Biomarker-based monitoring of orthodontically induced root resorption: Expression of cementum protein-1 in orthodontic patients with and without skeletal anchorage

Abstract

Objectives:

This study aimed to estimate and compare changes in the cementum-specific biomarker, Cementum Protein-1 (CEMP-1), in gingival crevicular fluid (GCF) and the severity of orthodontically induced root resorption (OIRR), assessed radiographically, in patients undergoing orthodontic retraction with or without midline mini-implants. A secondary objective was to evaluate the influence of overall treatment duration on the extent of orthodontic root resorption.

Materials and Methods:

This prospective cohort study consisted of 40 patients divided into two groups with equal gender distribution: Group 1 underwent retraction of anterior teeth following first premolar extraction, while Group 2 underwent simultaneous intrusion and retraction using midline mini-implants. GCF samples were collected at T1 (before treatment), T2 (beginning of retraction), T3 (3 months after start of retraction), and T4 (end of treatment). The samples were assayed using ELISA Kits specific for the biomarker. Radiovisiography (RVG) of the anterior maxillary teeth was obtained at T1, T3, and T4 to assess root resorption.

Results:

At T1, CEMP-1 levels were comparable in the two groups. The biomarker levels increased over time in both groups. At T4, Group 2 had significantly higher biomarker levels compared to Group 1. RVG at T4 revealed a statistically significant increase in grading of root resorption in both groups (P < 0.05) with a higher mean difference in Group 2 (2 ± 0.21).

Conclusion:

Elevated levels of CEMP-1 in GCF are associated with greater degrees of apical root resorption and are promising noninvasive prognostic biomarkers for monitoring OIRR in orthodontic patients. Moreover, an increase in duration of treatment was found to increase OIRR.

INTRODUCTION

Root resorption is an undesirable but unavoidable consequence of orthodontic treatment and can range from mild (0.4–1.5 mm) to severe root resorption (>2 mm). The prevalence of severe root resorption ranges from 2% to 5%, and this can have clinically significant consequences, like a reduced crown-to-root ratio, which may affect the long-term viability of the dentition.[1] Risk factors causing orthodontic root resorption have been categorised in scientific literature as general and local factors.[2,3]

Vertical maxillary excess (VME), marked by excessive gingival display (gummy smile) in individuals with normal lip length, affects approximately 7% of young adult males and 14% of young adult females.[4] The advent of temporary anchorage devices (TADs), such as mini-implants, has significantly expanded the possibilities of controlled tooth movement, particularly in complex cases involving anterior intrusion and retraction. However, these movements are among the most closely associated with increased risk of root resorption.[5] Striking a balance between achieving the desired dental movements and minimising damage to the root structure is crucial in managing cases of deep bite and gummy smile, emphasising the importance of closely monitoring the progression of orthodontic root resorption.[2] The present study focused on examining the specific impact of local factors, particularly intrusion and retraction during orthodontic treatment, on the severity of orthodontic root resorption.

Two-dimensional radiographs and CBCT are currently used to evaluate root resorption during orthodontic treatment.[6,7] Recently, there has been growing interest in using gingival crevicular fluid (GCF) to identify protein biomarkers linked to resorptive activity due to its non-invasive nature, ease of collection, and potential for early detection.[8] Biomarkers such as dentin sialoprotein (DSP) have shown promise in detecting OIRR.[2,9,10,11,12] Cementum Protein-1 (CEMP-1), a protein secreted by cementoblasts, is another potential marker of root remodelling and repair. To date, limited research has explored the temporal changes in GCF biomarker levels with root resorption severity during different phases of orthodontic treatment. The present study aimed to assess whether quantification of CEMP-1 in GCF correlated with the severity of OIRR, as measured on radiovisiographs, in Angle’s Class I malocclusion patients treated with extraction of maxillary first premolars—with or without midline mini-implant-assisted intrusion. A secondary objective was to examine the relationship between overall treatment duration and the extent of root resorption.

MATERIALS AND METHODS

Study Design: This prospective cohort study was approved by the Institutional Ethical Committee (IEC/MES/73/2022) and adhered to the Declaration of Helsinki 2013. The study was registered with the Clinical Trials Registry of India (CTRI/2022/12/048229). Written informed consent/assent was obtained from all subjects who participated in this study.

