In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)

In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)). Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA). Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l(-1)) were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l(-1) 2,4-D and 2 mg.l(-1) BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.

In vitro cytotoxicity of dental alloys and cpTi obtained by casting

Background: This study aimed to compare the cytotoxicity of base-metal dental alloys and to evaluate if the casting method could influence their cytotoxicity. Methods: Disks of base-metal dental alloys were cast by two methods: plasma, under argon atmosphere, injected by vacuum-pressure; and oxygen-gas flame, injected by centrifugation, except Ti-6Al-4V and commercially pure titanium (cpTi), cast only by plasma. SCC9 cells were cultured in culture media D-MEM/Ham`s F12 supplemented, at 37 degrees C in a humidified atmosphere of 5% carbon dioxide and 95% air, on the previously prepared disks. At subconfluence in wells without disks (control), cell number and viability were evaluated. Results: In plasma method, cpTi and Ti-6Al-4V were similar to control and presented higher number of cells than all other alloys, followed by Ni-Cr. In oxygen-gas name method, all alloys presented fewer cells than control. Ni-Cr presented more cells than any other alloy, followed by Co-Cr-Mo-W which presented more cells than Ni-Cr-Ti, Co-Cr-Mo, and Ni-Cr-Be. There were no significant differences between casting methods related to cell number. Cell viability was not affected by either chemical composition or casting methods. Conclusion: cpTi and Ti-6Al-4V were not cytotoxic while Ni-Cr-Be was the most cytotoxic among tested alloys. The casting method did not affect cytotoxicity of the alloys. (c) 2007 Wiley Periodicals, Inc.

In Vitro Evaluation of Acellular Dermal Matrix as a Three-Dimensional Scaffold for Gingival Fibroblasts Seeding

Background: Tissue engineering principles could improve the incorporation of acellular dermal matrix (ADM). The aim of this study is to verify if ADM is a suitable three-dimensional matrix for gingival fibroblasts and cancerous cells ingrowth, and also if cultured medium conditioned in ADM affect cellular behavior. Methods: Canine gingival fibroblasts (CGF), human gingival fibroblasts (HGF), and murine melanoma cell line (B16F10) were seeded on ADM for up to 14 days. The following parameters were assessed: morphology and distribution of CGF, HGF, and B16F10; CGF and HGF viability; and the effect of ADM conditioned medium (CM) on CGF viability. Results: Epifluorescence revealed that CGF were unevenly distributed on the ADM surface, showing no increase in cell number over the periods of study; HGF formed a monolayer on the ADM surface in a higher number at 14 days (P<0.05); B16F10 exhibited an increase in cell number within 7 days (P<0.05), and were mainly arranged in cell aggregates on the ADM, forming a continuous layer at 14 days. A higher percentage of cells on the ADM surface (P<0.05) compared to inside was observed for all cell types. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MU) values indicated higher cell viability in samples cultured with HGF compared to CGF (P=0.024). A significantly lower cell viability for CGF grown in CM compared to cells grown in non-CM was observed at 48 and 72 hours (P<0.05). Conclusions: ADM is not suitable as a three-dimensional matrix for gingival fibroblasts ingrowth. Gingival fibroblasts and highly proliferative cells as B16F10 can only be superficially located on ADM, and CGF are negatively affected by culture medium conditioned in ADM, reducing its viability. J Periodontol 2011;82:293-301.

Nitric oxide detection in cell culture exposed to LPS after Er:YAG laser irradiation

P>Aim To evaluate in vitro the effect of calcium hydroxide [Ca(OH)(2)] and Er:YAG laser on bacterial endotoxin [also known as lipopolysaccharide (LPS)] as determined by nitric oxide (NO) detection in J774 murine macrophage cell line culture. Methodology Samples of LPS solution (50 mu gmL-1), Ca(OH)(2) suspension (25 mg mL-1) and LPS suspension with Ca(OH)(2) were prepared. The studied groups were: I - LPS (control); II - LPS + Ca(OH)(2); III - LPS + Er:YAG laser (15 Hz 140 mJ); IV - LPS + Er:YAG laser (15 Hz 200 mJ); V - LPS + Er:YAG laser (15 Hz 250 mJ), VI - Pyrogen-free water; VII - Ca(OH)(2). Murine macrophage J774 cells were plated and 10 mu L of the samples were added to each well. The supernatants were collected for NO detection by the Griess reaction. Data were analysed statistically by one-way anova and Tukey`s test at 5% significance level. Results The mean and SE (in mu mol L-1) values of NO release were: I - 10.48 +/- 0.58, II - 6.41 +/- 0.90, III - 10.2 +/- 0.60, IV - 8.35 +/- 0.40, V - 10.40 +/- 0.53, VI - 3.75 +/- 0.70, VII - 6.44 +/- 0.60; and the values for the same experiment repeated after 1 week were: I - 21.20 +/- 1.50, II - 9.10 +/- 0.60, III - 19.50 +/- 1.00, IV - 18.50 +/- 0.60, V - 21.30 +/- 0.90, VI - 2.00 +/- 0.20, VII - 6.80 +/- 1.70. There was no significant difference (P > 0.05) between the control and the laser-treated groups (III, IV and V), or comparing groups II, VI and VII to each other (P > 0.05). Group I had significantly higher NO release than group II (P < 0.05). Groups II and VI had similar NO release (P > 0.05). Conclusions Calcium hydroxide inactivated the bacterial endotoxin (LPS) whereas none of the Er:YAG laser parameter settings had the same effectiveness.

