In vitro determination of the remineralizing potential and cytotoxicity of non-fluoride dental varnish containing bioactive glass, eggshell, and eggshell membrane.

PURPOSE: This study compares in vitro remineralization potential and cytotoxicity of fluoride-free varnish combinations containing bioactive glass, eggshell, and membrane powder and fluoride varnish formulations on artificial caries lesions. METHODS: Artificial caries lesions were formed in two windows on third molars. One of the windows was coated with one of the following varnish formulations: FV (fluoride varnish), F-BAGV (fluoride and bioactive glass containing varnish), BAGV (bioactive glass containing varnish), EPV (eggshell powder containing varnish), EMP-EPV (eggshell membrane protein and eggshell powder containing varnish), STMP-EMP-EPV (sodium trimetaphosphate-treated eggshell membrane protein and eggshell powder containing varnish). The samples were remineralized, then investigated under scanning electron microscopy, and elemental analyses were performed by X-ray dispersive analysis (EDX). In addition, the traditional colorimetric tetrazolium-based reduction assay (XTT) and the modern impedance-based real-time cell analysis system (RTCA) were used to investigate their cytotoxicity. RESULTS: The varnish applied area's Ca/P ratio was lower than stoichiometric hydroxyapatite except for EPV (1.66) and STMP-EMP-EPV (1.67) groups. Undiluted extracts of all varnishes, 1:2 dilutions of FV and F-BAGV groups were cytotoxic in XTT assay. In RTCA, the normalised cell index of the EMP-EPV and STMP-EMP-EPV groups was higher than the control group. CONCLUSION: Bioactive glass, eggshell, eggshell membrane proteins and STMP-treated eggshell and eggshell membrane protein containing varnish have similar remineralizing effect to fluoride-containing varnish on demineralized enamel. Integrating biological or bioactive components instead of fluoride into the dental varnishes might reduce cytotoxicity.

Real-Time Analysis of Antiproliferative Effects of Mouthwashes Containing Alcohol, Sodium Fluoride, Cetylpyridinium Chloride, and Chlorhexidine In Vitro.

OBJECTIVES: In this study, the cytotoxic responses of six different over-the-counter mouthwashes on L929 cells were analyzed by two different techniques: the traditional colorimetric tetrazolium-based reduction assay (MTT) and the modern impedance-based real-time cell analysis (RTCA) system to investigate their biocompatibility in vitro. Thus, the investigation of the antiproliferative effects of the specified materials via different techniques is vital to reach this goal. MATERIALS AND METHODS: First, L929 mouse fibroblasts were exposed to the dilutions of mouthwashes for 2 minutes. After incubation, the tetrazolium reduction method was used to assess the metabolic viability of cells measured by colorimetric MTT assay and morphological inspection of cells was performed via phase-contrast microscopy. Furthermore, the effect of each mouthwash on the proliferation, morphology, and adhesion of L929 cells was monitored continuously by a noninvasive and label-free RTCA system for 140 h. RESULTS: Our data showed that all of the mouthwashes had varying cytotoxic effects on fibroblasts compared to the control group in MTT assay. In addition to that, RTCA technology has provided the growth kinetic profiles that can be used to analyze if the treatment is causing antimitotic or DNA-damaging effect on cells. Thus, analysis via this system can tell us the mechanism of toxicity behind the cell growth inhibition in vitro. Here, we found that only mouthwash 1 moderately maintained the viability of the L929 cells, yet displaying antimitotic effects and the other mouthwashes (mouthwash 2-mouthwash 6) showed toxicity via DNA-damaging effects. CONCLUSIONS: Of the six types of mouthwash tested, the most biocompatible result was obtained from a mouthwash containing alcohol (i.e., mouthwash 1). On the other hand, sodium fluoride- (NaF-) and cetylpyridinium chloride- (CPC-) containing mouthwash (i.e., mouthwash 2) showed the most cytotoxic effect.

Effects of mouthwashes on color stability and surface roughness of three different resin-based composites.

