TITLE:

COMPARISON OF ORAL HEALTH STATUS, DENTAL CARIES EXPERIENCE AND SALIVARY BIOMARKERS IN TYPE 1 DIABETIC CHILDREN AND NON DIABETIC CHILDREN

INTRODUCTION:

Type 1 Diabetes mellitus (T1DM), also known as autoimmune diabetes, is a chronic disease characterized by insulin deficiency due to pancreatic B-cell loss and leads to hyperglycemia.
Type 1 Diabetes is usually present in individuals without a family history. Only 10-15% of the patients have a first or second-degree relative with the disease. However, the lifetime risk for developing Type 1 Diabetes Mellitus is significantly increased in relatives of patients, as about 6% of children, 5% of siblings and 50% of monozygotic twins present the disease compared to 0.4% prevalence of the general population.
People with diabetes in 1,000,000 are approximately 74,194.7 in India. According to 2021 census report, the 0-14 age group is 25.31% of the total population of India, among which, Type 1 diabetic children (0 to 14 years) in 1,000,000 are approximately 126.4 in India. In India, new cases of type 1 diabetes (0-14 years) in 1,000,000 are 19.2.
Higher prevalence of dental diseases like dental caries, xerostomia, gingival inflammation are reported for children with Type 1 diabetes when compared to systemically healthy children.
The present study attempted to compare the oral health status, dental caries experience, salivary flow rate, salivary pH, salivary glucose level, salivary sialic acid and total salivary antioxidant levels in Type 1 Diabetic Children and Non Diabetic Children.
Reason for selecting parameters:

• Healthy flow of saliva is essential for maintenance of both oral and general health. It provides a cleansing effect. Saliva may constitute a first line of defense against free radical mediated oxidative stress. Salivary flow is significantly reduced in type 1 diabetic children.
• Serum sialic acid levels are found to be raised in storage diseases, cardiovascular diseases and cancers. Free sialic acid levels are raised in type 1 diabetes.
• Harmful effects of oxygen are due to formation of Reactive Oxygen Species in diseased states. To protect us against the oxidizing action of free radicals, every individual has an army of antioxidants which fight against free radicals. Thus in any diseased state, free radicals produced are quite higher. Type 1 Diabetes is one such diseased state in which free radicals are increased significantly.
• The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction of different organs and impaired salivary gland functions leading to changes in saliva composition. In type 1 diabetes, salivary glucose levels get elevated. Increasing the level of glucose in saliva affects the activity of microorganisms.
• The Salivary Flow Rate, Salivary pH, Salivary Glucose, Salivary Sialic Acid, Salivary Total Anti-Oxidants Levels may be altered in Type 1 Diabetic patients and due to which, poor oral hygiene and increased dental caries experience may be seen.

Since diabetes has an influence on oral health, it is important for the dentist to be aware of newer advances in the field of diabetes and to recognize specific oral problems related to diabetes. Thus, the dentist becomes an important part of the healthcare team for the patients with diabetes. This study will help us to prevent deterioration of oral health status in Type 1 Diabetic Children by taking appropriate measures to caries and periodontal diseases.
Many researchers have tried to compare dental caries experience or gingival health status in Type 1 Diabetic Children and Non Diabetic Children but there is still a gap of knowledge to associate changes in salivary composition or salivary biomarkers to increased oral health problems in Type 1 Diabetic Children. Also, the association of diabetes and dental caries has received much less attention and the results have been controversial. Most studies are cross-sectional and show either higher, similar or lower caries prevalence among diabetic than among the controls. This study includes evaluation of both changes in salivary composition or salivary biomarkers and deteriorated oral health of Type 1 Diabetic Children.

OBJECTIVES:

1. To evaluate Oral Health Status, Dental Caries Experience, Salivary Flow Rate, Salivary pH, Salivary Glucose, Salivary Sialic Acid and Salivary Total Antioxidant Capacity in Type 1 Diabetic children and Non Diabetic children.
2. To compare Oral Health Status, Dental Caries Experience, Salivary Flow Rate, Salivary pH, Salivary Glucose, Salivary Sialic Acid and Salivary Total Antioxidants Capacity in Type 1 Diabetic children with Non Diabetic children.

RESEARCH QUESTION: Is there a difference between Oral Health Status, Dental Caries Experience, Salivary Biomarkers such as Salivary Flow Rate, Salivary pH, Salivary Glucose, Salivary Sialic Acid, Salivary Total Antioxidant Capacity in Type 1 Diabetic children and Non Diabetic children?

