Comparison of Changes in Dentin Sialoprotein Levels and Severity of Orthodontic Root Resorption in Conventional Versus Delayed Premolar Extraction A Prospective Cohort Study

1. INTRODUCTION, INCLUDING RATIONALE OF THE STUDY

Orthodontic root resorption (ORR) is a common undesirable consequence of orthodontic treatment. Dentin sialoprotein (DSP) is a potential biomarker for assessing root resorption severity. While premolar extractions are a standard practice in orthodontic treatment, the timing of extraction whether conventional or delayed can influence the magnitude of root resorption. However, comprehensive evidence comparing DSP levels and ORR severity in these two extraction protocols remains scarce. Understanding the relationship between the timing of premolar extractions, DSP changes, and root resorption severity is critical to optimizing treatment outcomes and minimizing adverse effects in orthodontic patients.

One of the common and undesirable sequelae associated with the orthodontic tooth movement is root resorption, clinically referred to as Orthodontic Root Resorption (ORR). Brezniak and Wasserstein described ORR as an unavoidable pathological process during orthodontic tooth movement, characterized by loss of material from the dentin or cementum of the root.Heavy orthodontic forces result in development of hyalinized areas in periodontal spaces. Cells and blood vessels from the surrounding healthy periodontium then remove these tissues, resulting in the loss of cementoid and mature collagen near the cementum. This disrupts the normal barriers protecting the root from resorption.

The incidence of ORR ranges from 44% to 91%, according to several studies.Though in most of the cases, only mild to moderate root resorption is experienced by the patient, which is clinically insignificant and does not affect dental health, severe cases can lead to tooth mobility and potentially jeopardize the success of orthodontic treatment. The likelihood of ORR is higher in anterior teeth compared to posterior teeth, with maxillary central and lateral incisors being the most affected, followed by canines. Within the dental arch, the severity of ORR follows the order: anterior maxilla, anterior mandible, posterior mandible, and posterior maxilla. Genetic characteristics, biological factors, orthodontic treatment techniques and age of patient all play a role in increasing the likelihood of root resorption. Orthodontic factors include magnitude, duration, and type of the orthodontic force applied. Studies suggest that pausing orthodontic forces during treatment may help reduce root resorption, likely due to cementum repair during inactive periods. Radiographic techniques are commonly used to diagnose root resorption, offering advantages such as wide accessibility, ease of use, and cost-effectiveness. However, the downside of this method includes difficulties in early detection, technique errors, and radiation exposure. Radiographs are also unable to show whether the progression of root resorption is active. While CT and CBCT offer improved sensitivity, their expense and increased radiation exposure make them unfeasible for routine use. Hence, a safer and more reliable alternative for diagnosing root resorption, especially in earlier stages, is essential.

GCF (Gingival Crevicular Fluid) is an inflammatory transudate that flows through the gingival crevice. The quantity and composition of the fluid varies depending on health of the periodontium. GCF contains a variety of biochemical and cellular factors that reflect the condition of the underlying periodontium.

Dentin Sialoprotein (DSP) is an N terminal cleavage product of dentine sialophosphoprotein (DSPP) and part of the small integrin-binding ligand N linked glycoprotein family. Immunolocalization studies reveal its presence in odontoblasts, dental pulp, predentine, and dentine, but not in bone, cartilage, ameloblasts, soft tissues, or other oral components, indicating its high specificity to dentin. Balducci et al. (2006) have confirmed the presence of DSP in GCF of patients diagnosed with both mild and severe resorption after at least one year of treatment with fixed appliances. Hence, in conclusion, DSP can serve as biomarker for detecting root resorption associated with the orthodontic treatment, providing an alternative to radiographic methods.

In orthodontics, therapeutic extractions are a common practice. Traditionally, these extractions are performed prior to the initiation of the treatment to simplify biomechanics, prevent roundtripping, and eliminate appliance interference during extractions.Certain other strategies recommend extracting the teeth at a later stage, prior to the retraction phase. Tooth removal also triggers the regional acceleratory phenomenon (RAP) in the localized area, sparking debate over the optimal timing for initiating space closure. Clinical evidence suggests that fresh extraction sites may be beneficial for tooth movement by leveraging a planned regional accelerated phenomenon shortly after extractions. Prolonged treatment can increase the risk of iatrogenic damage, such as root resorption, demineralization, and periodontal issues. Additionally, patient compliance may decline over time, leading to a higher likelihood of adverse effects and less favourable outcomes.

