Regenerative capacity of human dental pulp and apical papilla cells after treatment with a 3-antibiotic mixture.

INTRODUCTION: A 3-antibiotic combination (3Mix) has been widely used in regenerative endodontics. Recent studies recommend that a safe concentration of 3Mix is in the range of 0.39 µg/mL and 1 mg/mL because higher concentrations may limit tissue regeneration. The aim of this study was to determine the regenerative capacity of isolated human dental pulp cells (DPCs) and apical papilla cells (APCs) after a 7-day treatment with selected doses of 3Mix. METHODS: Primary human DPCs/APCs from the third passage were divided into control and experimental groups. In the control group, cells were cultured in regular complete media. In the experimental group, cells were cultured in complete media containing 0.39 µg/mL or 1 mg/mL of 3Mix for 7 days. After the treatment period, the media were changed, and the cells were further tested for proliferation and differentiation potential. For cell proliferation, a colorimetric qualification of 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide was used on days 1, 3, 5, and 7. For differentiation potential, a dentinogenic differentiation medium was added into treated cells and cultured for 7, 14, and 21 days. Results were analyzed using quantitative alizarin red S staining and real-time reverse-transcription polymerase chain reaction. RESULTS: After 7 days of treatment, 100% cell death was discovered in the 1-mg/mL 3Mix group. The proliferative capacity of 0.39 µg/mL 3Mix-treated DPCs and APCs was significantly lower than that of untreated cells at all time points (P < .05). Mineralized nodule formation was found both in the 3Mix-treated and control groups, but it was significantly less in the 3Mix-treated groups at 7, 14, and 21 days (P < .01). Quantitative reverse-transcription polymerase chain reaction showed no statistically significant difference (95% confidence interval) in bone sialoprotein, alkaline phosphatase, and dentin matrix protein 1 gene expression in either 3Mix-treated DPCs or APCs compared with control groups. CONCLUSIONS: One milligram per milliliter of 3Mix had strong toxicity to DPCs/APCs when applied for 7 days, whereas 0.39 µg/mL 3Mix showed no toxicity but still affected cell proliferation and mineralization potential. However, no differences in dentinogenic gene expressions were observed between the 3Mix-treated and untreated groups.

Davallialactone reduces inflammation and repairs dentinogenesis on glucose oxidase-induced stress in dental pulp cells.

INTRODUCTION: The chronic nature of diabetes mellitus (DM) raises the risk of oral complication diseases. In general, DM causes oxidative stress to organs. This study aimed to evaluate the cellular change of dental pulp cells against glucose oxidative stress by glucose oxidase with a high glucose state. The purpose of this study was to test the antioxidant character of davallialactone and to reduce the pathogenesis of dental pulp cells against glucose oxidative stress. METHODS: The glucose oxidase with a high glucose concentration was tested for hydroxy peroxide (H2O2) production, cellular toxicity, reactive oxygen species (ROS) formation, induction of inflammatory molecules and disturbance of dentin mineralization in human dental pulp cells. The anti-oxidant effect of Davallilactone was investigated to restore dental pulp cells' vitality and dentin mineralization via reduction of H2O2 production, cellular toxicity, ROS formation and inflammatory molecules. RESULTS: The treatment of glucose oxidase with a high glucose concentration increased H2O2 production, cellular toxicity, and inflammatory molecules and disturbed dentin mineralization by reducing pulp cell activity. However, davallialactone reduced H2O2 production, cellular toxicity, ROS formation, inflammatory molecules, and dentin mineralization disturbances even with a long-term glucose oxidative stress state. CONCLUSIONS: The results of this study imply that the development of oral complications is related to the irreversible damage of dental pulp cells by DM-induced oxidative stress. Davallialactone, a natural antioxidant, may be useful to treat complicated oral disease, representing an improvement for pulp vital therapy.

Expression status of mRNA for sex hormone receptors in human dental pulp cells and the response to sex hormones in the cells.