Effects of the Association between a Calcium Hydroxide Paste and 0.4% Chlorhexidine on the Development of the Osteogenic Phenotype In Vitro

The present study aimed to evaluate whether the association between a calcium hydroxide paste (Calen paste) and 0.4% chlorhexidine (CHX) affects the development of the osteogenic phenotype in vitro. With rat calvarial osteogenic cell cultures, the following parameters were assayed: cell morphology and viability, alkaline phosphatase activity, total protein content, bone sialoprotein immunolocalization, and mineralized nodule formation. Comparisons were carried out by using the nonparametric Kruskal-Wallis test (level of significance, 5%). The results showed that the association between Calen paste and 0.4% CHX did not affect the development of the osteogenic phenotype. No significant changes were observed in terms of cell shape, cell viability, alkaline phosphatase activity, and the total amount of bone-like nodule formation among control, Calen, or Calen + CHX groups. The strategy to combine Ca(OH)(2) and CHX to promote a desirable synergistic antibacterial effect during endodontic treatment in vivo might not significantly affect osteoblastic cell biology. (J Endod 2008;34:1485-1489)

In vitro antibacterial action of Tetraclean, MTAD and five experimental irrigation solutions

P>Aim To investigate the antibacterial effect of Tetraclean, MTAD and five experimental irrigants using both direct exposure test with planktonic cultures and mixed-species in vitro biofilm model. Methodology Tetraclean, MTAD and five experimental solutions that were modifications of existing formulae including MTAD + 0.01% cetrimide (CTR), MTAD + 0.1% CTR, MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 (Tween 80 replaced by 0.1% CTR) and MTAD-D (MTAD without the Tween 80 and no CTR added) were used as disinfectants in the experiments. In the direct exposure test, a suspension of Enterococcus faecalis was mixed with each of the solutions. After 0.5, 1, 3 and 10 min, an inactivator was added and the number of surviving bacteria was calculated. A mixed-species biofilm from subgingival plaque bacteria was grown in brain heart infusion broth in anaerobic conditions on synthetic hydroxyapatite discs. Two-week-old biofilms were exposed to the solutions for 0.5, 1 and 3 min. The samples were observed by confocal laser scanning microscopy after bacterial viability staining. The scans were quantitatively analysed, and the volume of killed cells of all cells was calculated for each medicament. Results Tetraclean and MTAC-2 (0.1% CTR) killed planktonic E. faecalis in < 30 s. Complete killing of bacteria required 1 min by MTAC-1, 3 min by MTAD + 0.1% CTR and 10 min by MTAD, MTAD-D and MTAD + 0.01% CTR. In the biofilm test, there were significant differences in microbial killing between the different solutions and times of exposure (P < 0.005). MTAC-2 showed the best performance, killing 71% of the biofilm bacteria in 3 min, followed by MTAC-1 and Tetraclean. MTAD and the three MTAD modifications demonstrated the lowest antibacterial activity. Conclusion Tetraclean was more effective than MTAD against E. faecalis in planktonic culture and in mixed-species in vitro biofilm. CTR improved the antimicrobial properties of the solutions, whereas Tween 80 seemed to have a neutral or negative impact on their antimicrobial effectiveness.

