells was significantly increased from 30 days to 60 days of culture (p &amp;lt;0.0001). No difference in AMH expression was observed, while AR expression in Sertoli cells was induced during culture. Alpha-SMA expression was detected in the PTMCs surrounding the seminiferous tubules. The two different culture conditions, with and without retinoic acid in the culture media, did not affect cell survival or maturation. Limitations, reasons for caution The small number of testicular biopsies available is a limitation. Wider implications of the findings Our organotypic culture conditions support the long-term survival of germ cells in testicular tissue from infant boys with cryptorchidism. Thus, further studies are needed to induce the maturation of germ cells under similar experimental conditions. Trial registration number not applicable

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.

Physicochemical properties and osteoclastogenesis for three premixed calcium silicate-based sealers post set.

Solubility, pH, ion release, cytotoxicity, and osteoclastogenesis inhibition in bone marrow-derived monocyte macrophages (BMMs) were evaluated in EndoSequence BC Sealer (END), Bio-C Sealer (BC), and Sealer Plus BC (SPBC). pH was determined after immersion of the sealers in deionized water (DW) and Minimum Essential Medium Alpha (α-MEM). Solubility was obtained by mass loss. Ion release was measured by using X-ray fluorescence spectroscopy (XRF). Cytotoxicity was evaluated by MTT assay. Inhibition of osteoclastogenesis was evaluated by tartrate-resistant acid phosphatase (TRAP). Data were analyzed using the t-test, ANOVA and Tukey/Dunnett's post-hoc tests (α = 0.05). END had the highest pH in DW (p < 0.05), and BC, in α-MEM (p < 0.05). Solubility in DW was the lowest for SPBC (p < 0.005). The highest calcium release was observed for BC in DW at 12 h (p < 0.05), and in α-MEM at 12 and 24 h (p < 0.05). The lowest toxicity was detected for END (p < 0.05). BC had the highest inhibitory effect on osteoclasts (p < 0.05). Overall, the highest solubility and pH values were found in DW. However, the calcium silicate-based sealer showed higher solubility than the ISO standards. Calcium release was the highest for BC. END showed the highest cell viability, and BC, the highest osteoclast inhibition.

The reaggregation of normal granulosa-cumulus cells and mouse oocytes with polycystic ovarian syndrome in vitro: An experimental study.

BackgroundThe dialogue between oocytes and their surrounding cells plays a major role in the progress of oocyte meiosis and their developmental potential.ObjectiveThis study aimed to evaluate the effect of co-culture of normal granulosa-cumulus cells (GCCs) with oocytes from polycystic ovarian syndrome (PCOS) mice.Materials and MethodsNormal GCCs were collected from 10 virgin adult Naval Medical Research Institute female mice (30-35 gr, 7-8 wk old), and were cultured in an alpha-minimum essential medium supplemented with 5% fetal calf serum for 24-48 hr (110 cells/well). Then, germinal-vesicle oocytes from PCOS mice were cultured in the presence of cultured normal GCCs (experimental group) and without GCCs (control group). The maturation rate and quality of the PCOS oocytes were examined by evaluating TFAM and Cx43 gene expression (real-time PCR) and the connection among PCOS oocytes and normal GCCs after 24 hr of culture.ResultsThe co-culture of normal GCCs and PCOS oocytes in the experimental group led to the formation of a complex called a PCOS oocyte-normal GCCs complex. The maturation rate of these complexes was significantly increased compared to that of the control group (p 0.001). A significant difference was also found in the expression of Cx43 (p 0.001) and TFAM (p 0.05) genes in the experimental group compared with the control group. The connection between PCOS oocytes and normal GCCs was observed in the scanning electron microscope images.ConclusionCo-culture with normal GCCs improves the capacity of PCOS oocytes to enter meiosis, which may result in the promotion of assisted reproduction techniques.

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.

Calcium Hydroxide Increases Human Umbilical Cord Mesenchymal Stem Cells Expressions of Apoptotic Protease-Activating Factor-1, Caspase-3 and Caspase-9.