 Novelty

This study explores a novel perspective by investigating the association between DSP levels and severity of ORR in cases involving conventional versus delayed premolar extractions. A distinctive aspect of this research is its focus on differences in rate of the tooth movement during retraction between these two extraction timings and its consequent effect on orthodontic root resorption, an area that has been inadequately explored in previous studies. By comparing biochemical markers like DSP in GCF with radiographic parameters, this study aims to provide a thorough understanding of how extraction timing influences both the biological response and efficiency of space closure.

2. RESEARCH QUESTION

How do changes in dentin sialoprotein (DSP) levels in GCF compare with the severity and progression of orthodontic root resorption in patients with Angle’s Class I malocclusion with bimaxillary dentoalveolar protrusion undergoing two therapeutic extraction protocols—conventional therapeutic extraction and delayed extraction, at three time periods, in the Department of Orthodontics, MES Dental College?

  3. AIM AND OBJECTIVES

To compare the changes in DSP levels in GCF with severity of ORR using RVG (Radiovisiography) at 3 points in patients with Angle’s Class I bimaxillary protrusion undergoing two therapeutic extraction protocols conventional therapeutic extraction and delayed therapeutic extraction protocols, who report to the Department of Orthodontics and Dentofacial Orthopaedics, MES Dental College, Perinthalmanna.

 OBJECTIVE

To compare the changes in DSP and severity of orthodontic root resorption assessed using RVG.

 4. STUDY DESIGN

A Prospective Cohort Study

 5. SETTING

The study will be conducted among patients with Angle’s Class I bimaxillary dentoalveoloar protrusion malocclusion who report to the Department of Orthodontics and Dentofacial Orthopaedics, MES Dental College, Perinthalmanna seeking fixed orthodontic treatment.

 6. SAMPLING

a. Sample Size Calculation

Variable used for sample size calculations - DSP

n = 6 per group, there will be a minimum of 6 patients in each group

 b. Inclusion Criteria

•           Patients with complete dentition.

•           Patients in the age group 18 – 25 yrs.

•           Patients should be having healthy gingival and periodontal status.

•           Patients with no radiographic signs of root resorption.

 c. Exclusion Criteria

•           Missing molars or any of the anterior teeth.

•           Previous history of orthodontic treatment.

•           Erupted or unerupted supernumeraries in the maxillary anterior segment.

•           Impacted canines, Endodontically treated teeth.

•           Upper anterior crowding  greater than or equal to 4mm.

•           Periodontitis

•           History of avulsion or replantation.

•           History of prolonged use of systemic steroids and NSAIDs

•           History of systemic disorders

d. Sampling Procedure

Sampling will be done by consecutive sampling methods from the patients reporting to the Department of Orthodontics, MES Dental College, Perinthalmanna, who satisfy the inclusion and exclusion criteria.

 Study Period

10th September  2025 to 31st December 2026

 7. METHODOLOGY

a. Methods Of Data Collection

Patients with Angle’s Class I bimaxillary proclination will be selected based on specific inclusion and exclusion criteria. Using consecutive sampling, they will be divided into two groups: conventional therapeutic extraction (Group 1) and delayed therapeutic extraction (Group 2). In Group 1, the first premolars will be extracted immediately before the initiation of orthodontic treatment. Following this, orthodontic treatment will begin for both groups using 0.022” prescription Ormco Mini Diamond Brackets. The anterior segment levelling and alignment will be performed using a standardized sequence of NiTi wires: 0.014”, 0.016” x 0.022”, 0.017” x 0.025”, and 0.019” x 0.025”, with each wire retained for 4 weeks. After this alignment of 6 months duration, subsequently the retraction mechanics will be initiated by placing 0.019” x 0.025” stainless steel working wires for an additional 4 weeks. At the completion of this stage, Group 2 patients will undergo therapeutic extraction of the first premolars. Retraction for both groups will then be carried out with active tiebacks (ORMCO – 0.010” SS ligature wire) using a force of 200g with 0.19”x 0.25” stainless steel wires, with monthly activations. Oral hygiene will be maintained throughout the treatment through periodic oral prophylaxis.Treatment of patients in both groups will be done by the same faculty (VA) and data will be collected by the principal investigator for the purpose of the observational study.