OBJECTIVE: The aim of this study is to evaluate the effects of different types of mouthwashes (Klorhexidin, Curasept ADS 205, Meridol, Listerine Cool Citrus) on the surface roughness and color changes of a microhybrid (Point 4), a bulk fill (SonicFill), and a nanohybrid (Nova Compo-N) resin-based composite (RBC). MATERIALS AND METHODS: Disk-shaped specimens were prepared from tested RBCs and divided into four subgroups which immersed in four different types of mouthwashes. The specimens were subjected to immersion cycles in the mouthwashes and artificial saliva (n = 8). Each cycle consisted of complete immersion in a mouthwash for 21 min and afterwards in saliva for 12 h at 37°C, and this cycle was repeated 8 times. The surface roughness was evaluated using a profilometer and coloration was evaluated using a spectrophotometer before and after immersion time. One-way analysis of variance (ANOVA) for the evaluation of surface roughness data was performed, and interrelation between groups was identified with the Sheffe's multiple comparison test. RESULTS: There were no significant differences between the Ra values of the RBCs before and after immersion in mouthwashes (P > 0.05). There were significant differences between ΔE value of the SF and NCN groups before and after immersion time (P < 0.05). CONCLUSION: Mouthwashes contribute to oral health, especially in patients at high risk of caries. However, in such patients, patient-specific recommendations should be made when using mouthwashes due to the large number of composite fillings.

Efficacy of using erbium, chromium-doped: Yttrium scandium gallium garnet laser-treated dentine in a dentine barrier test device.

OBJECTIVES: The aim of this study was to evaluate the efficacy of using erbium, chromium-doped:yttrium scandium gallium garnet (Er,Cr:YSGG) laser-treated dentine in a dentine barrier test device. MATERIALS AND METHODS: The test materials (G-Bond™ and Vitrebond™) were applied onto laser-treated or laser-untreated dentine discs. After 24 h of exposure with perfusion of the test chamber, cell survival was evaluated based on enzyme activity and compared to a nontoxic control material. The mean of the control was set to 100% viability. Data were analyzed using the one-way analysis of variance and the Tukey's honest significant difference tests. RESULTS: The responses of bovine pulp-derived cells after exposure to G-Bond and Vitrebond on Er,Cr:YSGG laser-treated and laser-untreated dentin were statistically different from negative control group (P < 0.05). CONCLUSION: Er,Cr:YSGG laser treatment was not successful enough in decreasing the cytotoxic effects of the dental materials. Different parameters of Er,Cr:YSGG laser or different laser types could be investigated as an alternative to minimizing the cytotoxic effects of dental materials.

Cytotoxicity evaluation of dentin contacting materials with dentin barrier test device using erbium-doped yttrium, aluminum, and garnet laser-treated dentin.

OBJECTIVES: The effect of dentin contacting materials on three-dimensional cultures of pulp-derived cells was evaluated in a dentin barrier test device using erbium-doped yttrium, aluminum, and garnet (Er:YAG) laser-treated dentin. METHODS: The test materials (iBond(®), G-Bond™, and Vitrebond™) were applied on laser-treated or untreated dentin discs. After 24 h of exposure with perfusion of the test chamber, cell survival was evaluated by enzyme activity and related to a nontoxic control material. The mean values of control tissues were set to represent 100% viability. Data were analyzed using Kruskal-Wallis and Mann-Whitney U test. RESULTS: Vitrebond was the most toxic material for both laser-treated and untreated dentin. On untreated dentin, G-bond was cytotoxic to the pulp-derived cells (p < 0.05), and iBond was similar to the negative control group (p > 0.05). However, G-Bond and iBond were not cytotoxic when they were applied to Er:YAG laser-treated dentin (p > 0.05). CONCLUSION: Er:YAG laser treatment of dentin may protect the pulp cells from toxic substances of dentin contacting restorative materials; however, this effect is material related. Taking into consideration the limitations of this in vitro study, the Er:YAG laser treatment of dentin before restoration might be an option for decreasing the cytotoxic effects of the dental materials. Further research is required for clinical applications.

Effects of current provisional restoration materials on the viability of fibroblasts.

OBJECTIVES: The aim of the present study was to evaluate the cytotoxic effects of three different provisional restoration materials on fibroblasts. Two bis-acrylic based [Tempofit Duomix (Detax), Protemp 3 Garant (3M ESPE)] and one urethan dimethacrylate [Revotek LC (GC Corporation)] based provisional restoration materials used. METHODS: Materials were prepared according to the manufacturers' instructions in standard teflon disks (2x5 mm) and four samples were extracted in 7 ml of Basal Medium Eagle with 10% new born calf serum and 100 mg/ml penicillin/streptomycin for 24 hours. The L929 fibroblast cells were plated (25.000 cells/ml) in well plates, and maintained in a CO(2) incubator at 37 degrees C for 24h. After 24 hours, the incubation medium was replaced by the immersed medium in which the samples were stored and the L929 fibroblasts were incubated in contact with eluates for 24 hours at 37 degrees C for 24h. The fibroblast cell viability was analyzed by measuring the mitochondrial activity with the methyltetrazolium test (MTT). Twelve well used for each specimen and experiment repeated for two times. The data was statistically analyzed by Mann-Whitney U tests. RESULTS: The results showed that, Revotek LC and Protemp 3 Garant were not cytotoxic for fibroblast cells when compared to control group (P>.05). However, Tempofit duomix was cytotoxic for L929 fibroblasts when compared to control group and other tested materials (P<.05). CONCLUSIONS: Taking into consideration the limitations of an in vitro study, our study indicate that provisional restoration materials might have cytotoxic effects on fibroblasts and should be selected carefully for clinical applications.