PurposeCalcium hydroxide is a gold standard dental material generally used for pulpal and periapical therapy including regenerative endodontic procedures because of its positive properties. However, evaluation about this material on stem cells is limited. Human umbilical cord mesenchymal stem cells (HUCMSCs) are potential to be used in regenerative therapy. Regenerative therapy needs a sustainable cell supply to maintain its regenerative capacity. The aim of this study was to ascertain the apoptosis result of calcium hydroxide on HUCMSCs through the expression of apoptotic protease-activating factor-1 (APAF-1), caspase-3, and caspase-9.Materials and MethodsThis study used a thawed frozen stock of passage 5 HUCMSCs, grown in minimum essential medium (MEM) alpha containing calcium hydroxide at concentration of 0.1 microgram/mL for 1, 3 and 7 days. Polyclonal antibody with fluorescence isothiocyanate (FITC) label was used to evaluate the expressions. APAF-1, caspase-3, and caspase-9 expressions were recorded and compared on every observation day using fluorescence microscope. Analysis of variance was performed to analyze the significance among the results of treatment groups. The results were concluded significant if p<0.05.ResultsThe addition of calcium hydroxide in MEM alpha medium increases HUCMSCs expression of APAF-1, caspase-3 and caspase-9 significantly, compared to the control group without calcium hydroxide (p<0.05) in all the times. Day 1 showed the lowest increase followed by higher expressions on day 3 and day 7.ConclusionHUCMSCs express increased APAF-1, caspase-3 and caspase-9 after in-vitro calcium hydroxide exposure. This should be considered when using calcium hydroxide on HUCMSCs for regenerative procedures with regard to other positive properties.

In vitro growth culture in a medium with reduced sodium chloride improves maturation and developmental competence of pig oocytes derived from small antral follicles

The objective of this study was to evaluate the effects of reducing the sodium chloride content in in vitro growth (IVG) medium to 61.6 mM on in vitro maturation (IVM) and embryonic development of pig oocytes derived from small antral follicles (SAF) less than 3 mm in diameter. SAF oocytes were cultured for 2 days to induce IVG in alpha-minimal essential medium with 108 mM NaCl (αMEM-108) or porcine zygote medium (PZM) containing 61.6 mM (PZM-61.6) or 108 mM (PZM-108) NaCl. These media were further supplemented with 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 10% (v/v) fetal bovine serum. After IVG culture, oocytes were matured for 44 h in our standard IVM medium. The IVG culture in PZM-61.6 significantly increased nuclear maturation (88.0 ± 2.2%) of SAF oocytes compared to that in PZM-108 (77.3 ± 3.9%) or αMEM-108 (75.9 ± 3.8%). After parthenogenesis (PA), the proportions of blastocysts, based on the number of metaphase II (MII) oocytes, induced for PA were not different among IVG oocytes cultured in PZM-61.6 (50.2 ± 3.0%), PZM-108 (46.8 ± 2.9%), or αMEM-108 (45.6 ± 2.9%). The IVM oocytes derived from IVG in PZM-61.6 showed increased perivitelline space (PVS) (12.1 ± 0.6 μm) and intra-oocyte glutathione (GSH) content (1.19 ± 0.04 pixels/oocyte) compared to PVS (8.0 ± 0.5 and 7.4 ± 0.4 μm) and GSH (1.03 ± 0.04 and 1.00 ± 0.04 pixels/oocyte) of oocytes derived from PZM-108 and αMEM-108, respectively. The IVG culture in PZM-61.6 stimulated meiotic resumption after IVG and faster nuclear progression after IVM than that in αMEM-108. After somatic cell nuclear transfer (SCNT), the blastocyst formation of SAF oocytes grown in PZM-61.6 (17.8 ± 3.3%) was higher than that of oocytes grown in PZM-108 (7.5 ± 2.7%) but not different from that of oocytes in αMEM-108 (11.4 ± 3.4%). Regardless of the different osmotic pressures, nuclear maturation was significantly increased by IVG culture in PZM with reduced NaCl (86.8 ± 2.3 and 84.9 ± 4.2% in PZM-61.6 and PZM-61.6 with sorbitol, respectively) than in PZM-108 (70.5 ± 3.4%). Blastocyst formation was not affected by the differences in NaCl content and osmotic pressure of the IVG medium, whereas the mean number of cells in blastocysts was significantly higher following IVG culture in PZM-61.6 than in the other groups. In conclusion, the results demonstrate that, following SCNT in pigs, IVG culture of SAF oocytes in a medium with a reduced NaCl concentration stimulates oocyte maturation and improves subsequent embryonic development.

