Effect of propolis on human fibroblasts from the pulp and periodontal ligament

Propolis, a flavonoid-rich product of honey comb, exhibits antibacterial and anti-inflammatory properties. In this study, we examined the tolerance of fibroblasts of the periodontal ligament (PDL) and dental pulp to propolis and compared with that of calcium hydroxide in vitro. Cells from human dental pulp and PDL were obtained from healthy third molars and subjected to various concentrations of propolis (0-20 mg/ml) and calcium hydroxide (0-250 mg/ml). The cell viability after propolis treatment was analyzed by crystal violet staining of the cells followed by spectrophotometric analysis. Data revealed that exposure of PDL cells or pulp fibroblasts to 4 mg/ml or lower concentrations of propolis resulted in >75% viability of cells. On the contrary, calcium hydroxide 0.4 mg/ml was cytotoxic and <25% of the cells were found to be viable. Further investigations may find propolis to be a possible alternative for an intracanal antimicrobial agent.     

Histologic evaluation of the effect of formocresol and glutaraldehyde on the periapical tissues after endodontic treatment

The root canals of twelve pairs of human incisors without hard tissue or periodontal disease were treated with either formocresol or glutaraldehyde. The periapical reactions were compared histologically after periods varying between 6 and 8 weeks. A formocresol dressing always resulted in irritation of the periapical tissues, whereas little or no irritation was found after treatment with glutaraldehyde. It can be concluded that, after a vital pulpectomy, treatment of root canals with glutaraldehyde is faster than, as efficient as, and less irritating than treatment with formocresol.     

Short-term reactions of human dental pulp to formocresol and its components – a clinical-experimental study

Abstract – Pulps of 58 human premolars were amputated under standardized conditions, and standardized amounts of dressings containing 4 or 19% formaldehyde solutions, 35% cresol or formocresol, were applied to the wound surface. Tissue reactions were studied after 4, 8 or 16 d using enzyme histochermical and routine histologic techniques. The results showed that formaldehyde fixed the tissue in vivo , the extent of fixation being dependent on the concentration of formaldehyde. Fixation did not reach the apical foramen. Formaldehyde also caused vascular changes at varying distances from the fixed tissue, indicating transport of formaldehyde via the vessels, Cresol caused necrosis and vascular changes limited mainly to the upper portion of the root pulp. Application of formocresol resulted in devitalization of the whole or almost the whole of the root pulp within 4 d, accompanied by complex vascular changes, depending mainly on the formaldehyde component. Root pulp exposed to formocresol for 5 min only showed partial devitalization due to compromised blood flow, while practically no fixation occurred. The inflammatory response to formocresol and its components was remarkably limited.     

Distribution of 14C-formaldehyde after pulpotomy with formocresol.

Pulpotomies were performed on rhesus monkeys with use of formocresol to determine if there was uptake of 14C-formaldehyde into the systemic circulation after formocresol pulpotomies. Five-minute exposure of pulpal tissue to the 14C-formocresol resulted in the systemic absorption of approximately 1% of the dose. Two hours of exposure of pulp tissue to the 14C-formocresol did not increase the systemic absorption. Multiple sequential pulpotomies resulted in proportionately higher systemic absorption of 14C-formaldehyde. Application of 131I to pulpotomy sites indicated that formocresol compromises the microcirculation of the dental pulp. Autoradiography disclosed extensive concentrations of 14C-formaldehyde in the pulp, dentin, periodontal ligament, and bone.     

Antibody formation to dog pulp tissue altered by formocresol uithin the root canal.

After pulpal extirpation of twenty teeth in each of five dogs, these animals were primarily immunized intramuscularly by combining formocresol with the dog's own pulp (three dogs), saline solution with pulp (one dog) and injecting sheep erythrocytes (one dog). A sixth dog was used as a control for the Arthus skin test. Secondary immunizations were accomplished via the root canal every 7 days over a 28-day period. Arthus skin-test reactions demonstrated less of a response to the formocresol alone than when the dogs' pulp was conbined with this material. In vitro analysis of hemagglutinating antibody titer showed a tremendous increase when pulp was incubated with formocresol as compared to the saline-treated pulp. Therefore, dogs' pulp tissue became antigenically altered by the formocresol recognized by the host, and a specific humoral response resulted.     

Biochemical effects of formocresol on bovine pulp tissue.