Sample size estimation: Sampling was conducted using the consecutive sampling method from the patients reporting to the Department of Orthodontics at our Institute. Based on a standard deviation of 0.05, an effect size of 0.097, a significance level of 5%, and a power of 90%, while accounting for a 20% anticipated loss to follow-up, a minimum of seven patients was required in each group.[10] Forty patients, with Angle’s Class I malocclusion with bimaxillary dentoalveolar protrusion requiring therapeutic extraction of maxillary first premolars, were divided into two groups with equal gender distribution (Group 1 and Group 2), as illustrated in Figure 1. Group 1 patients underwent retraction of anterior teeth following first premolar extraction. Group 2 patients (with vertical maxillary excess) underwent simultaneous intrusion and retraction using midline mini-implants.

Figure 1.

Study design flow chart

Selection of Participants: The inclusion criteria comprised patients aged 15 to 25 years with a complete dentition excluding third molars, good gingival and periodontal health, and no radiographic evidence of root resorption. Exclusion criteria included the history of prior orthodontic treatment, presence of supernumerary teeth in the maxillary anterior region, impacted canines, endodontically treated teeth, upper anterior crowding of 4 mm or more, and any history of systemic diseases or long-term medication use.

Method: At the initiation of orthodontic treatment, all patients received thorough oral prophylaxis and standardised oral hygiene instructions. Both groups underwent fixed orthodontic treatment using a pre-adjusted edgewise appliance (PEA) with 0.022” slot MBT prescription brackets (ORMCO Mini 2000) following the extraction of all four first premolars. A uniform bonding protocol was employed across all participants.[13]

Initial alignment was achieved using 0.016” Nickel Titanium (NiTi) archwires, which were sequentially followed by 0.017” × 0.025” NiTi, 0.019” × 0.025” NiTi, and ultimately 0.019” × 0.025” stainless-steel posted archwires for anterior en-masse retraction. A retraction force of 200 g, measured using a Dontrix gauge, was delivered in both groups using active tie-backs. In Group 2, after alignment, a self-drilling mini-implant (Implant Genesis, 1.4 × 8 mm) was inserted between the root apices of the maxillary central incisors, positioned 6–8 mm from the alveolar crest along the midline.[14] A light intrusive force of 60 g was applied using an elastomeric chain attached to the midline mini-implant and measured with a Dontrix gauge for each patient; this force was maintained during every visit until the completion of retraction.[15] All clinical procedures were performed by a single investigator (VA) to ensure consistency. Patients were reinforced with oral hygiene instructions and received a mid-treatment oral prophylaxis.[13]

Outcome assessment: The specific time-points for outcome assessment (collection of GCF and taking RVG) are illustrated in Figure 1.

Gingival Crevicular Fluid (GCF) Collection: GCF samples were obtained from the maxillary incisors after isolating the area with cotton rolls to prevent contamination. PerioPaper strips (Oraflow Inc., Hewlett, New York, USA) were gently inserted into the mesio-buccal and disto-buccal sulci of each incisor and left in place for 30 seconds. Strips that showed any signs of blood contamination were excluded from the analysis.[10]

Following collection, the strips were placed into sterile 1.5 ml Eppendorf vials containing phosphate-buffered saline. The samples were centrifuged at 4°C for 10 minutes and stored at –80°C in the Department of Microbiology of our affiliated Medical College until further processing. Biomarker concentrations were analysed using a sandwich enzyme-linked immunosorbent assay (ELISA) with a 96-well kit (Origin Diagnostics and Research), adhering strictly to the manufacturer’s instructions. The levels of the biomarker were quantified by comparing the optical density (OD) values against the standard curve.

Radiographic Assessment (RVG): Digital periapical radiographs of the maxillary incisors were captured using a radiovisiography (RVG) system (RVG-4, Trophy Radiologie S.A., Croissy-Beaubourg, France) with exposure parameters set at 65 kVp, 8 mA, and an exposure time of 0.2 seconds. To ensure reproducibility, an RVG sensor positioner was employed, and the paralleling cone technique was utilised to maintain a standardised object-to-sensor distance.

Apical root resorption was evaluated on a 5-point scale (Grades 0–4) using the Modified Malmgren Grading Criteria [Table 1].[16,17] Grading was performed by a single calibrated investigator to ensure consistency. Each maxillary incisor was assessed individually for orthodontic root resorption, and the mean grade of all four incisors was calculated for analysis.

Table 1.

Modified Malmgren grading scale – Index for grading root resorption

Grade	Description
0	Absence of resorption
1	Irregularity in the apical root contour, maintaining the original root length
2	Resorption of up to 2 mm of the root length
3	Resorption from 2 mm up to 1/3 of the root length
4	Severe root resorption, above 1/3 of the root length

Additionally, the total duration of active orthodontic treatment (in months) was recorded for each participant, from the start of appliance placement to debonding. This variable was analysed to determine its correlation with the severity of root resorption, thereby addressing the secondary objective of the study.