Cytotoxicity and Bioactivity of Calcium Silicate-based Cements in a Culture of Stem Cells from the Apical Papilla.

The present in vitro study evaluated the cytotoxicity and bioactivity of commonly-used calcium silicate-based cements in a culture of stem cells from the apical papilla (SCAPs). NeoMTA Plus (Avalon Biomed), BiodentineTM (Septodont) and MTA HP Repair (Angelus) cements were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and sulphorhodamine-B (SRB) viability assays. Cells were seeded (1*104 cells mL-1) in 96-well plates and exposed to 1:4 diluted extract in 24 h and 72 h. For the analysis of bioactivity, alkaline phosphatase (ALP) enzyme activity and Alizarin Red S (AZR) were assessed after 24 h of cell culture in 12-well plates (1*104 cells mL-1), where cells were exposed to 1:4 diluted extract on days 1 and 7. Minimum Essential Eagle's Medium alpha modification was used as control. ANOVA and Tukey's post hoc test were used to compare the different cements at each experimental time point. No significant differences were found between the cements and the control specimens on MTT at 24 h and 72 h (P>0.05); however, the calcium silicate-based cement materials showed higher cell viability compared to the control group (P<0.05). In the 24-h SRB, NeoMTA Plus showed lower cell viability than BiodentineTM and MTA HP Repair (P<0.05), with all groups similar to the control group (P>0.05). Compared to 24-h results, only NeoMTA Plus presented increased cell viability at 72 h (P<0.05). ALP activity was similar across the materials at 1 day (P>0.05). ALP activity was higher for BiodentineTM when compared to NeoMTA Plus (P<0.05), nevertheless, it was similar to MTA HP Repair and control groups (P>0.05) at 7 days. At 1- and 7-day periods of AZR assay, BiodentineTM presented higher levels of mineralized nodule formation (P<0.05). All evaluated calcium silicate-based cements demonstrated cell viability and bioactivity, suggesting that these (bio)materials may be indicated for use in regenerative dentine-pulp complex procedures.

P0906SALUBRINAL IMPROVES OSTEOCLAST DIFFERENTIATION AND VASCULAR CALCIFICATION IN UREMIC MILLENNIUM THROUGH INHIBITION OF THE ENDOPLASMIC RETICULUM (ER) STRESS CONDITION