OBJECTIVES: Sex hormone receptors are reported to be present in human dental pulp (HDP) cells. The purpose of this study was to examine the biological significance of oestrogen and androgen receptors (ER and AR, respectively) in HDP cells. DESIGN: We isolated HDP cells expressing ER- and AR-mRNAs and investigated the expression status of the receptors and the response to sex hormones in the cells. RESULTS: HDP cells expressing ER- and/or AR-mRNAs had the ability to form alizarin red S-positive nodules in which calcium and phosphorus were deposited in vitro and to differentiate into odontoblasts-like cells and dentine-like tissue in vivo. Individual clones isolated from HDP cells exhibited a different expression pattern of mRNA for ER and AR. Some clones expressed ERα- and/or ERß-mRNAs and the others coexpressed ER- and AR-mRNAs. Using the Ingenuity software, we found that 17ß-estradiol (E2) and dihydrotestosterone (DHT) could act directly on HDP cells through ER-or androgen signalling-mediated mechanisms. E2 or DHT stimulated the mRNA expression for genes related to odontogenesis of dentine-containing teeth and odontoblast differentiation, suggesting that ER and AR in HDP cells may be involved in dentinogenesis. CONCLUSIONS: Our findings provide new insights into the biological significance of sex hormone receptors in HDP cells.

Effects of protein deficiency induced by raw soy with and without sucrose on dentine formation and dentinal caries in young rats.

OBJECTIVES: The effects of protein deficiency and sucrose on formation and mineralization of dentine and dental caries in the molars of young rats were investigated. MATERIALS AND METHODS: Two groups of weaning Wistar rats received raw soy flour to induce protein deficiency with sucrose or starch as the carbohydrate source; the other two groups received skim milk powder as the protein source with sucrose or starch as the carbohydrate source. At the onset, tetracycline was injected to mark the dentine formed at that moment. After 6 weeks, lower molars were sectioned sagittally, and the areas of dentine formation and dentinal caries developed during the experiment were quantified separately in the first and second molars. Dental caries was also classified according to Schiff's reaction. Calcium (Ca), phosphorus (P) and total mineral elements were analyzed using an electron probe microanalyzer. RESULTS: Rats in both protein-deficient groups and normal protein sucrose group had significantly larger areas of dentine formed compared with rats fed with normal protein starch diet. Ca, P and total mineral elements in dentine were significantly reduced by normal protein sucrose diet. P content was significantly reduced in dentine of rats in protein-deficient sucrose group. Rats in normal protein sucrose group had significantly more and larger dentinal caries lesions than in any other group. CONCLUSION: This study suggests that protein deficiency induced by soy prevents the progression of dental caries even in highly cariogenic environment.

The effect of a high-sucrose diet on dentin formation and dental caries in hyperinsulinemic rats.

A high-sucrose diet decreases dentin formation and its minerals, but the mechanisms behind the effect are largely unknown. We studied the combined and separate effects of sucrose and insulin on dentin formation and mineral metabolism in growing rats. At weaning, animals were randomized into 4 groups: control/sucrose diets both with and without external insulin (1 U/x 100 g body weight daily). After 4 weeks, we measured areas of dentin formation, numbers and areas of dentinal caries lesions, and serum and urine glucose, insulin, Ca, Na, K, and P. Exogenous insulin increased serum and urine insulin levels and decreased serum glucose level, but did not affect dentin formation or dentinal caries lesion formation or progression. A high-sucrose diet decreased dentin formation independently of insulin. The differences in serum and urine minerals between the groups were minor. The findings confirm that sucrose-diet-induced reduction in dentinogenesis is independent of insulin and loss of minerals in urine.

Effects of a high sucrose diet and intragastric sucrose feeding on the dentinogenesis, dental caries, and mineral excretion of the young rat.