Statistical Analysis: To minimise potential bias and maintain operator blinding during treatment, each participant was assigned a unique identification number. All data collection (GCF and radiographs) and assessments were performed using these coded identifiers. Outcome data were anonymised and coded prior to statistical analysis to reduce interpretation bias. The collected data were tabulated using Microsoft Excel. All statistical analyses were performed using IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY), with a significance threshold set at P < 0.05. The intra-class correlation coefficient (ICC) was used to assess the intra-examiner reliability and repeatability of the radiographic measurements. Normality of the data was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated using Levene’s test. Descriptive statistics were conducted to evaluate the mean and standard deviation of each group. Inferential statistics to find out the difference between the groups was done using Independent T-test, and within group analysis was done using Repeated Measures ANOVA comparing the multiple time-points. The Pearson Correlation Analysis and linear regression plots were used to assess the relationship of severity of orthodontically induced root resorption with CEMP-1 and duration of treatment.

RESULTS

Demographic characteristics: A total of 40 participants were included in the analysis, divided into two groups with equal gender distribution (Group 1 and Group 2). Participants ranged in age from 15 to 25 years, and the mean age of patients in Group 1 and Group 2 was 18.1 ± 2.13 and 18.2 ± 3.11 years, respectively [Table 2]. The independent-samples t-test revealed no statistically significant difference in the mean age between the two groups (P > 0.05), confirming age-matching between cohorts. The intra-class correlation coefficients (ICCs) for the recorded measurements ranged between 0.798 and 0.994, reflecting excellent intra-operator consistency and reliability in data acquisition. Shapiro–Wilk tests indicated that the variables were normally distributed (P > 0.05).

Table 2.

Comparison of variables and root resorption at different time-points

Variable Time-points		Mean±SD		P for inter-group comparison
		Group 1	Group 2
Age group	15-25	18.1±2.13	18.2±3.11	0.24†
Duration		17.25±1.29	19.55±0.83	0.00618*
CEMP-1 (ng/mL)	T1	1.8±1.27	1.82±1.15	0.96†
	T2	9.53±1.44	9.36±1.78	0.74†
	T3	370.97±49.7	436.23±43.92	0.000072*
	T4	346.58±53.82	482.3±43.92	<0.0001*
P for intragroup comparison		<0.0001*	<0.0001*
RVG-Grading	T1	0	0
	T3	1.2±0.32	1.35±0.43	0.22†
	T4	1.73±0.28	2±0.21	0.0014*
P for intragroup comparison		<0.0001*	<0.0001*
Root Resorption Severity	Not Severe (n) (Grade <2)	12	4	0.010*
	Severe (n) (Grade >2)	8	16

*Significant at P<0.05. ^†Not statistically significant

Analysis of CEMP-1: Descriptive analysis of CEMP-1 levels across four time points (T1 to T4) revealed distinct temporal patterns in both groups [Table 2]. CEMP-1 levels demonstrated a marked increase over time in both groups, with Group 1 showing means from 1.80 ± 1.27 ng/mL at T1 to 346.58 ± 53.82 ng/mL at T4, and Group 2 from 1.82 ± 1.15 ng/mL at T1 to 482.30 ± 43.92 ng/mL at T4. At T1 and T2, the CEMP-1 levels were not significantly different between the groups. There was a statistically significant increase in CEMP-1 levels in Group 2 compared to Group 1 (P < 0.0001) at both T3 and T4, suggesting greater cementum remodelling activity in response to more complex tooth movements such as intrusion [Table 2 and Figure 2].

Figure 2.

CEMP-1 at different time-points in Groups 1 and 2

Pearson correlation analysis between CEMP-1 and grading of orthodontically induced root resorption showed a strong positive correlation (r = 0.65), suggesting that higher levels of CEMP-1 are associated with increased root resorption [Figure 3].

Figure 3.

Regression plot for CEMP-1 and grading of root resorption

Comparison of grades of root resorption: Grading represents the severity or extent of root resorption. At T1, grading of root resorption was consistently zero across all participants, while T3 and T4 showed some variation between groups. The mean difference of grading of root resorption was higher in Group 2 at T4 (2 ± 0.21) compared to Group 1 (1.73 ± 0.28) [Table 2]. Post-treatment RVG at T4 revealed a statistically significant increase in root resorption in both groups (P < 0.05).

