Acid-base balance, dentinogenesis and dental caries

Experimental studies in rats

Tuula Bäckman

Institute of Dentistry

Abstract

High-sucrose diet and metabolic acidosis have some similar effects on bone and they both reduce the formation of dentine. This series of experiments was conducted in order to get information about the effects of acidosis and alkalosis on dentine during primary dentinogenesis and also to ascertain if high-sucrose diet affects dentine formation via acidosis. Chronic metabolic acidosis (0.25 mol/L of NH4Cl in drinking water), chronic metabolic alkalosis (0.25 mol/L of NaHCO3 in drinking water) and chronic respiratory alkalosis (atmospheric pressure equivalent to an altitude of 3000 m) were induced in the rats immediately after weaning for 6 and 7 weeks. One subgroup from each of the main groups was fed a high-sucrose (43%) diet and one a standard maintenance diet, each ad libitum. The control groups had the same diets, but normal drinking water and atmospheric pressure. All the rats were injected with tetracycline (to mark the onset of the experiment in dentine) and inoculated orally with Streptococcus sobrinus. The acid-base status was verified by blood gas analysis at the end of the experiments. After sacrifice, fissure caries was scored with Schiff reagent and the areas of dentinal lesions and tetracycline-marked new dentine were measured from sagittally sectioned mandibular molars. The mineral elements (Ca, Mg, F, Na, P and total mineral contents) of the dentine formed before and during the experiment were measured with an electron probe microanalyzer.

With the high-sucrose diet, respiratory alkalosis and metabolic acidosis promoted the initiation and progression of caries while metabolic alkalosis slightly retarded it. With the standard diet, all the experimental conditions slowed the rate of dentine formation and metabolic acidosis had the most pronounced effect. The mineral analysis revealed a totally different pattern of mineralization when the rats with metabolic acidosis (increased calcium and total mineral content) were compared to the previously reported rats with a high-sucrose diet (decreased calcium and total mineral content). Besides this, metabolic alkalosis did not correct the effects of the dietary sucrose on dentine formation and blood gas analysis showed no acid-base disturbances in the sucrose diet group. Therefore, a high amount of sucrose in the diet slows the rate of dentine formation and reduces the ability of teeth to resist caries attack by mechanisms different from those of metabolic acidosis. Nevertheless, metabolic acidosis was found to be the most harmful state of disturbance in acid-base balance for the teeth of young rats, especially with a diet containing a high amount of sucrose.