Previous studies show that a high sucrose diet reduces the rate of primary dentinogenesis and increases dental caries, although their cause-effect relationship is still obscure. The purpose of this study was to explore whether the effect of sucrose load on the dentinogenesis and dental caries of young rat molars is mediated by systemic (intragastric) or by systemic and local (dietary) factors. At weaning (19 days), animals were randomized into the control, intragastric sucrose, and dietary sucrose groups for 4 weeks. The areas of dentin appositions and dentinal caries lesions were measured planimetrically. Caries was also determined with Shiffs staining and the width of predentin by histology. Urinary Ca, K, and Na levels were measured by flame photometry, urinary P levels using an UV method, and serum insulin levels using radioimmunoassay. Systemic and local sucrose load reduced dentin appositions and intragastric sucrose increased urinary Ca excretion. No differences in the width of predentin were noticed. Only dietary sucrose enhanced the occurrence and progression of caries. The present findings show that sucrose load reduces dentinogenesis by impairing the synthesis of dentin matrix, but also point out the crucial importance of the local sucrose challenge in the initiation of dental caries.

The effect of sucrose diet of rat dams on the dentine apposition and dental caries of their pups.

The purpose was to examine whether a sucrose diet in rat dams reduces dentine apposition and enhances dental caries in their pups. Sprague-Dawley rat pups were randomized into four groups on the day of birth. During the lactation period three dams received a standard rodent diet and three a diet containing 41% sucrose. At the age of 3 weeks the pups were weaned and given an intraperitoneal injection of oxytetracycline hydrochloride. During the experimental period, half of the pups of control dams received a sucrose diet (Cnt-Suc pups) and half of the pups of sucrose dams received a standard diet (Suc-Cnt pups). The pups in the other halves received the same diet as given to their dams during lactation (Cnt-Cnt and Suc-Suc pups). Urine samples were collected from dams and pups during the test periods. After 4 weeks the pups were killed, their blood was collected and their jaws sectioned sagittally. Dentine apposition was determined planimetrically and dental caries using Schiffs staining. Ca, K and Na of serum and urine were measured flame photometrically and P using an ultraviolet method. The lactational sucrose diet was associated with decreased P and Na excretion in the dams, and also with reduced dentine apposition in their pups. Reduced dentine apposition and induced Ca excretion with decreased P, K and Na excretions were observed in Suc-Suc pups and Cnt-Suc pups during the experiment period. Fewer intact teeth with more enamel and dentinal caries lesions occurred in sucrose-exposed groups (Suc-Cnt, Cnt-Suc and Suc-Suc pups). It was concluded that the sucrose diet given to dams during lactation changed the function of the pulp dentine complex of their pups leading to reduced dentinogenesis, but also predisposed to a reduction of dentinogenesis in pups during the experimental period and enhanced the occurrence of dental caries.

Effect of capsaicin treatment or inferior alveolar nerve resection on dentine formation and calcitonin gene-related peptide- and substance P-immunoreactive nerve fibres in rat molar pulp.

The aim of this study was to investigate whether decreased sensory innervation induced by capsaicin treatment or axotomy of the inferior alveolar nerve has an effect upon dentine formation in the rat first molar. Dentine formation was visualized by intravital injection of Procion brilliant Red H8BS and denervation was verified immunohistochemically for the neuropeptides calcitonin gene-related peptide (CGRP) and substance P. The observation times were 6 weeks for the capsaicin-treated group and 11 days for the axotomized group. Capsaicin injections caused a consistent reduction in numbers of CGRP- and substance P-immunoreactive fibres in the pulps and a somewhat smaller reduction in the periodontal tissues. Unilateral axotomy of the inferior alveolar nerve induced an almost complete loss of immunoreactive fibres in the pulp and in the mesial gingiva of the first molar. Dentine formation at the mesial pulp horn and at the central pulp floor was significantly reduced in both groups compared to controls. The results suggest that sensory neuropeptides such as CGRP and substance P may play a part in dentine formation.

Dentin formation after corticosteroid treatment. A clinical study and an experimental study on rats.