Describing orthodontic root resorption of less than 2 mm as Grade 2 and below (less severe) and resorption greater than 2 mm as Grades 3 and 4 (more severe) is consistent with the grading of orthodontic root resorption as shown in Table 1. Chi-square test was conducted to compare the distribution of grading of root resorption severity between Group 1 and Group 2. The results revealed a statistically significant difference between the groups (χ² = 6.67, df = 1, P = 0.010). Group 2 exhibited a higher proportion of severe resorption cases (80%) compared to Group 1 (40%). These findings suggest that participants in Group 2 were significantly more likely to experience greater root resorption, highlighting the potential impact of treatment mechanics or biological response associated with that group.

Duration: The box plot [Figure 4] shows Group 2 has significantly longer treatment duration (19.55±0.83 months) compared to Group 1 (17.25±1.29 months). The Pearson correlation analysis revealed a strong positive correlation which was statistically significant (r = 0.716, P < 0.001) between duration of treatment and severity of root resorption. The regression model explains 51% of the variance, with severity increasing by 0.087 units per unit increase in duration of treatment [Figure 5].

Figure 4.

Box plot depicting duration of treatment in Groups 1 and 2

Figure 5.

Regression plot for duration of treatment and orthodontic root resorption

DISCUSSION

This prospective cohort study investigated the association between gingival crevicular fluid (GCF) levels of Cementum Protein-1 (CEMP-1) and the severity of orthodontically induced root resorption (OIRR) in patients undergoing anterior retraction, with or without midline mini-implant-assisted intrusion.

Data collection was performed at four distinct time-points in both groups, each corresponding to critical phases of the orthodontic treatment process. These time-points were selected for their clinical relevance as they represent key milestones that reflect changes in biological response and treatment progression.

Despite advancements in techniques and materials, the occurrence and severity of root resorption remain unpredictable, posing challenges in managing vertical maxillary excess and gummy smiles.[18] Intrusion is considered a potential trigger for root resorption among the different types of tooth movements.[19] Maxillary incisors were chosen due to their susceptibility to resorption and the ease of imaging.[9,10,20,21,22] Burstone CR[23] proposed an optimum force of 20 g for intruding anterior teeth, while Gianelly AA and Goldman HM[24] recommended a range of 15–50 g for smaller teeth. In the present study, elastomeric chains were used to deliver 60 g of intrusive force, equating to approximately 10–15 g of force per tooth.[15]

As defined by Taba M et al.,[25] a biomarker is a measurable substance that objectively reflects normal biological processes, pathological changes, or responses to therapeutic interventions. Recently, there has been increasing interest in identifying biomarkers linked to orthodontic root resorption to improve its early detection, monitoring, and management. CEMP-1, a cementoblast-derived protein, plays a role in cementogenesis and has been proposed as a biomarker for root remodelling.[26,27] Our findings align with prior studies which reported elevated expression of CEMP-1 in response to orthodontic forces and during periods of active root remodelling.[9] The rise in CEMP-1 concentrations suggests active tissue response to root surface injury and potential reparative activity during and after mechanical stress.

Descriptive and repeated measures ANOVA analyses revealed significant intra-group changes in CEMP-1 levels across time points, with Group 2 (mini-implant) showing higher levels at T4. This trend aligned with greater root resorption in Group 2, suggesting that CEMP-1 plays an active role in remodelling. At T3, the CEMP-1 levels increased in both groups, but at T4, CEMP-1 levels reduced in Group 1 but increased in Group 2. A possible explanation may be the continuing root resorption in Group 2 patients who were undergoing simultaneous intrusion and retraction. Pearson correlation analysis confirmed a positive association between CEMP-1 levels and resorption severity.

The most popular radiographic test for identifying resorption is the periapical x-ray. Compared to panoramic and cephalometric radiographs, Sameshima GT and Asgarifar KO state that periapical radiographs have less distortion and overlap and display finer detail, making it possible to see anatomical details like the cementoenamel junction.[22,28] Advanced imaging techniques such as micro-CT and CBCT offer superior accuracy for three-dimensional assessment of root resorption.[29] The use of two-dimensional RVG instead of cone-beam computed tomography (CBCT) in this study is justified based on practical considerations such as reduced radiation exposure, lower cost, and greater clinical accessibility. However, it is important to acknowledge the inherent limitations of RVG, primarily its lower diagnostic sensitivity due to the superimposition of anatomical structures in two-dimensional imaging.

Grading the root resorption [Table 2] revealed that there were no significant differences in baseline scores of RVG between the two groups. There were significant intra- and inter-group changes at T3 and T4, with Group 2 showing higher severity, supporting the role of additional intrusive force in exacerbating OIRR.[14] This observation aligns with previous research by Linge BO and Linge L,[30,31] which suggests that intrusion may induce greater root resorption compared to overall orthodontic treatment.