Effect of Three Methods for Cleaning Dentures on Biofilms Formed In Vitro on Acrylic Resin

Purpose: The aim of this study was to evaluate the effect of three denture hygiene methods against different microbial biofilms formed on acrylic resin specimens. Materials and methods: The set (sterile stainless steel basket and specimens) was contaminated (37 degrees C for 48 hours) by a microbial inoculum with 106 colony-forming units (CFU)/ml (standard strains: Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Candida albicans, Pseudomonas aeruginosa, and Enterococcus faecalis; field strains: S. mutans, C. albicans, C. glabrata, and C. tropicalis). After inoculation, specimens were cleansed by the following methods: (1) chemical: immersion in an alkaline peroxide solution (Bonyplus tablets) for 5 minutes; (2) mechanical: brushing with a dentifrice for removable prostheses (Dentu Creme) for 20 seconds; and (3) a combination of chemical and mechanical methods. Specimens were applied onto a Petri plate with appropriate culture medium for 10 minutes. Afterward, the specimens were removed and the plates incubated at 37 degrees C for 48 hours. Results: Chemical, mechanical, and combination methods showed no significant difference in the reduction of CFU for S. aureus, S. mutans (ATCC and field strain), and P. aeruginosa. Mechanical and combination methods were similar and more effective than the chemical method for E. faecalis, C. albicans (ATCC and field strain), and C. glabrata. The combination method was better than the chemical method for E. coli and C. tropicalis, and the mechanical method showed intermediate results. Conclusion: The three denture hygiene methods showed different effects depending on the type of microbial biofilms formed on acrylic base resin specimens.

In vitro inhibitory effect of trichostatin A on canine grade 3 mast cell tumor

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M -phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.

A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome

Oocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological. criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

Influence of nitric oxide during maturation on bovine oocyte meiosis and embryo development in vitro

The effect of s-nitroso-N-acetyl-1,1-penicillamine (SNAP, a nitric oxide donor) during in vitro maturation (IVM) on nuclear maturation and embryo development was investigated. The effect of increasing nitric oxide (NO) during prematuration or maturation, or both, on embryo development was also assessed. 10(-3) M SNAP nearly blocked oocytes reaching metaphase II (MII) (7%, P < 0.05) while 10(-5) M SNAP showed intermediate proportions (55%). For 10(-7) M SNAP and controls (without SNAP), MII percentages were similar (72% for both, P > 0.05), but superior to the other treatment groups (P < 0.05). Blastocyst development, however, was not affected (38% for all treatments, P < 0.05). TUNEL-positive cells in hatched blastocysts (Day 9) increased when IVM included 10(-5) M SNAP (8 v. 3 to 4 cells in the other treatments, P > 0.05), without affecting total cell numbers (240 to 291 cells, P > 0.05). When oocytes were prematured followed by IVM with or without 10(-7) M SNAP, during either culture period or both, blastocyst development was similar (26 to 40%, P > 0.05). When SNAP was included during both prematuration and IVM, the proportion of Day 9 hatched embryos increased (28% v. 14 to 19% in the other treatments, P < 0.05). Apoptotic cells, however, increased when SNAP was included (6 to 10 cells) in comparison to prematuration and maturation without SNAP (3 cells, P < 0.05). NO may be involved in meiotic progression and apoptosis during embryo development.

Propagation, growth, and carbohydrates of Dendrobium Second Love (Orchidaceae) in vitro as affected by sucrose, light, and dark

In general, plant material grown in vitro has low photosynthetic ability to achieve positive carbon balances. Therefore, a continuous supply of carbohydrates from the culture medium is required, and sucrose has been the most commonly used carbon source. In this paper, we investigate the effects of different sucrose concentrations and the presence and absence of light on the endogenous levels of soluble carbohydrates and starch as well as on the proliferation and growth of Dendrobium Second Love (Orchidaceae) in vitro. The possibility of using etiolated stem segments as a means for micropropagating this hybrid was also verified. The results obtained indicated that the presence and absence of light and the sucrose concentrations used influenced the amounts of soluble carbohydrates and starch and the proliferation of D. Second Love shoots and roots. An increase in sucrose concentration caused a progressive increase in the amounts of total carbohydrates and starch. Under both light conditions, sucrose was the main sugar found in the shoots followed by glucose and fructose. The addition of sucrose to the culture medium up to 2% and 4% was advantageous to the number of shoots produced per explant and the root longitudinal growth in the presence and absence of light, respectively. Shoot and root dry matter and the number of roots formed per explant increased as sucrose concentration was raised up to 6% in both light treatments. The use of dark-grown shoot segments proved to be a useful and reliable alternative for the micropropagation of this hybrid.