The overall aim of this series of studies was to determine whether the extensive narrowing of the dental pulp chamber seen in dental radiographs of patients with end stage renal disease was related to the method of treatment of the renal disease. The treatments studied were immunosuppression, renal transplantation and hemodialysis. A strong correlation was found between narrowing of the dental pulp chamber and renal transplantation. In the immunosuppression group and in the renal transplantation group, the patients received immunosuppressive drugs, mainly corticosteroids and azathioprine. Significantly higher doses of corticosteroids had been given to patients showing narrowing of the dental pulp chamber than to patients without such narrowing. The total plasma clearance of the corticosteroids received was measured among transplanted patients. The total plasma clearance was lower in patients with narrowing of the dental pulp chamber than in patients without narrowing. The morphology of the corticosteroid induced dentin in premolars from five deceased patients who had received one or more renal transplants each was compared with that in extracted teeth from five healthy individuals. The results of this study showed that dentin formation after high dose corticosteroid treatment in humans seems to start with a widening of the predentin zone, followed by extensive mineralisation causing substantial reduction of the dental pulp chamber within a few years after the start of treatment. In an experimental study on rats, it was shown that intravenous administration of high doses of corticosteroids induced dentin formation along the pulp chamber walls in the molars. Energy dispersive X-ray microanalysis was used for estimation of the calcium and phosphorus content of the dentin in rat teeth. The calcium and phosphorus composition of the corticosteroid induced dentin was found to be equal to that of normal dentin in control rat molars, which might imply that the mineralisation process was not affected by the corticosteroid treatment and thus the quality of the corticosteroid induced dentin might be equal to normal dentin regarding the calcium and phosphorus composition. In a scanning microscopy study of the morphology of the corticosteroid induced dentin in rat incisors, it was found to be equal to that of normally formed dentin.

Effects of xylitol and carbohydrate diets on dental caries, dentine formation and mineralization in young rats.

Female Wistar rats were weaned at the age of 3 weeks and fed for 7 weeks either a high-sucrose diet, a non-cariogenic raw potato-starch diet, a high-sucrose diet with 5% xylitol supplement, a raw potato-starch diet with 5% xylitol supplement or a non-cariogenic, commercial, powdered rat food (Ewos R3) for reference. A low xylitol concentration reduced the progression and severity of carious lesions but did not affect dentine apposition or the width of predentine in rats fed high-carbohydrate diets. Widening of the predentine zone in rats fed a high-sucrose diet might reflect disturbed mineralization, which could not be explained by serum ionized calcium or phosphate ion levels and which could not be corrected by low xylitol concentrations. It is concluded that the reduced area of dentinal carious lesions after low xylitol supplementation is not dependent on dentine formation or mineralization, but rather on direct effects in the mouth.

Changes in the plasma electrolytes and metabolites of the rat following acute exposure to sodium fluoride and strontium chloride.

Acute exposure of rats to strontium or fluoride by i.p. injection of sodium fluoride or strontium chloride resulted in a systemic response in which changes occurred in the plasma electrolytes and metabolites. Strontium resulted in a rapid but temporary hypercalcaemia while fluoride produced a temporary hypocalcaemia. There was no significant hypophosphataemia after fluoride and only a transient hypophosphataemia with strontium. There was some indication of kidney damage and a general stress response following fluoride injection. These results do not support the hypothesis that interglobular dentine is associated with hypophosphataemia or hypoplastic enamel with hypocalcaemia and are in conflict with the observation that the formation of interglobular dentine following the injection of lead acetate is associated with hyperphosphataemia and hypercalcaemia.

Dentinogenic activity of allogenic plasma fibronectin on dog dental pulp.

The response of ectomesenchymal cells of dog dental pulp to implantation of Millipore filters supplemented with bovine plasma fibronectin was evaluated after observation periods of one or four weeks. Two concentrations of plasma fibronectin were used (0.2 and 1 mg/mL). Experiments also included implants treated with control solutions (PBS or 1 mg/mL of dog albumin). Formation of a layer of elongated, polarized cells was demonstrated in direct contact with the implants treated with 1 mg/mL of plasma fibronectin solution, after one week post-operatively. Microfilamentous organization and orientation of rough endoplasmic reticulum was observed mainly in the supranuclear zone of the polarized cells. Implants treated with the same solution were consistently surrounded by a thick layer of dentinal matrix after four weeks of their exposure to pulp sites. Implants treated with control solutions or with the low concentration of fibronectin never showed any sign of cell polarization and matrix synthesis. These data provide evidence that the pulp cells can express their odontoblastic phenotype in response to a surface containing concentrated fibronectin (even allogenic), without the need of other molecules as exogenous inductive factors.