GnRH or eCG treatment fails to restore reproductive function in GnRH immunized ewes.

This study was designed to evaluate the potential of using eCG or GnRH in restoring reproductive functions in GnRH immunized ewes. Thirty-three multiparous Kivircik ewes were randomly assigned into either control group (n=11) or immunization group (n=22). Ewes were immunized against GnRH by injecting with a cocktail of ovalbumin-LHRH-7 (ovalbumin-GnRH-7) and thioredoxin-LHRH-7 (thioredoxin-GnRH-7) fusion proteins generated by recombinant DNA technology in April. 500 IU eCG or 0.008 mg GnRH analogue was used to induce ovulations. Serum GnRH antibodies were present in animals of the immunized group beginning the second week after the first immunization and maintained throughout the study (14 months). Immunization caused anestrus in immunized ewes. eCG or GnRH analogue administration given after 14 days progestagen (20 mg fluorogestone acetate, FGA) treatment during breeding season (mid July) did not induce ovulation in these ewes. Two more attempts with single or multiple eCG injections failed to induce ovulation in this group as well. It appears that the gonadotropin stimulation was not of adequate time since neither eCG nor GnRH administration was able to restore reproductive function in immunized animals. The immunization effect lasted more than a year. These results suggest that GnRH immunization exerts its effect via the hypothalamo-pituitary axis and that more than such stimulation is required to overcome the reproductive suppression.

LHRH fusion protein immunization alters testicular development, ultrasonographic and histological appearance of ram testis.

This study was designed to evaluate the effectiveness of a luteinizing hormone releasing hormone (LHRH) fusion protein immunization on reproductive traits in ram lambs including the changes in histology and ultrasonographic appearance of testis. Thirty native ram lambs at 19 weeks of age were divided into control (C, n = 10), immunization (I, n = 10) and castration (E, n = 10) groups. Animals in immunization group were immunized against LHRH using ovalbumin-LHRH-7 (OL) protein generated by recombinant DNA technology as a primary and a booster injection at 19 and 23 weeks of age respectively. Animals were bled via jugular venepuncture at 2-week intervals to determine LHRH antibody and testosterone concentrations. Bi-weekly ultrasonographic examination of the testes was performed to determine the changes in ultrasonographic appearance as the age increased. Biopsied testicular tissues taken at 19, 29 and 41 weeks age were also evaluated. At 41 weeks of age, animals were slaughtered. Semen and epididymis were evaluated for the presence of sperm cells. Testicular development and sperm production were suppressed in the immunized animals. Semineferous tubule diameters decreased, basal membrane of the tubule was thickened and hyalinized in immunized ram lambs. While testes of control animals gained their normal ultrasonographic appearance as the age increased, immunized animals had uniform hypoechogenic testicular structure as observed at 19 weeks of age until slaughter. Simultaneous histological and ultrasonographic evaluations indicated that the changes in testicular histology could partly be monitored via ultrasonographic imaging. Nevertheless, it is difficult to claim that ultrasonographic image reflects the exact changes in such instances. In conclusion, these results indicate that recombinant OL fusion protein is effective in immunocastration in ram lambs and has a potential to be used as an alternative to physical castration. Further research studies should be conducted to help assess reproductive status of testes from ultrasound images.

Changes in testicular development, ultrasonographic and histological appearance of the testis in buck kids immunized against LHRH using recombinant LHRH fusion protein.

