Cytotoxic effects and pulpal response caused by a mineral trioxide aggregate formulation and calcium hydroxide

Purpose: To evaluate: the in vivo pulpal response after pulpotomy with different capping agents. In addition, the in vitro cytotoxic effects of both materials were assessed by applying them on culture of pulp cells. Methods: For the in vivo test, the coronal pulp of 28 teeth of dogs was mechanically removed and the root pulps were capped with the following dental materials: Group 1: Pro-Root NITA (PRMTA); and Group 2 (control): calcium hydroxide saline paste (CH). After 60 days, the animals were sacrificed and the teeth processed for histological analysis. In the in vitro test, experimental extracts obtained from both capping agents were applied on the cultured MDPC-23 odontoblast-like cells. Results: In the root pulps capped with PRMTA or CH, coagulation necrosis partially replaced by dystrophic calcification as well as tubular dentin matrix laid down by elongated pulp cells was observed. None or mild inflammatory response occurred beneath the capped pulpal wound. Regarding the pulpal response, PRMTA and CH presented no statistical difference. However, the teeth capped CH presented greater healthy pulp loss which resulted in convex shape of the hard barrier than PRMTA. When applied on the cultured cells, it was demonstrated that PRMTA and CH solutions decreased the cell metabolic activity by 9.9% and 29.4%, respectively. CH caused higher cytotoxic effects to the MDPC-23 cells as well as deeper healthy pulp tissue loss than PRMTA. However, similar sequence of healing occurred after pulpotomy with both dental materials.

Determination of para-Chloroaniline and Reactive Oxygen Species in Chlorhexidine and Chlorhexidine Associated with Calcium Hydroxide

The aim of this study was to determine whether para-chloroaniline (PCA) and/or reactive oxygen species (ROS) are generated by chlorhexidine (CHX) alone or after CHX is mixed with calcium hydroxide at different time points. Mass spectrometry was performed to detect PCA in samples of 0.2% CHX and Ca(OH)2 mixed with 0.2% CHX. High-performance liquid chromatography was used to confirm the presence of CHX in the mixture with Ca(OH)2. The samples were analyzed immediately after mixing and after 7 and 14 days. During the intervals of the experiment, the samples were maintained at 36.5 degrees C and 95% relative humidity. PCA was detected in the 0.2% CHX solution after 14 days. The mixture of CHX with Ca(CH)2 liberated ROS at all time points, but no traces of CHX were present in the mixture as a result of immediate degradation of the CHX. (J Endod 2008;34:1508-1514)

Histopathological and histomicrobiological study of root canal therapy medication, comparison of calcium hydroxide versus gutta-percha with zinc oxide/eugenol in the teeth of dogs

The presence of microorganisms in dental structures with experimentally induced necrosis was evaluated. The materials were tested to evaluate their antimicrobial activity and tissue repair efficacy. Four dogs were used in this experiment, with a total of 64 roots of premolar teeth, divided into three groups. The root canals of Group I were filled with gutta-percha and zinc oxide/eugenol cement; Group II were filled with calcium hydroxide, and Group III were not filled. All animals were clinically and radiographically examined 15 days after surgery andthen again every subsequent 15 days until 120 days, when the teeth were extracted en bloc.Histopathological analysis showed inflammatory infiltration, cement and bone resorption andnecrotic tissue in the apical delta in different proportions. Histomicrobiological analysis showedthe presence of microorganisms inside the teeth structures, with different concentrationsaccording to the treatment used. There was statistical significance between the groups(p>0.05). Gutta-percha with zinc oxide/eugenol demonstrated good antimicrobial activity;calcium hydroxide was not efficient. The conclusion of this study is that gutta-percha withzinc oxide/eugenol is the better protocol for filling root canals in dogs.

Microbial culture and checkerboard DNA-DNA hybridization assessment of bacteria in root canals of primary teeth pre- and post-endodontic therapy with a calcium hydroxide/chlorhexidine paste

Aim. To investigate the root canal microbiota of primary teeth with apical periodontitis and the in vivo antimicrobial effects of a calcium hydroxide/chlorhexidine paste used as root canal dressing. Design. Baseline samples were collected from 30 root canals of primary teeth with apical periodontitis. Then, the root canals were filled with a calcium hydroxide paste containing 1% chlorhexidine for 14 days and the second bacteriologic samples were taken prior to root canal filling. Samples were submitted to microbiologic culture procedure to detect root canal bacteria and processed for checkerboard DNA-DNA hybridization. Results. Baseline microbial culture revealed high prevalence and cfu number of anaerobic, black-pigmented bacteroides, Streptococcus, and aerobic microorganisms. Following root canal dressing, the overall number of cfu was dramatically diminished compared to initial contamination (P < 0.05), although prevalence did not change (P > 0.05). Of 35 probes used for checkerboard DNA-DNA hybridization, 31 (88.57%) were present at baseline, and following root canal dressing, the number of positive probes reduced to 13 (37.14%). Similarly, the number of bacterial cells diminished folowing application of calcium hydroxide/chlorhexidine root canal dressing (P = 0.006). Conclusion. Apical periodontitis is caused by a polymicrobial infection, and a calcium hydroxide/chlorhexidine paste is effective in reducing the number of bacteria inside root canals when applied as a root canal dressing.

Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1-and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)(2), in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)(2) treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)(2) were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)(2)-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)(2)-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)(2) and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)(2) stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)(2) stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)(2)-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)(2) promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.

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)

Effect of a calcium hydroxide-based paste associated to chlorhexidine on RAW 264.7 macrophage cell line culture

Objective. The objective of this study was to evaluate the effect of a calcium hydroxide Ca(OH)(2)-based paste (Calen) associated or not to 0.4% chlorhexidine digluconate (CHX) on RAW 264.7 macrophage cell line culture. Study design. The cell viability (MTT assay), immunostimulating properties (NO dosage), and anti-inflammatory properties (NO, TNF-alpha, and IL-1 alpha dosage) were evaluated after cell exposure to the materials. Data were analyzed statistically by Kruskal-Wallis test at 5% significance level. Results. There was low immunostimulating activity of the Calen paste associated or not to 0.4% CHX in the different materials` concentrations evaluated (P > .05). Anti-inflammatory activity with inhibition of NO and cytokine (TNF-alpha and IL1-alpha) release was observed only with Ca(OH)(2) + CHX at the highest concentration (25 mu g/mL). Conclusion. As the Calen paste associated to 0.4% CHX did not alter cell viability or the immunostimulating and anti-inflammatory properties, the addition of CHX brought no benefits to the Ca(OH)(2)-based paste with regard to the tested parameters. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:e44-e51)

Study of the pozzolanic reaction kinetics in sugar cane bagasse-clay ash/calcium hydroxide system: kinetic parameters and pozzolanic activity

This paper presents a study of the pozzolanic reaction kinetics between calcium hydroxide and a mixture of sugar cane bagasse with 20 and 30% of clay, burned at 800 and 1000 degrees C (SCBCA) by electrical conductivity measurements. A kinetic-diffusive model produced in previous studies by some of the authors was used. The model was fitted to the experimental data, which allowed the computation of the kinetic parameters of the pozzolanic reaction (reaction rate constant and free energy of activation) that rigorously characterised the pozzolanic activity of the materials. The results show that SCBCA demonstrated reactivity and good pozzolanic qualities in the range 800-1000 degrees C.

Histopathological and histomicrobiological study of root canal therapy medication, comparison of calcium hydroxide versus gutta-percha with zinc oxide/eugenol in the teeth of dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Short-term vs long-term calcium hydroxide therapy after immediate tooth replantation: a histomorphometric study in monkey's teeth

Endodontic treatment is an important step of tooth replantation protocols, but the ideal moment for definitive obturation of replanted teeth has not yet been established. In this study, a histomorphometric analysis was undertaken to evaluate the repair process on immediate replantation of monkeys teeth after calcium hydroxide (CH) therapy for 1 and 6 months followed by root canal filling with a CH-based sealer (Sealapex (R)). The maxillary and mandibular lateral incisors of five female Cebus apella monkeys were extracted, kept in sterile saline for 15 min, replanted and splinted with stainless steel orthodontic wire and composite resin for 10 days. In Group I (control), definitive root canal filling was performed before tooth extraction. In Groups II and III, CH therapy started after removal of splint, and definitive root canal filling was performed 1 and 6 months later, respectively. The animals were euthanized 9 months after replantation, and specimens were processed for histomorphometric analysis. In all groups, epithelial attachment occurred at the cementoenamel junction or very close to this region; the areas of resorption on root surface had small extension and depth and were repaired by newly formed cementum; and the periodontal ligament was organized. Statistical analysis of the scores obtained for the histomorphometric parameters did not show any statistically significant difference (P = 0.1221) among the groups. The results suggests that when endodontic treatment is initiated 10 days after immediate replantation and an antibiotic regimen is associated, definitive root canal filling can be performed after a short-term CH therapy.

Mechanism of calcium hydroxide-induced neutrophil migration into air-pouch cavity

The aim of this study was to investigate cellular migration induced by calcium hydroxide to air-pouch cavities in mice. The migration was more specific to neutrophil and was dose and time dependent (peaking 96 h after stimulation). This migration was inhibited by pretreatment with thalidomide, indomethacin, MK886, meloxicam, dexamethasone, MK886 associated with indomethacin, and MK886 associated with indomethacin and dexamethasone. The air-pouch exudate from animals stimulated with calcium hydroxide showed an increase of leukotriene-B4 (LTB4), interleukin-1 beta, tumor necrosis factor alpha (TNF-alpha), cytokine-induced neutrophil chemoattractant (KC), and macrophage inflammatory protein 2 (MIP-2) release. Pretreatment with 3% thioglycollate increased the macrophage population in the air pouch but did not change neutrophil migration. Depleting the resident mast cells through chronic pretreatment with compound 48/80 did not alter neutrophil migration in response to calcium hydroxide. It was possible to conclude that calcium hydroxide-induced neutrophil migration to the air-pouch cavity in mice is mediated by LTB4, TNF-alpha, KC, MIP-2, and prostaglandins, but it was not dependent on macrophages or mast cells.