Table of Contents
Acknowledgements
Abbreviations
1. Introduction
2. Review of the literature
2.1. Acid-base balance
2.2. Causes of disturbances in acid-base balance
2.2.1. Causes of metabolic acidosis
2.2.2. Causes of metabolic alkalosis
2.2.3. Causes of respiratory acidosis
2.2.4. Causes of respiratory alkalosis
2.3. Maintaining of acid-base balance
2.3.1. Role of lungs in maintaining acid-base balance
2.3.2. Role of buffers in maintaining acid-base balance
2.3.3. Role of kidneys in maintaining acid-base balance
2.4. Acid-base balance and bone
2.4.1. Metabolic acidosis and bone
2.4.2. Respiratory acidosis and bone
2.4.3. Metabolic alkalosis and bone
2.5. Acid-base balance and teeth
2.6. High-sucrose diet and dentine
2.7. Dentinogenesis
3. Working hypothesis and aims of the study
4. Materials and methods
4.1. Maintenance of the rats
4.2. Diets
4.3. Conduct of the experiment
4.3.1. Induction of metabolic acidosis
4.3.2. Induction of metabolic alkalosis
4.3.3. Induction of respiratory alkalosis
4.3.4. Control rats (normalosis)
4.4. Anesthesia and blood samples
4.5. Preparation and analyses of the tooth samples
4.5.1. Quantification of dentine apposition
4.5.2. Mineral analysis (EPMA)
4.5.3. Caries scoring
4.6. Pilot studies
4.7. Statistical analyses
4.7.1. Statistics in blood gas analysis
4.7.2. Statistics in measuring dentine formation
4.7.3. Statistics in mineral analysis
4.7.4. Statistics in measuring caries
5. Results
5.1. Pilot studies
5.2. General health
5.3. Blood properties
5.3.1. Metabolic acidosis
5.3.2. Metabolic alkalosis
5.3.3. Respiratory alkalosis
5.4. Dentine formation
5.5. Dentine minerals
5.6. Caries
5.6.1. Areas of dentinal caries
5.6.2. Caries scoring
6. Discussion
6.1. General health
6.2. Acid-base balance
6.3. Dentine formation
6.4. Mineral analysis
6.5. Caries
7. Conclusions
References
List of Tables
1. Compositions of the diets.
2. The nutritional values of the diets.
3. Grouping and treatment of the rats in the experiments.
4. Results of the pilot studies on the effects of metabolic acidosis and alkalosis. Increase (+) or decrease (-) of the weight gain of the rats, and dentine formation and dentinal caries in the 1st, 2nd and 3rd molars given in percentages compared to the controls. All the groups were fed the high-sucrose diet (see Table 1).
5. Mean weight gain (in grams) during the periods of the ordinary experimental studies. The groups are arranged according to the weight of the males.
6. Mean dentine formation in three molars in square micrometers x103 during the experimental period.
7. Median (md) dentine mineral contents in dentine formed before (a) and during (b) the experimental period, given in weight percentages. Minimum, median and maximum of the difference between mineral contents in dentine formed during and before the experiment. P-values for comparisons between b versus a and between the experimental groups (2, 4, 6) versus the control group (8).
8. Areas of dentinal caries lesions. Total numbers of the fissures examined (n) and percentages of the fissures with dentinal caries lesions. Minimum, maximum and mean of the lesion area in each group, given in square micrometers (calculated only from the fissures with lesion). The experimental groups are arranged according to the number and the mean size of the lesion.
9. Results of Schiff staining of the caries lesions. Percentages are given of the fissures with no caries (N) and lesions of different depths (A, T, B, C). The groups are arranged according to Table 8.
10. Minimum, median and maximum of the sum scores of the caries lesions in each group (Schiff staining). Healthy fissures (N) were scored as 0, enamel lesions (A) as 1 and dentinal lesions (T, B, C) as 7 to emphasize the difference between enamel and dentinal lesions. The groups arranged according to Tables 8 and 9.
List of Figures
1. Schematic drawing of the dentine (left) and the bone (right). Gray area = mineralized tissue. Striped gray area = unmineralized new dentine (predentine) / bone (osteoid). Left: carious dentine and cariogenic bacteriae up, odontoblast cells (pulp) down. Odontoblast processes in tubules. Right: Active (round) and passive (flat) osteoblasts surround the bone. Osteocytes are located inside the bone. They are communicating via the bone canaliculi with each other and with the osteoblasts. In left upper corner, multinucleated osteoclast resorbs the bone.
2. Sectioning of the rat mandible.
3. Schematic drawing of sagittally sectioned mandibular molars of rat. Wide black line = enamel. Grey area = dentine apposition measured. Dotted line = tetracycline-marked onset of dentine formation during the experiment.
4. Back-scattered electron image (COMPO) of rat"s first and second mandibular molar showing the spots from where the mineral elements were measured with EPMA.
5. Photomicrograph of the crown of the third molar. The dentinal caries lesion is seen as a fluorescent area (surrounded by dotted line). Fluorescent tetracycline line shows the onset of dentine apposition during the experiment.
6. Blood pH in the experimental groups. Each 7-week group is included in the corresponding 6-week group: m-acid-stan (groups 2 and 9), m-alk-stan (4 and 10) and norm-stan groups (8 and 11) are combined. In box plots the box presents 1st and 3rd (upper and lower) quartiles with the median value inbetween. The whiskers give the lowest and highest values. Extreme values are marked as circles. n = number of blood samples.
8. Blood base excess (B.E.) in each group given in mEq/L. For groups and box plot presentation: see Fig. 6.
8. Blood bicarbonate (HCO3-) given in mmol/L. For groups and box plot presentation: see Fig. 6.
9. Blood carbon dioxide partial pressure (pCO2) given in kPa. For groups and box plot presentation: see Fig. 6.
10. Blood oxygen partial pressure (pO2) given in kPa. For groups and box plot presentation: see Fig. 6.
11. Dentine formation in square micrometers in the second molars in the six week"s experiments. The box presents the 1st and 3rd (upper and lower) quartiles with the median value inbetween. The whiskers give the lowest and highest values. n = number of the teeth.
12. Dentine formation in square micrometers in the second molars in the seven weeks"s experiments. For box plot presentation: see Fig. 11.
13. Areas of dentinal caries in the second molars of the rats in the groups 1, 3, 5 and 7. Extreme values are marked as circles and outline values as asterisks. For box plot presentation: see Fig. 11.
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