[Morphological changes in the lower incisor of suckling rats induced by large dose of aspirin].

Large doses of aspirin (200 mg/kg or 400 mg/kg, s.c) caused marked hypocalcemia in suckling rats, two hours after administration. The hypocalcemic effect was more evident in two week old rats than in one week old ones. Although the mechanism of aspirin-induced hypocalcemia is not clear at this moment, the drug can be a useful tool for inducing experimental hypocalcemia in suckling rats, besides hormonal and/or nutritional controls. In this report, large doses of aspirin were administrated to new born rats, once at one week after birth, or twice at one and two weeks after birth. The morphological changes of the lower incisor were examined using computer programs which have been developed for the analysis of plane curves such as the traces of the side view of the incisor. Aspirin administration shortened the length of the lower incisor and its labial trace. The width of the incisor, especially in the middle, was also diminished by aspirin administration. These observations suggest that the drug not only induced hypocalcemia in suckling rats but also to some extent suppressed the activity of odontoblasts which produce the dentin of the incisor. Analysis of curvature variance, calculated with the labial trace of the lower incisor, also suggested that large doses of aspirin had two effects. It suppressed mineralization of the teeth through its hypocalcemic effect, and it inhibited synthesis of the collagenous matrix. The computer programs applied in this study have proved useful in determining and analyzing morphological changes of bio-materials which are difficult to measure directly.

The effect of lead acetate on dentine formation in the rat.

The intravenous injection of lead acetate produces a response in dentine by formation of the so-called lead line. This is associated with a rapid but temporary rise in serum calcium and phosphorus. Previous studies have suggested that lead replaces calcium and phosphorus in the apatite lattice. In this study the lead line was investigated in the scanning electron microscope using backscattered electron imaging and was shown to consist of continuous hypomineralized interglobular spaces within the dentine. Dentine formation was disrupted for at least a week after injection of lead ions. With energy dispersive analysis by X-rays, no localized concentration of lead was detectable within the 'lead line'. It is suggested that the lead line results from a direct effect of lead on the odontoblasts and other hard tissue-forming cells producing a rapid loss of intracellular calcium temporarily displaced by lead ions and a subsequent disturbance of local calcium metabolism.

Effects on pulp and dentin of iontophoresis of sodium fluoride on exposed roots in dogs.

Experiments were performed on young permanent teeth in which there was demonstrable active dentin formation. Following surgical exposure of roots and removal of cementum with scalers, 1 percent sodium fluoride in aqueous solution was applied either topically or with direct-current iontophoresis. Two levels of current, therapeutic or five times therapeutic, were used. Observation periods were 7 and 80 days. There were no demonstrable histologic or ultrastructural alterations of the underlying pulp; nor was there a resultant change in the rate of dentin formation. While exposure of root surface dentin alone (negative control) produced no alterations, grinding the surface (positive control) caused noticeable changes in dentin, odontoblasts, and pulp.

Acute and protracted effects of vinblastine on odontoblasts and dentinogenesis in rat incisors.

The effects of a large dose of vinblastine sulfate (2 mg/kg body weight) on proliferating odontoblast precursors and secretory odontoblasts in the continuously growing rat incisor were studied. The rats were killed 6 h, 24 h, 3 d and 7 d after vinblastine injection. Most cells in the proliferating zone contained arrested mitoses, or had perished after 24 h. After 3 and 7 d, the odontoblasts derived from this zone were reduced in number, and showed altered cell shapes. The odontoblasts had produced irregular dentin. The secretory odontoblasts had displaced nuclei and altered cell shapes after 24 h. Those most affected were opposite early mineralized dentin. In some incisors the cells had perished. In the protracted experiments almost all the odontoblasts were changed and had produced abnormal dentin. In the early mineralized dentin area, accumulations of cells were present after 3 d, and osteodentin-like material after 7 d.

The effect of vincristine on dentinogenesis in the rat incisor.

A preliminary report on the histomorphologic effects of vincristine on rat incisor dentinogenesis is presented. Niche-like areas of osteodentin and a faint incremental line in the dentin were the main derangements observed after single injections of the drug. The severity of the lesions was dose-dependent.