RESEARCH HYPOTHESIS:

• Null hypothesis: There is no significant difference in Oral Health Status, Dental Caries Experience, Salivary Biomarkers such as Salivary Flow Rate, Salivary pH, Salivary Glucose, Salivary Sialic Acid, Salivary Total Antioxidant Capacity in Type 1 Diabetic children when compared to Non Diabetic children.
• Alternative hypothesis: There is a significant difference in Oral Health Status, Dental Caries Experience, Salivary Biomarkers such as Salivary Flow Rate, Salivary pH, Salivary Glucose, Salivary Sialic Acid, Salivary Total Antioxidant Capacity in Type 1 Diabetic children when compared to Non Diabetic children.

METHODOLOGY:

• STUDY TYPE: Analytical Study
• STUDY DESIGN: Case Control Study
• SAMPLE SIZE: 50 Case (Type 1 Diabetic children) and 50 Control (Non Diabetic children)

Group 1 (Study Group): 50 Case (Type 1 Diabetic children) (5 to 12 years)
Group 2 (Control Group): 50 Control (Non Diabetic children) (5 to 12 years)

INCLUSION CRITERIA FOR CASE:

• Type 1 diabetic diagnosed children.
• Age group of 5 to 12 years.
• Male and female children.
• Children whose guardians are providing informed consent.

EXCLUSION CRITERIA FOR CASE:

• Presence of other systemic disease/s.
• Patient with cognitive impairment.

INCLUSION CRITERIA FOR CONTROL:

• Non diabetic children.
• Age group of 5 to 12 years.
• Male and female children.
• Children whose guardians are providing informed consent.

EXCLUSION CRITERIA FOR CONTROL:

• Presence of other systemic disease/s.
• Patient with cognitive impairment.

MATERIALS TO BE USED:

1. Disposable gloves
2. Disposable Mask
3. Protective eyewear
4. Plane mouth mirror – no.5
5. Williams graduated probe – no.15/734
6. Explorer – no.23 shepherd’s hook
7. Modified Who oral health assessment form 4
8. Eppendorf tube for Saliva collection
9. Measuring cylinder for salivary flow rate measurement
10. pH meter
11. Test tubes
12. Pipettes
13. Graph papers
14. Glass Beakers
15. Distilled water

Chemicals for tests:

• Salivary glucose estimation:
• 3,5-Dinitrosalicylic acid
• Sodium hydroxide
• Sodium potassium tartarate
• Standard glucose
• Estimation salivary total antioxidant capacity:
• Ammonium molybdate
• Sodium phosphate
• Sulfuric acid
• Ascorbic acid (vit C)
• Alpha – tocopherol (vit A)
• Salivary sialic acid estimation:
• Ninhydrin Reagent
• Glacial acetic acid
• Acetone
• Sulfuric acid

Study Conduct:
Information Dissemination: Information brochures will be provided to the parents, explaining the disease and its correlation with oral health in simple and local language.

Informed Consent Process: Written informed consent will be obtained in the local language with a verbal explanation provided to the child’s parents. The consent will be easy to understand and include information about clinical checkups for oral health status, dental caries index, and steps involved in saliva collection. Children whose parents agree to sign the informed consent will be included in the study.

A thorough examination will be performed that will include demographic data and a complete intra-oral examination. Oral health and dental health status will be examined using the following indices:

DENTAL CARIES EXPERIENCE:

Using a Plain Mouth Mirror no. 5 and Explorer no. 23, dental caries will be recorded and scored.

Primary Teeth (Scores) | Permanent Teeth (Scores) | Status

• A: 0 | Sound
• B: 1 | Caries
• C: 2 | Filled with caries
• D: 3 | Filled, no caries
• E: 4 | Missing due to caries
• -: 5 | Missing for other reasons
• F: 6 | Fissure Sealant
• G: 7 | Fixed Dental Prosthesis
• -: 8 | Unerupted
• -: 9 | Not Recorded

DMFT (Decayed, Missing, Filled Teeth) is derived directly from the data in the boxes:

• D component: Teeth coded 1 or 2 (B or C).
• M component: Teeth coded 4 or 5 (E).
• F component: Teeth coded 3 or 6 (D or F).

DMFT Calculation: DMFT = D + M + F.

ORAL HEALTH STATUS:

Using Williams Graduated Probe no. 15/734, oral health status will be recorded and scored.

Gingival Bleeding Scores:

• 0: Absence of bleeding
• 1: Presence of bleeding
• 9: Tooth Excluded
• X: Tooth not present

Pocket Score:

• 0: Absence of condition
• 1: Pocket 4-5 mm
• 2: Pocket 6 mm or more
• 9: Tooth excluded
• X: Tooth not present

Collection of Saliva:

Saliva will be collected in the morning hours (8 am to 10 am), considering circadian rhythms. The patient will be instructed not to eat or drink anything 1 hour before collection.