Calf pulp was treated with full-strength formocresol, diluted formocresol, or saline for 4 hours. After washing and homogenization, the water extractable supernates were analyzed for total amino acid, carbohydrate, and hydroxyproline content. Additional samples were tested against trypsin, pepsin, collagenase, and hyaluronidase. Other tissue samples treated with 1/5, 1/10, and 1/25 dilutions of formocresol were subjected to trypsin and collagenase. The control tissue gave 50 per cent more extractable material, which contained over 300 per cent more total amino acids and hydroxyproline but only slightly more carbohydrate than the treated tissue. Formocresol treatment produced an 80 to 90 per cent reduction in reactivity to trypsin, pepsin, and collagenase but little change from hyaluronidase action. The increase in reactivity of the tissue to enzyme hydrolysis paralleled the increase in dilution of formocresol. These results indicate a profound effect on the protein fraction of pulp exposed to full-strength formocresol.     

Comparison of mineral trioxide aggregate and formocresol as pulp-capping agents in pulpotomized primary teeth

PURPOSE: The aim of this study was to use clinical, radiographic, and histologic examinations to compare the relative success of gray mineral trioxide aggregate (MTA), white MTA, and formocresol as pulp dressings in pulpotomized primary teeth. METHODS: Twenty-four children, each with at least 3 primary molars requiring pulpotomy, were selected for this study's clinical and radiographic portion. An additional 15 carious primary teeth planned for serial extraction were selected for this study's histologic portion. All selected teeth were evenly divided into 3 test groups and treated with pulpotomies. Gray MTA was used as the pulp dressing for one third of the teeth, white MTA was the dressing for one third, and the remaining one third were treated with formocresol. The treated teeth selected for the clinical and radiographic evaluations were monitored periodically for 12 months. The treated teeth selected for histologic study were monitored periodically and extracted 6 months postoperatively. RESULTS: Four children with 12 pulpotomized teeth failed to return for any follow-up evaluations in the clinical and radiographic study. Of the remaining 60 teeth in 20 patients, 1 tooth (gray MTA) exfoliated normally and 6 teeth (4 white MTA and 2 formocresol) failed due to abscesses. The remaining 53 teeth appeared to be clinically and radiographically successful 12 months postoperatively. Pulp canal obliteration was a radiographic finding in 11 teeth treated with gray MTA and 1 tooth treated with white MTA. In the histologic study, both types of MTA successfully induced thick dentin bridge formation at the amputation sites, while formocresol induced thin, poorly calcified dentin. Teeth treated with gray MTA demonstrated pulp architecture nearest to normal pulp by preserving the odontoblastic layer and delicate fibrocellular matrix, yet few inflammatory cells or isolated calcified bodies were seen. Teeth treated with white MTA showed a denser fibrotic pattern, with more isolated calcifications in the pulp tissue along with secondary dentin formation. CONCLUSIONS: Gray MTA appears to be superior to white MTA and formocresol as a pulp dressing for pulpotomized primary teeth.     

Cultured pulp fibroblasts: are they suitable for in vitro cytotoxicity testing?

The use of cell cultures to test the biocompatibility of dental materials is gaining in importance. Any cytotoxic effects that restorative materials may have will be on the dental pulp and for that reason cultured pulp cells should be the model of choice for biocompatibility testing. The aim of this investigation was to study the growth and morphologic characteristics and toxic response of human pulp lines and to compare these parameters to those of human buccal mucosa fibroblasts. Twenty-one specimens of pulp tissue and six from buccal mucosa were cultured using standard techniques. Six pulp cell lines were cultured successfully as were all six from the buccal mucosa specimens. From these specimens, 12 growth curves were computed. To study the morphology of the cultured cells, they were observed microscopically and classified into three morphological types: slender elongated cells (type I), epithelioid shaped cells (type II) and large stellate cells (type III). Their numbers and proportions were determined for each cell line and compared statistically. To gauge sensitivity to toxic materials, cells were exposed to concentrations of arecoline. An analysis of the growth curves showed no statistical difference between pulp cells and buccal mucosa cells; the slopes of the curves, however, differed significantly between individual cell lines, and these individual differences were greater among pulp cell lines. The morphology of the pulp and mucosa fibroblasts was similar microscopically. There was no significant difference between the number and proportion of the cell types in the two groups, but there were significant differences between the individual cell lines. Pulp cells showed a greater inhibition of growth when exposed to arecoline. Because pulp fibroblasts are difficult to culture, their reported survival rate is poor; due to the differences that exist between individual cell lines, we conclude that pulp cells when used as single cell lines or even pooled may not be ideal for testing biocompatibility, especially if reproducibility is a prerequisite. Any evaluation will require tests on not one, but several cell lines in order to minimize the effect of inter-cell-line differences. Their greater sensitivity to toxic substances, on the other hand, may show that pulp cells could be more sensitive indicators of cytotoxicity.     