This study was designed to evaluate the effectiveness of recombinant Ovalbumin-LHRL (OL) immunization on changes in testicular size, histological appearance and testosterone production in buck kids. Thirty native buck kids at 18 weeks of age were divided into three groups, control (n = 10), immunization (n = 10) and castration (n = 10) groups. Immunized animals received OL protein generated by recombinant DNA technology. Ultrasonographic and histological examinations of the testes were performed. Animals were slaughtered at 44 weeks of age. Semen and epididymides were evaluated for the presence of sperm cells. Immunized animals generated anti-LHRH antibodies. Testosterone production, testicular and accessory glands development and sperm production were suppressed in the immunized animals (p < 0.01). Semineferous tubule diameters decreased (p < 0.01), basal membrane of the tubule was thickened and hyalinized in immunized kids. Immunization affected ultrasonographic appearance of the testes drastically. While testes of control animals gained their normal ultrasonographic appearance as the age increased, immunized animals had uniform hypoechogenic testicular structure as observed at 18 weeks of age until slaughter. Simultaneous histological and ultrasonographic evaluations indicated that the changes in testicular histology could partly be monitored via ultrasonographic imaging; nevertheless, it is difficult to claim that ultrasonographic image reflects the exact changes in such instances. In conclusion, these results indicate that recombinant OL fusion protein is effective in immunocastration in buck kids and has a potential to be used as an alternative to physical castration. Further researches should be conducted to help assessing reproductive status of testes from ultrasound images.

LHRH antagonist decreases LH and progesterone secretion but does not alter length of estrous cycle in heifers.

The objective of this study was to evaluate the suppressive effect of an LHRH antagonist, Cetrorelix SB-75 (SB-75), on secretion of LH, FSH and ovarian function in beef heifers. In Exp. 1, heifers were treated with a single dose of 10 microg/kg body weight intramuscularly on d 3 of the estrous cycle. In Exp. 2, heifers received either a single injection (100 microg/kg) of SB-75 on d 3 of the estrous cycle or multiple injections of 20 microg/kg on d 3, 4, 5, 6, and 7. Serum LH, but not FSH, was suppressed from one to several days. However, neither FSH nor progesterone was significantly altered. In Exp. 3, heifers received an injection vehicle (5% mannitol) or 100 microg/kg BW of SB-75 on d 1 of the estrous cycle (30 h after first observed standing estrus). Injection of SB-75 suppressed LH pulse frequency on d 3, 5, and 7 (P < 0.001). The mean LH concentrations in the SB-75 treatment groups were lower on d 3 (P < 0.01) and 5 (P < 0.05). There were no differences (P > 0.1) between the two groups in the mean concentrations of LH on d 1, 7, or 14. Treatment did not affect the secretion pattern or concentration of FSH. Injection of SB-75 did not alter estradiol-173 concentrations (P > 0.1). Treatment reduced corpus luteum (CL) function as indicated by lower progesterone production. However, the length of the estrous cycle was not shortened. These data show that the CL can form and survive in the face of depressed LH concentrations during the early stages of the estrous cycle.

The effects of recombinant LHRH fusion proteins on testicular development and histology in ram lambs.

Two recombinant luteinizing hormone releasing hormone (LHRH) fusion proteins were evaluated for their effectiveness in suppression of testicular development and histology by injecting together. Recombinant fusion proteins, ovalbumin-LHRH-7 and thioredoxin-LHRH-7, were generated using recombinant DNA technology and expressed in E. Coli. Eleven ram lambs ranked by age and body weight were randomly assigned to receive either ovalbumin and thioredoxin recombinant protein mixture (control group, n = 5) or ovalbumin-LHRH-7 and thioredoxin-LHRH-7 recombinant fusion protein mixture, anti-LHRH vaccine, (immunization group, n = 6). Animals in each group were weaned at 17 wk of age and were injected (primary immunization) with either mixture at 18 wk of age. Both groups received a booster immunization 8 wks later (26 wk of age). Scrotal circumference, scrotal length, testicular diameter and testicular length were measured in both groups every other week. All animals were slaughtered at 36 wk of age. Immediately after slaughter, a small testicular tissue was taken and processed for histological examination. In the ram lambs in immunization group scrotal circumference and testicular diameter increased steadily until second booster and then remained as a plateau until the end of the experiment. The differences in scrotal circumferences and testicular diameter were significant between the two groups during the last three weeks of the study (p < 0.05). There were no differences in testicular and scrotal length throughout the study (p > 0.05). Seminiferous tubules lost their regular shape and were decreased in diameter in immunization group. Although a few spermatozoa were seen in some tubules, in general, there were atrophy of the seminiferous tubules and loss of spermatogenesis, nevertheless, it seemed that animals in this group were potentially fertile.