Abstract Background and Aims Chronic kidney disease (CKD) is a globally increasing health problem especially in aged era. CKD-mineral and bone disorder (CKD-MBD) becomes a principal consequence of CKD, which includes mineral abnormalities, vascular calcifications (VC) and renal osteodystrophy (ROD), and patients eventually present with fractures and cardiovascular disease (CVD) (Figure 1). Thus, bone loss in CKD patients relates closely with cardiovascular calcification. The endoplasmic reticulum (ER) stress is triggered by many clinical conditions unique to CKD, including uremic toxins, chronic inflammation, metabolic acidosis, proteinuria, malnutrition, etc. In our study, we hope to evaluate the ER stress inhibitor, salubrinal as targeted therapy for CKD related bone loss and vascular calcification. Methods: Isolation of primary osteoclasts from long bone primary cell culture: Femoral and tibia bone from 8-week-old Sprague–Dawley rats were removed and used as primary osteoclast cultures. Osteoclast precursor cells were seeded in 96-well plates (2.0 x 104 cells/well) and cultured for 6 days in alpha minimum essential medium (α-MEM) supplemented with 10% FBS, 50 ng/ml M-CSF and 50 ng/ml RANKL in the absence or presence of uremic toxins. The whole process of osteoclast development in cell cultures was divided into first M-CSF–dependent growth of osteoclast progenitor’s phase and latter phase is RANKL induced terminal differentiation phase. The experiment is performed with the approval of the Laboratory Animal Center of the Taipei Medical University in Taipei, Taiwan. Primary Human Aorta Vascular Smooth Muscle Cell Culture: Primary human aorta vascular smooth muscle cells (VSMC) were isolated by ScienCell Research Laboratories, Inc. (Carlsbad, CA) and purchased through Innoprot (Derio, Spain). We examined the role of uremic toxin on ER stress and autophagy using osteoclasts and vascular smooth muscle cells cultured with uremic toxins including PTH, p-cresylsulfate (pCS) and p-cresylglucuronide (pCG), indoxyl sulfate, asymmetric dimethylarginine (ADMA), and homocysteine. The cells are treated with salubrinal with or without toxins and see how ER stress and autophagy effects on survival and differentiation of osteoclasts and VSMC cells. Results We found that ER stress and autophagy related mRNA were increased in osteoclasts using GEO database analysis, revealed that ER stress and autophagy play an important role in osteoclast differentiation. Further, we revealed that uremic toxins increased the autophagy and ER stress status and increased the osteoclasts differentiation. Treatment of the osteoclasts under uremic millennium with salubrinal, an ER stress inhibitor, inhibited the ER stress and osteoclast differentiation. Uremic toxins also increased the ER stress in human vascular smooth muscle cells (VSMCs). After treated with salubrinal, the calcification and proliferation improved in these cells. Conclusion ER stress increases osteoclast differentiation and vascular calcification in uremic millennium, and salubrinal improves these conditions through inhibition of ER stress. Thus, salubrinal probably might be used as targeted therapy for vascular calcification and bone loss in CKD patients.

The effect of conditioned media on mouse oocytes ultrastructure following in vitro maturation

Optimizing the culture condition and vitrification process during in vitro maturation (IVM) of oocyte has been controversial. This study aimed to evaluate the effect conditioned medium (CM) enriched with human mesenchymal stem cells (hUCM) derived from bone marrow and umbilical cord blood on the ultrastructure of immature mouse oocytes during IVM. Three hundred ninety-two germinal vesicle (GV) oocytes were obtained from mouse ovaries. They were divided into fresh IVM (fIVM) and vitrified IVM (vIVM) groups and cultivated in either alpha Minimum Essential Medium (α-MEM) (control) or CMs supplemented with supernatants derived from cultures of bone marrow or umbilical cord mesenchyme stem cells (MSCs). Then the maturation rate was evaluated in the experimental groups. Twelve in vivo fresh mature oocytes were randomly obtained from uterine tubes as the control group. Finally, 43 matured IVM oocytes were randomly selected for ultra-structural evaluations using transmission electron microscopy (TEM). Oocyte maturation rates were significantly higher in fIVM than vIVM groups. The highest maturation rate was observed in the fIVM umbilical cord MSCs while the lowest rate was related to α-MEM vIVM (86.44% vs 72.72%). There was an increase in the number of mitochondria–vesicle (MV) complexes in IVM groups. Also, the distribution of cortical granules showed the highest decline in α-MEM vIVM groups. The highest aggregations of mitochondria–SER (M-SER) were observed in fIVM umbilical cords. Oolemma had an irregular border with small microvilli in vIVM groups. The Zona pellucida was darker in the vIVM than fIVM groups. In conclusion, umbilical cord MSCs showed a superior efficacy than bone marrow cells for supporting oocyte maturation and conserving its ultrastructure.

Effect of different extracts and fractions of Senecio biafrae (Oliv. &Hiern) J. Moore on in vivo and in vitro parameters of folliculogenesis in experimental animals.