Effect of formaldehyde-containing drugs on human dental pulp evaluated by enzyme histochemical technique

abstract — The in vivo effect of formaldehyde on pulp tissue in short-term studies cannot be established by using routine histologic techniques because the tissue is exposed to a fixative in vivo as well as during the histologic preparation. The pulps of permanent pre-molars were amputated and zinc oxide with 4% formaldehyde or formocresol was used as wound dressing. The observation periods varied from 1 to 16 d. After extraction the teeth were freeze-sectioned, freeze-dried and then incubated for histochemical demonstration of some oxidative and hydrolytic enzymes. A demarcated border between apically stained and cervically nonstained pulp tissue was found in sections incubated for oxidative enzymes. When formocresol, which has a high concentration of formaldehyde, was used, the border was situated closer to the apex. This was also the case when the observation period was increased. The incubation for lactate dehydrogenase gave a high staining intensity. Thus the use of frozen sections in combination with the histochemical method for the demonstration of lactate dehydrogenase appears to be suitable for the study of the penetration of formaldehyde in pulp tissue in short-term studies.     

Effect of formaldehyde-containing drugs on human dental pulp evaluated by enzyme histochemical technique.

The in vivo effect of formaldehyde on pulp tissue in short-term studies cannot be established by using routine histologic techniques because the tissue is exposed to a fixative in vivo as well as during the histologic preparation. The pulps of permanent premolars were amputated and zinc oxide with 4% formaldehyde or formocresol was used as wound dressing. The observation periods varied from 1 to 16 d. After extraction the teeth were freeze-sectioned, freeze-dried and then incubated for histochemical demonstration of some oxidative and hydrolytic enzymes. A demarcated border between apically stained and cervically nonstained pulp tissue was found in sections incubated for oxidative enzymes. When formocresol, which has a high concentration of formaldehyde, was used, the border was situated closer to the apex. This was also the case when the observation period was increased. The incubation for lactate dehydrogenase gave a high staining intensity. Thus the use of frozen sections in combination with the histochemical method for the demonstration of lactate dehydrogenase appears to be suitable for the study of the penetration of formaldehyde in pulp tissue in short-term studies.     

Involvement of oxidative stress in mutagenicity and apoptosis caused by dental resin monomers in cell cultures

OBJECTIVE: This investigation studied the possibility that apoptosis as well as mutagenicity induced by resin monomers are mediated by oxidative stress. METHODS: A range of dilutions of three resin monomers (GMA, TEGDMA, and HEMA) was added to culture medium (DMEM/10% FBS), of V79-4 fibroblasts and RPC-C2A pulp cells for 24 h. Their cytotoxic effects were measured by a colorimetric functional assay (MTT). Chromosomal aberration induced by the resin monomers was investigated by counting micronuclei in V79-4 cells. The effects of the resin monomers on DNA fragmentation were viewed by agarose gel electrophoresis of DNA, isolated from RPC-C2A pulp cells that were treated by resin compounds. Resin monomer-induced apoptosis was further confirmed by flow cytometry (staining with both annexin V-FITC and PI). RESULTS: All monomers exhibited a dose-dependent cytotoxic effect, and the ranking of the cytotoxicity based on TC50 was GMA > TEGDMA > HEMA. The resin monomer-induced cytotoxicity was significantly decreased by co-treatment with N-acetylcystein (NAC), an antioxidant. The authors also confirmed a dose-dependent genotoxicity of the resin monomers that had induced micronucleated cells in V79-4 fibroblasts. Similar to the effects on cytotoxicity, NAC reduced the numbers of micronuclei in comparison with those generated by the resin monomers. The preventive effects of NAC were also observed in monomer-induced apoptosis in RPC-C2A cells. A DNA ladder pattern, characteristic of apoptosis, was shown at cytotoxic concentrations, but NAC blocked the resin monomer-mediated DNA fragmentation. The preventive effects of NAC on apoptosis were confirmed by Annexin V staining. Cells exposed to 300 microM GMA, 7 mM TEGDMA, or 14 mM HEMA for 24 h showed a significant increase in apoptotic cells, while NAC co-treatment caused a reduction in apoptotic cells compared to controls. SIGNIFICANCE: These findings suggest that glutathione depletion and oxidative stress are responsible for GMA, TEGDMA, and HEMA-induced mutagenicity and apoptosis.     