Group 1 – Patients with Angle’s Class I bimaxillary dentoalveolar protrusion malocclusion who will be treated with fixed orthodontic treatment for retraction of anterior teeth following conventional therapeutic extraction of maxillary first premolar teeth.

Group 2 – Patients with Angle’s Class I bimaxillary dentoalveolar protrusion malocclusion who will be treated with fixed orthodontic treatment for retraction of anterior teeth following delayed therapeutic extraction of maxillary first premolar teeth.

 GCF Collection

All samples will be collected after thorough cleansing of the oral cavity. The maxillary anterior teeth (12, 11, 21, and 22), from which samples will be obtained, will first be isolated with cotton rolls and dried with air. GCF collection will be done by placing PerioPaper strips (Oraflow Inc., Hewlett, New York, USA) into the gingival sulcus of maxillary central incisors and lateral incisors. The strips will be placed into the mesio-buccal and disto-buccal aspects of the gingival sulcus to the point of slight resistance and kept in position for 30 seconds. Strips that are contaminated with blood will be excluded from the analysis. The time points of sample collection are outlined in Table-1, and all samples will consistently be taken from the same sites. The PerioPaper strips containing GCF will be stored in sterile Eppendorf vials containing phosphate-buffered saline. These vials will be centrifuged for 10 minutes at 4°C and then stored at -80°C in the Department of Microbiology, MES Medical College, Perinthalmanna, until the collection of all samples is complete. Each aliquot will be thawed to room temperature before testing. The samples will be analyzed using an ELISA kit (96T) from Origin Diagnostics and Research, Kollam, Kerala. All assay procedures will be done according to the manufacturer’s protocol. Quantitative analysis will be conducted at the conclusion of the assay in the Department of Microbiology, MES Medical College, Perinthalmanna.

RVG

RVG will be taken for the maxillary anterior teeth (12, 11, 21, and 22) using the parallelling cone technique and the images will be analyzed. The Modified Malmgren Grading Criteria(12,13)will be applied to assess extent of the external apical root resorption.

 Gingival crevicular fluid (GCF) and radiographic (RVG) assessments were done at different time points. Both GCF and RVG were recorded before treatment (T1). At the beginning of retraction, six months into treatment (T2), only GCF was assessed. Finally, at the end of one year (T3), both GCF and RVG were recorded again.

 Outcome Measurement

In this study, the severity of orthodontic root resorption will be assessed using the Modified Malmgren grading scale according to Beck and Harris, based on RVG. For each grade of root resorption, the number of patients corresponding to that grade will be identified, and the mean DSP value of these patients will be calculated at the same time point as the RVG assessment. This process will be repeated across all grades for both Group 1 and Group 2 separately to analyse whether increasing root resorption severity corresponds to significant changes in mean DSP levels.

 8. DATA ANALYSIS PLAN

List Of Variables and Their Measurement Methods with Standardization Techniques

·        Independent Variables

·        Group 1 - Conventional Therapeutic Extraction

·        Group 2 - Delayed Therapeutic Extraction

·        Dependent Variables

·        Dentin Sialoprotein (DSP) levels

·        Severity of orthodontic root resorption as seen on an RVG

List of Variable Wise Statistical Tests to Be Used for Data Analysis

The data will be entered into Microsoft Excel and processed using SPSS. Quantitative variables will be presented as percentages with mean and standard deviation. All continuous variables will be evaluated regarding normality using the Shapiro- Wilk test. According to the normality tests, parametric (t-test) and non-parametric (Mann Whitney tests) will be used to compare variations in the levels of DSP and grading of radiographs in the two groups. P less than 0.05 will be considered statistically significant.