Senecio biafrae is a medicinal plant widely used in traditional medicine to cure female infertility. Some effects have been pharmacologically demonstrated on immature female rats but in vivo and in vitro investigations are still necessary for determining its mechanism of action. The aim of the present study was to evaluate the estrogenic and FSH-like effects of the plant extracts and fractions on some fertility parameters in immature female rats and on in vitro survival and growth of swine preantral follicles. 21-23 days old female Wistar rats orally received extracts and fractions of S. biafrae at 0, 8 and 64mg/kg doses over 20 days. The LH, FSH, estradiol and progesterone serum levels were evaluated as well as the ovarian cholesterol, uterus and ovaries masses and proteins. The numbers of follicles at different developmental stages were recorded in ovarian cortexes after histology. Slices of swine ovarian cortexes were cultured along 1 or 7 days in alpha-minimum essential medium (α-MEM) and fixed for morphological analysis of preantral follicles. The fresh control, cultured control (CIV control) and different Senecio biafrae-treated ovarian fragments were analyzed for preantral follicles development. Treatments that showed the best follicle growth in culture were submitted to AgNOR test. The aqueous and MeOH/CH2Cl2 extracts as well as the ethyl acetate and hexane fractions of S. biafrae were submitted to the HPLC for analysis of polyphenolic secondary metabolites. Ovarian and uterine proteins were significantly high (p<0.01) in animals treated with the two dosages of ethyl acetate and n-butanol fractions. The same result was recorded with uterine proteins in animals treated with the hexane fraction. The FSH level significantly dropped with all ethanolic extract doses and with the 64mg/kg dosage of the methanol/methylene chloride (MeOH/CH2Cl2) extract while LH was reduced (p<0.01) in almost all the treated groups. Estradiol level was significantly increased (p<0.001) in the three groups receiving the extracts, but reduced (p<0.001) in the three groups receiving the fractions of the plant. The progesterone level increased with almost all the treated groups. Primary and secondary follicles augmented (p<0.01) in MeOH/CH2Cl2 extract and n-butanol fraction while tertiary follicles increased with the same extract and the ethyl acetate fraction (p<0.05). Treatments with aqueous and ethanolic extracts as well as ethyl acetate fraction led to a significant increase (p<0.05) in the number of morphologically normal follicles after 7 days of culture as compared to the CIV control. The number of AgNOR dots per follicle was significantly low (p<0.05) in all cultured groups as compared to the fresh control, except the ethyl acetate 2.8ng/ml dosage. The same observation was done with AgNOR dots per cell in the 2.8ng/ml dosage aqueous extract-treated fragments. The phenolic compounds mainly encountered in the plant, independently of the extract or fraction are apigenin, eugenol and rutin. Extracts and fractions of S. biafrae have an important FSH-like effect which induces follicular survival and growth.

Cytotoxicity of Calcium Hydroxide on Human Umbilical Cord Mesenchymal Stem Cells

Objective: To examine the cytotoxicity of calcium hydroxide on human umbilical cord mesenchymal stem cells (HUCMSC) to understand the characteristics for use in regenerative dentistry procedures especially regenerative endodontics. Material and Methods: HUCMSC was isolated, cultured, and confirmed by flow cytometry. The biological characteristics, such as cell morphology, proliferation, and protein expression, were screened. To check the cytotoxicity, HUCMSC was cultured and divided into two groups, the control group (cultured in minimum essential medium (MEM) alpha) and calcium hydroxide group (cultured in MEM alpha and calcium hydroxide). Methyl-thiazole-tetrazolium (MTT) assay was done on different concentrations of calcium hydroxide (0.39 to 25 µg/mL) and the cells were observed and counted. One-way ANOVA test was used with a significance level set at 5% . Results: Flow cytometric analysis confirmed positive of CD73, CD90, CD105, negative of CD45 and CD34. A significant difference was found between the concentration of 6.25 and 3.125 µg/mL (p=0.004). There was no significant difference among 6.25, 12.5 and 25 µg/mL concentrations. There was also no significant difference among 0.39, 0.78, 1.56, and 3.125 µg/mL concentrations . Conclusion: Even though calcium hydroxide is a medicament of choice in clinical endodontics, it decreases the viability of HUCMSC. The lower the concentration of calcium hydroxide, the higher the viability of HUCMSC.