• Unstimulated saliva (5 ml) will be collected in a funnel inserted into a beaker, allowing the patient to drool passively.
• After collection, the saliva will be transferred into plastic containers, labeled, sealed, and transported in a biohazardous bag to prevent contamination.

A) Estimation of Salivary Flow Rate:

The unstimulated salivary flow rate (USF) will be calculated using the formula:

USF = Total volume of collected saliva / Time period for collection of saliva.

The saliva will be stored at -80 C in a deep freezer at the College of Biosciences and Technology, Loni.

B) Salivary pH:

1. Salivary pH will be measured using a pH meter (Thermo Orion).
2. 1 ml of saliva will be transferred to a glass beaker.
3. The tip of the pH meter will be dipped into the saliva.
4. The pH value will be displayed on the meter.

Interpretation of Results:

• pH 0 to 6.9: Acidic
• pH 7: Neutral
• pH above 7: Basic

C) Salivary Sialic Acid:

1. Sialic acid will react with ninhydrin in an acidic medium to form a colored product, which can be measured spectrophotometrically at 470 nm.
2. A precipitate will form after ethanol treatment. To this precipitate, 1.0 ml of distilled water, 1.0 ml of glacial acetic acid, and 1.0 ml of acidic ninhydrin reagent will be added.
3. The reaction mixture will be heated for 10 minutes in a boiling water bath. After cooling, absorbance will be measured at 470 nm.
4. A standard sialic acid curve will be generated for comparison.

D) Salivary Total Antioxidant Capacity:

1. An aliquot of 0.1 ml of saliva will be combined with 1 ml of a reagent solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate.
2. The tube will be incubated at 95 C for 90 minutes.
3. After cooling, the absorbance will be measured at 695 nm.
4. The assay is based on the reduction of phosphate-Mo (VI) to phosphate Mo (V) and the subsequent formation of a bluish-green color complex.

E) Salivary Glucose Test:

1. DNSA Method will be used to estimate reducing sugars.
2. A standard curve will be created using standard solutions and the test sample will be compared.
3. The optical density (O.D.) at 540 nm will be measured.
4. The glucose concentration will be determined using the standard graph.

Statistical Analysis:

• Data will be analyzed using SPSS software v26.0.
• A significance level of 5% will be considered.
• Normality tests (Kolmogorov-Smirnov) will be performed. Parametric tests will be applied to normally distributed data, and non-parametric tests will be applied to skewed data.
• Demographic characteristics will be presented using descriptive statistics.
• Comparisons will be made using independent t-tests or Mann-Whitney tests for salivary biomarkers and other measures.
• Chi-square tests will be used for comparing dental caries, gingival bleeding, and pocket scores.

Amendment of Protocol:

No changes will be made to the study procedures without mutual agreement from the investigator, physician, and ethical committee.

Confidentiality:

Patient identity will be kept confidential. Data will only be available to the investigator and regulatory authorities, with any breach of confidentiality reviewed by the investigator and ethical committee.

Ethical Considerations:

• Ethical approval will be obtained before commencing the study from the institutional ethical committee.
• Informed consent will be obtained from all participants and their guardians.

Implications:

This study aims to enhance the oral health care of Type 1 Diabetic children by raising awareness of the connection between salivary biomarkers and oral health. Improved dental care will contribute to better overall health for these children.

References:

1. Katsarou A, Gudbjörnsdottir S, Rawshani A et al. Type 1 diabetes mellitus. Nat Rev Dis Primers. 2017 March.
2. Paschou SA, et al. On type 1 diabetes mellitus pathogenesis. Endocr Connect. 2018 Jan;7(1):R38-R46.
3. IDF Diabetes Atlas 2021.
4. Rai K, Hegde AM, et al. Dental caries and salivary alterations in Type I Diabetes. J Clin Pediatr Dent. 2011 Winter;36(2):181-4.
5. Hegde AM, Joshi S, et al. Evaluation of oral hygiene status in acute lymphoblastic leukemic (ALL) children. J Clin Pediatr Dent. 2011 Spring;35(3):319-23.
6. Joshi S, Hegde AM, et al. Evaluation of salivary sialic acid levels in acute lymphoblastic leukemic children. J Clin Pediatr Dent. 2013 Spring;37(3):309-13.
7. Kenzaburoh Yao, Toshihiko Ubuka, et al. Direct determination of bound sialic acids. Analytical Biochemistry. 1989.
8. Prieto P, Pineda M, et al. Spectrophotometric quantitation of antioxidant capacity. Anal Biochem. 1999 May 1;269(2):337-41.