Bactericidal and cytotoxic effects of sodium hypochlorite and sodium dichloroisocyanurate solutions in vitro

The antimicrobial and cytotoxic effects of sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) were evaluated and compared in vitro. The minimal inhibitory concentration and minimal bactericidal concentration of NaOCl and NaDCC were tested for Streptococcus sobrinus, Streptococcus salivarius, Enterococcus faecalis, and Streptococcus mutans. The cytotoxic effect was assessed by using human fibroblast tissue culture. Survival rate was assessed by a protein determination method. Results showed that the minimal inhibitory concentration and minimal bactericidal concentration values of NaOCl and NaDCC for the tested bacteria were in a similar range. NaDCC in concentrations higher than 0.02%, and NaOCl in concentrations higher than 0.01% were lethal to fibroblasts. In conclusion it seems that both agents were very effective in killing bacteria, and their cytotoxicity to fibroblasts in tissue culture was similar.     

4种牙本质粘结剂对牙髓成纤维细胞毒性作用的比较

目的:对比研究4种第七代牙本质粘结剂对人牙髓成纤维细胞的毒性作用,初步探讨其生物安全性。方法:组织块培养法原代培养人牙髓成纤维细胞,免疫组织化学染色SP法鉴定细胞来源,采用四甲基偶氮唑盐比色法和浸提液法,双盲观察4种新一代牙本质粘结剂(G-Bond、i-Bond、xeno V、Clearfil S3Bond)的不同体积分数浸提液(12.5%、25%、50%、100%)作用不同时间(24、48、72 h)对人牙髓成纤维细胞的毒性作用。结果:4种牙本质粘结剂不同体积分数浸提液的作用下,人牙髓成纤维细胞形态均发生不同程度的变化。24、48 h时,xeno V细胞毒性影响较小,i-Bond细胞毒性影响较大,G-Bond与Clearfil S3 Bond细胞毒性无明显差异;72 h时,4种牙本质粘结剂对细胞毒性影响无明显差异。结论:4种牙本质粘结剂毒性趋于0～2级,随着作用时间延长,细胞毒性无明显差异。     

Morphologic and enzyme histochemical observations on the pulp of human primary molars 3 to 5 years after formocresol treatment.

The state of the pulp of twenty-seven primary teeth treated by formocresol pulpotomy (clinically and radiographically successful) was assessed 3 to 5 years after treatment. A wide variation was found in the pulpal condition, from normal pulp tissue to total necrosis. Resorption and apposition of hard tissue were common findings. Five teeth were freeze-sectioned and incubated for histochemical demonstration of oxidative enzymes. The pulps of two teeth were vital; two teeth had necrotic areas subjacent to the amputation paste; and one pulp was totally necrotic. Six teeth were extracted 5 minutes after formocresol pulpotomy and incubated for demonstration of oxidative enzymes. An unstained zone, 1 to 2 mm. deep, was seen in all incubated sections. In conclusion, it seems that the formocresol method should be regarded only as a means to keep primary teeth with pulp exposures functioning for a relativley short period of time.     

The induction of oxidative stress, cytotoxicity, and genotoxicity by dental adhesives.

OBJECTIVES: Polymerized dental resin materials release residual monomers that may interact with pulp tissues. We hypothesized that dental adhesives might cause cytotoxicity in pulp cells via the generation of reactive oxygen species (ROS), which may also contribute to genotoxic effects in vitro. METHODS: For cytotoxicity testing, transformed human pulp-derived cells were exposed to extracts of primers and bonding agents of Clearfil SE bond, Clearfil Protect bond, AdheSE, Prompt L-Pop, and Excite for 24h. The cytotoxicity of the same materials was also analyzed in a dentin barrier test device using three-dimensional pulp cell cultures. The generation of ROS in monolayer cultures was measured after a 1h exposure period by flow cytometry (FACS), and genotoxicity as indicated by the formation of micronuclei was determined in V79 cells after a 24h exposure period. RESULTS: The dentin primers and bonding agents decrease cell survival in a dose-related manner. Cytotoxicity of bonding agents based on concentrations which caused 50% cell death (EC50) were ranked as follows: Excite (0.16 mg/ml)>AdheSE bond (0.30 mg/ml)>Clearfil Protect bond (0.35 mg/ml)>Clearfil SE bond (0.37 mg/ml), and Prompt L-Pop bond (0.68 mg/ml). Dentin primers were about 10-fold less effective. In contrast, no cytotoxic effects of the dental adhesives were observed in a dentin barrier test device. Yet, all dental adhesives increased the amounts of ROS about fivefold in pulp cells in a dose-related manner, and, again, the bonding agents were more efficient than the dentin primers. Finally, the number of micronuclei was increased about sixfold by extracts of the AdheSE primer. SIGNIFICANCE: Our results suggest that the cytotoxic potencies demonstrated by these materials might be of clinical relevance, since all dental adhesives disturbed the cellular redox state of pulp cells in monolayer cultures. As a result, the concentrations of biologically active ingredients of some of the agents may be high enough to modify pulp cell metabolism when the materials are used in deep cavities or directly contact pulp tissue.     