Life history, morphological variability and growth rates of the life phases of Gracilaria tenuistipitata (Rhodophyta: Gracilariales) in vitro

Gracilaria tenuistipitata, a species of commercial interest, is becoming a model organism for studies on red algal physiology and molecular biology as it can be grown easily in vitro under a broad range of conditions. Most of the experiments carried out around the world have been based on a tetrasporophytic clone isolated in our laboratory from a specimen collected in China. Here we describe the life history of this species, give anatomic details of the reproductive structures, illustrate the morphological variability of tetraspore progeny and compare the growth rate of gametophytic and sporophytic thalli. Tetrasporophytic branches showed higher growth rates than gametophytic branches.

In Vitro Propagation of Heliconia bihai (L.) L. from Zygotic Embryos

(In vitro Propagation of Heliconia bihai L. from Zygotic Embryos). The internal morphology of embryos from immature and mature fruits of Hcliconia bihai (L.) L. cv. Lobster Claw Two was examined. Embryos were inoculated into MS media (full MS and 1/2 MS) and GA(1) (0.2.5 and 5 mg L(-1)) with either sucrose or glucose. These plantlets were then replicated and transferred to MS medium (full MS or 1/2 MS) with 0 or 2.5 mg L(-1) BAP and their multiplication was evaluated 30 and 45 days after inoculation. The genetic variability of the multiplied plants was estimated using isoenzyme analyses. The internal morphology of the mature embryos revealed their tissues to be in more advanced stages of differentiation than immature embryos. In the conversion phase, 85% of the inoculated embryos developed into plants in the 1/2 MS medium with sucrose, in contrast to only 41% of the embryos that were cultivated with glucose. In the multiplication phase, plants cultivated in 1/2 MS medium with 2.5 mg L(-1) BAP demonstrated more buds. Isoenzyme analyses showed pattern changes in terms of the color intensity and the migration of some of the bands. These results may be associated with differences in the ages of the mother plants and of the plantlets obtained in vitro.

Ethylene and polyamine production patterns during in vitro shoot organogenesis of two passion fruit species as affected by polyamines and their inhibitor

Levels of ethylene and polyamines (PAs) were measured during organogenesis of hypocotyl explants of two species of passion fruit (Passiflora cincinnata Masters and Passiflora edulis Sims f. flavicarpa Degener `FB-100`) to better understand the relationships of these regulators and their influence on cell differentiation and morphogenesis. Moreover, histological investigation of shoot ontogenesis was conducted to characterize the different events involved in cell redifferentiation and regulation of PA and ethylene levels. A delay was observed in morphogenic responses of P. edulis f. flavicarpa as compared to P. cincinnata, and these changes coincided with production of elevated levels of polyamine and ethylene levels. During differentiation, cells showed high rates of expansion and elongation, and high ethylene levels were associated with high PA levels, suggesting that the two biosynthesis pathways were highly regulated. Moreover, their interaction might be an important factor for determining cell differentiation. The addition of PAs to the culture medium did not promote organogenesis; however, the incorporation of the PA inhibitor methylglyoxal bisguanylhydrazone in the culture medium reduced shoot bud differentiation, suggesting the need to maintaining a minimum level of PAs for morphogenic events to take place.

In vitro effect of low-level laser on odontoblast-like cells

The aim of this study was to evaluate the metabolism of odontoblast-like MDPC-23 cells subjected to direct LLL irradiation. The cells were seeded (20,000 cells/well) in 24-well plates and incubated for 24 hours at 37 degrees C. After this period, the culture medium (DMEM) was replaced by fresh DMEM supplemented with 2 or 5% (stress induction by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to laser doses of 2, 4, 10, 15 and 25 J/cm(2) from a near infrared InGaAsP diode laser prototype (LASERTable; 780 +/- 3 nm, 40 mW). One control group (sham irradiation) was established for each experimental condition (laser dose x FBS supplementation). Three and 72 hours after the last irradiation, cells were analyzed with respect to metabolism, morphology, total protein expression and alkaline phosphatase (ALP) activity. Higher metabolism and total protein expression were observed 72 hours after the last irradiation at the doses of 15 and 25 J/cm(2) (Mann-Whitney; p<0.05). Higher ALP activity was obtained with 5% FBS when the cells were irradiated with doses of 2 and 10 J/cm(2). For the dose of 25 J/cm(2), the highest ALP activity was observed with 10% FBS. It was concluded that the LLLT parameters used in this study stimulated the metabolic activity of the MDPC-23 cells, especially at the doses of 15 and 25 J/cm(2).