Calcium Hydroxide Mixed With Camphoric p-Monochlorophenol or Chlorhexidine in Delayed Tooth Replantation

This study evaluated the repair process after delayed replantation of rat teeth, using calcium hydroxide (Ca(OH)(2)) mixed with camphorated p-monochlorophenol (CMCP), chlorhexidine 2% (CHX), or saline as temporary root canal dressing to prevent and/or control inflammatory radicular resorption. Thirty Wistar rats (Rattus norvegicus albinos) had their right upper incisor extracted, which was bench-dried for 60 minutes. The dental papilla, the enamel organ, the dental pulp, and the periodontal ligament were removed. The teeth were immersed in 2% acidulated-phosphate sodium fluoride solution for 10 minutes. The root canals were dried with absorbent paper cones and divided into 3 groups of 10 animals according to root canal dressing used: group 1: Ca(OH)(2) + saline, group 2: Ca(OH)(2) + CMCP, and group 3: Ca(OH)(2) + CHX 2%. Before replanting, the teeth sockets were irrigated with saline. Histological analysis revealed the presence of inflammatory resorption, replacement resorption, and ankylosis in all 3 groups. Statistical analysis showed a significant difference between group 3 and the other groups. The use of Ca(OH)(2) mixed with CMCP or CHX did not show an advantage over the use of Ca(OH)(2) mixed with saline in preventing and/or controlling inflammatory resorption in delayed replantation of rat teeth.

Effect of calcium hydroxide-based materials on periapical tissue healing and orthodontic root resorption of endodontically treated teeth in dogs

This study evaluated periapical tissue healing and orthodontic root resorption of endodontically treated teeth sealed with calcium hydroxide in dogs. The sample consisted of three contralateral pairs of maxillary incisors and two contralateral pairs of mandibular incisors in each of two dogs using a split mouth design. After biomechanical preparation of the teeth in the first group (n = 10), a Ca(OH)(2) dressing was placed for 14 days before root canal filling with Ca(OH)(2)-based sealer (Sealapex) and gutta-percha points. In the second group (n = 10), root canals were obturated immediately after the mechanical preparation with gutta-percha points and zinc oxide and eugenol (ZOE)-based sealer (Endofill). After completion of endodontic treatment, the teeth were moved with an orthodontic appliance with a calibrated force of 200 g, reactivated every 21 days. After 105 days, the animals were killed and the teeth were removed upon completion of active treatment, without a period of recovery, and prepared for histomorphological analysis. All sections of each tooth were graded subjectively on a scale from one to four to obtain the average of the 16 histomorphological parameters analysed. Evaluation of the differences between the two treatment protocols was made with Mann-Whitney U-test. It was observed that the teeth treated with Ca(OH)(2)-based materials provided better outcomes (P = 5%), with complete repair of all root resorption areas, high rate of biological closure of the main canal and apical accessory canals by newly formed cementum, less intense and extensive chronic inflammatory infiltrate, and better organization of the periodontal ligament. Under the tested conditions, Ca(OH)(2)-based materials had a favourable action on periapical tissue healing and repair of orthodontic root resorption in endodontically treated dogs' teeth.

Periapical tissue reactions to calcium hydroxide and MTA after external root resorption as a sequela of delayed tooth replantation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of the healing process in delayed tooth replantation after root canal filling with calcium hydroxide, Sealapex and Endofill: a microscopic study in rats

The major concern in the therapeutics of tooth replantation refers to the occurrence of root resorption and different approaches have been proposed to prevent or treat these complications. The purpose of this study was to evaluate tissue response to delayed replantation of anterior rat teeth treated endodontically using calcium hydroxide, Sealapex, and Endofill without the placement of gutta-percha cones. Thirty rats had their right upper incisor extracted and maintained in dry storage for 60 min. After removal of the dental papilla, enamel organ, pulp tissue, and periodontal ligament remnants, the teeth were immersed in 2% sodium fluoride phosphate acidulated, pH 5.5, for 10 min. The root canals were dried with absorbent paper points and the teeth were assigned to three groups (n = 10) according to the filling material. Group I - calcium hydroxide and propyleneglycol paste, Group II - Sealapex, and Group III - Endofill. The sockets were irrigated with saline and the teeth were replanted. Replacement resorption, inflammatory resorption and ankylosis were observed in all groups. Although the occurrence of inflammatory resorption was less frequent in Group I, there were no statistically significant differences among the groups. It may be concluded that compared to the paste, filling the root canals with Sealapex and Endofill sealers without the placement of gutta-percha cones did not provide better results.