Cytotoxic and nongenotoxic effects of phenolic compounds in human pulp cell cultures

Phenolic compounds are widely used in clinical dentistry as sedatives for the dental pulp, as disinfectants for caries, and as root canal medications. The pathobiological effects of various phenolic compounds on human dental pulp fibroblasts were investigated with Hoechst 33258 fluorescence assay and DNA precipitation assay. All phenolic compounds showed cytotoxicity in Hoechst 33258 fluorescence assay by inhibiting cellular DNA in a concentration-dependent manner. The 50% inhibition concentrations required to decrease the cellular DNA contents by guaiacol, phenol, eugenol, and thymol were 9.8, 4.5, 0.9, and 0.5 mM, respectively. However these phenolic compounds did not cause DNA single-strand breaks in cultured human pulp fibroblasts. These results indicate that phenolic compounds are cytotoxic agents but are without genotoxic effects on human pulp fibroblasts in vitro. However care should be taken to reduce the possibility of pulpal as well as periapical irritations from inadvertent extrusion of these substances in clinical usage.     

Pulpal tissue reaction to buffered glutaraldehyde.

Pulpal tissue changes following pulpotomies with 2% w/v buffered glutaraldehyde in primary teeth were observed. A 3 minute single application of 2% w/v buffered glutaraldehyde was able to produce effective surface fixation. Limited penetration of the medicament left the remaining pulp tissue unaffected. The zone of fixation did not proceed apically. With time, macrophages and fibroblasts appear apical to the zone of fixation indicating the onset of replacement resorption.     

Antibacterial properties of dilute formocresol and eugenol and propylene glycol

Formocresol and eugenol are the two nonspecific intracanal medicaments commonly used in endodontic practice. Both have high tissue irritation potential when used in conventional strength. Propylene glycol is an alcohol that is injectable and itself possesses significant antibacterial action. It is a popular vehicle and hence was used to modify the two drugs. Standard bacteriologic methods were employed to test the antibacterial action of these lower concentrations of the two drugs against four test organisms. The investigations indicate that formocresol at as low as 10 to 20% and eugenol at 75% are bactericidal in action and hence may be useful at these concentrations for clinical use. Evaluation of these lower concentrations is warranted for possible clinical use. Propylene glycol, which possesses antibacterial action and is remarkably innocuous to tissues, appears to be a suitable vehicle for dilution of formocresol and eugenol.     

An in vitro study with various vehicles of diffusion of formocresol and its components

abstract — Pulpotomy in primary teeth using the formocresol method results in varying degrees of devitalization of the root pulp. The extent of this devitalization depends on, among other things, the ability of the components of formocresol to leave the dressing. The purpose of this study was to evaluate the rate and duration of diffusion of the components of formocresol when incorporated in different vehicles. The antimicrobial effect of the drugs was used to assess the diffusion of the components of formocresol in blood agar, with a sensitive microorganism as an indicator. Evaluation of the MICs assessed in broth medium for formalin, formocresol and cresol, respectively, and the zone size of growth in hibition on blood agar from these components when incorporated in ZnO or ZnO-eugenol cement, suggested that the initial zone of inhibition from formocresol was due mainly to the diffusion of formaldehyde. Cresol diffused more slowly from the dressing. The presence of eugenol in the dressing, as in ZnO-eugenol cement, gave smaller initial release of formaldehyde, formocresol and cresol compared with the release from ZnO, but more prolonged diffusion. A higher initial release of formaldehyde was obtained when the formocresol was incorporated in ZnO alone compared with ZnO-eugenol cement or Pharmatec®.