Chi-square analysis showed 80% of Group 2 patients exhibited severe resorption (Grade >2), compared to 40% in Group 1. This stark difference highlights the influence of treatment mechanics, particularly the magnitude and direction of orthodontic forces, on the biological response of the periodontal ligament and root surface. Prior studies have similarly linked intrusive forces, particularly with skeletal anchorage, to higher OIRR risk. For instance, Dermaut LR and De Munck A[5] demonstrated increased resorption with controlled intrusion using heavier forces, while Zhou Y et al.[32] observed that simultaneous intrusion and retraction increased apical resorption compared to retraction alone.

The greater resorption in Group 2 likely stems from the combined intrusion–retraction forces, which concentrate stress at the apical third of the root—an area susceptible to hyalinisation and osteoclastic activity. Chan and Darendeliler[28] confirmed that intrusive forces cause more OIRR than tipping or bodily movement. Additionally, Apajalahti et al.[33] also noted greater apical damage with vertical control devices like mini-implants. Additionally, extended force duration in Group 2 may have intensified root damage, consistent with findings from Smale I et al.,[34] regarding significant incisor movement and thin cortical bone. Harris and Baker[20] emphasised age-related variability in root resorption risk during orthodontic treatment, highlighting the importance of individualised treatment planning and monitoring. This emphasises the importance of monitoring root resorption during orthodontic interventions. Contrary to this, Parayaruthottam P and Antony V[35] detailed the successful intrusion of maxillary incisors without adverse effects on the periodontium or dentition by employing meticulous orthodontic techniques.

Conversely, Group 1 experienced less severe resorption, supporting the role of force direction and magnitude in tissue response. Individual factors such as root morphology and bone density also likely influenced the extent of resorption, emphasizing the need for personalised treatment planning and ongoing monitoring.

A notable observation in this study is the significant association between treatment duration and root resorption severity. Patients who underwent longer treatment exhibited greater radiographic root shortening. This is supported by the findings of Sameshima and Sinclair,[36] who identified treatment duration as one of the most consistent risk factors for OIRR in their large-scale retrospective study. Similarly, Levander and Malmgren[17] emphasised the importance of minimising treatment time to reduce the cumulative exposure of dental and periodontal tissues to orthodontic forces. These findings support the hypothesis that prolonged exposure to orthodontic forces contributes to the risk of root structure loss. However, additional factors such as force magnitude, direction of movement, and individual biological variability must also be considered in future analyses.

Clinical Implications and Future Directions: Elevated CEMP-1 levels in cases with skeletal anchorage suggest its potential as a non-invasive biomarker for monitoring resorption. Pre-treatment risk assessment and periodic imaging combined with biochemical markers could help customise force application and prevent irreversible damage.

Limitations and future scope: The study’s modest sample size and use of 2D imaging may limit the generalisability and sensitivity of results. Future research should incorporate broader biomarker panels and advanced imaging for a more comprehensive evaluation of resorptive processes.

CONCLUSION

This prospective cohort study highlights a significant association between the progressive increase in Cementum Protein-1 (CEMP-1) levels in gingival crevicular fluid (GCF) and the severity of orthodontically-induced root resorption (OIRR), as confirmed through radiographic assessment. Notably, patients who underwent simultaneous intrusion and retraction with midline mini-implants exhibited higher levels of CEMP-1 and greater degrees of root resorption compared to those treated with conventional retraction mechanics. These findings suggest that intrusive forces, particularly when combined with skeletal anchorage, place increased biomechanical stress on the apical region of the roots, thereby elevating the risk of resorptive activity.

In addition, the study demonstrates a positive correlation between the duration of orthodontic treatment and the extent of root resorption, reinforcing the importance of minimising treatment time and applying optimal, biologically compatible forces. Prolonged orthodontic treatment appears to sustain inflammatory and remodelling responses within the periodontal ligament, leading to more pronounced tissue damage over time.

The consistent pattern of elevated CEMP-1 levels alongside radiographic evidence of resorption positions this protein as a promising, non-invasive biomarker for early detection and monitoring of OIRR. Integrating biomarker analysis into orthodontic practice may enable clinicians to identify patients at higher risk of root resorption, tailor biomechanical strategies accordingly, and intervene proactively to preserve dental and periodontal health.

Overall, this study highlights the need for a biologically informed approach to orthodontic treatment planning, combining precise force application with biomarker surveillance to enhance both treatment efficacy and patient safety. This approach enhances patient safety, improves treatment outcomes, and embodies the principles of precision medicine in orthodontics.[37]

Patient release

Images obtained does not contain identifiable patients or human subjects.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was supported by the Indian Orthodontic Society-Research Fund 2022-23 (IOS-RF-LM).