Non-Destructive 3D Quantitative Analysis of Residual Periodontal Ligament on Extracted Human Premolars Using Intraoral Scanning

Summary

This protocol details a non-destructive, quantitative 3D digital method for evaluating residual periodontal ligament on extracted teeth. Using intraoral scanning and reverse-engineering software, we established an ex vivo workflow for periodontal assessment, providing a methodological basis for comparing extraction techniques in preclinical research.

Abstract

Intentional tooth replantation is a valuable treatment for preserving natural teeth with endodontic lesions. Its success critically depends on the viability and structural integrity of the residual periodontal ligament (PDL). Traditional evaluation methods, such as histological sectioning or two-dimensional photography, are either destructive or limited by projection errors. To address these limitations, this protocol presents a non-destructive, quantitative method for evaluating residual PDL using high-precision intraoral scanning. Extracted teeth were stained to visualize the PDL tissue and three-dimensionally scanned to generate high-fidelity digital models. The data were analyzed using reverse engineering software, where a region of interest was defined, and residual PDL coverage was quantified via color thresholding. This approach calculated the true 3D coverage area of the PDL without compromising the biological sample. To evaluate the sensitivity and utility of this protocol, it was applied to compare minimally invasive (MI) extractions using periotomes against conventional forceps extractions. Digital analysis revealed that the MI method preserved significantly more PDL tissue (61.99%) than the conventional method (50.46%). Furthermore, a subsequent cell viability assay corroborated the digital findings, demonstrating significantly higher metabolic activity in the MI group. Ultimately, this work establishes a proof-of-concept workflow for ex vivo quantitative PDL assessment, providing a robust methodological basis for comparing extraction techniques in preclinical research.

Introduction

Intentional tooth replantation is increasingly recognized as a viable, last-resort treatment modality for preserving natural teeth that would otherwise be deemed hopeless. It is indicated for managing persistent periapical lesions, inaccessible canal perforations, or extensive resorptive lesions that cannot be managed non-surgically^1,2. The standard procedure involves the intentional extraction of the affected tooth, followed by extraoral visual inspection, therapeutic intervention (including root-end resection, preparation, and root-end filling), and subsequent replantation into the original socket^1,2.

The presence and integrity of viable residual periodontal ligament (PDL) tissue on the root surface are the most critical prognostic factors determining the pattern of healing following tooth replantation^3,4. A healthy, intact PDL barrier is essential for re-establishing physiological mobility and preventing complications such as replacement resorption (ankylosis) or inflammatory root resorption. However, the avulsion of a tooth from the alveolar socket, whether traumatic or intentional, inherently disrupts the PDL fiber attachment and severs the neurovascular supply⁵. Consequently, the biological reconstruction of the PDL is crucial for the periodontal healing of replanted teeth³. Extensive research has established that maximizing the vitality and volume of residual PDL tissue during extraoral clinical procedures constitutes a critical prognostic factor, and its importance for the long-term functional survival of replanted teeth is well documented in the literature^6,7.

Given that extraction inherently disrupts PDL attachment, the choice of extraction technique directly influences the volume and viability of residual PDL tissue remaining on the root surface. Conventional extraction with forceps applies rotational and traction forces that can crush the PDL against the alveolar bone or strip it through friction. To minimize this trauma, minimally invasive (MI) extraction methods have been developed. Previous studies have reported that MI methods can significantly reduce surgical trauma to the periodontium of the affected teeth⁸. Techniques utilizing periotomes to sever the PDL attachment, or vertical extraction systems that avoid leverage forces, have been developed to preserve the residual PDL on the root surface. For example, case reports have documented improved clinical outcomes in intentional tooth replantation and surgical extrusion when MI systems were employed^9,10. These findings suggest that minimizing extraction traumas may improve the retention of residual PDL tissue on the root surface.

However, despite clinical consensus on the benefits of atraumatic extraction, there is limited direct evidence quantifying the extent of residual PDL tissue preserved when using MI methods compared with conventional techniques. This gap in knowledge stems largely from the limitations of available evaluation methodologies. Existing research predominantly employs subjective or destructive evaluation methods to assess the residual PDL on the root surface. Histological sectioning remains the standard for cellular analysis¹¹, but it is inherently destructive, rendering the sample unusable for further studies. Alternatively, staining combined with two-dimensional (2D) photographing has been used to characterize residual PDL¹². However, these methods are inadequate for quantifying residual PDL tissue of the entire root surface. A 2D photograph cannot accurately map the surface area of a complex, curved three-dimensional root; it inevitably introduces projection errors and distortion, leading to inaccurate quantifications.

In recent years, intraoral digital scanning technology has advanced significantly, offering high precision and rapid data acquisition. The digital method has been employed in previous studies to measure impressions and soft-tissue changes, or to assess tooth wear in young individuals¹³. Applications have even expanded to complex volumetric analyses, such as evaluating facial swelling in oral surgery¹⁴. These technologies allow for the creation of high-fidelity three-dimensional (3D) models that can be manipulated and measured in reverse engineering software, offering a potential solution for non-destructive, quantitative analysis. However, to date, the potential application of intraoral scanning for assessing the residual PDL of extracted teeth has not yet been explored.

The overarching goal of this protocol is to establish a standardized, non-destructive workflow that combines biological staining with high-precision intraoral scanning and 3D reverse engineering. By creating high-fidelity digital models with color-textured surface maps, this method allows for more objective, reproducible measurement of residual PDL across the entire root surface. This study applies the proposed workflow to compare the residual PDL coverage of two extraction techniques, periotome-assisted MI extraction versus conventional forceps extraction, in human premolars. It is hypothesized that the MI method preserves a greater proportion of residual PDL coverage than the conventional method.

Importantly, this protocol is designed strictly as an ex vivo research tool for laboratory or preclinical settings. The procedure involves staining, air-drying, digital scanning, and software-based segmentation, with a total analysis time that is incompatible with intraoperative use during intentional replantation, where extraoral time must be minimized. Consequently, this method is appropriate for (i) comparing the PDL-preserving efficacy of different extraction techniques under controlled conditions; (ii) training and calibration in educational settings; and (iii) validation studies linking digital PDL coverage metrics to biological outcomes. Its applicability to multi-rooted teeth with complex anatomies requires further investigation.

Protocol

The research protocol was performed in compliance with the guidelines of the Ethics Committee of the Peking University School of Stomatology (PKUSSIRB-202054022).

1. Patient selection and group allocation

1. Select patients requiring extraction of premolars for orthodontic purposes. Ensure premolars meet the inclusion criteria with the absence of caries or periodontal disease.
2. Exclude patients with high blood pressure, diabetes, history of smoking, or those aged less than 16 years or greater than 45 years.
3. Randomly allocate the premolars into two experimental groups: the Control Group (conventional extraction) and the Minimally Invasive (MI) Group (periotome-assisted extraction).
4. Randomly assign the teeth from each group to either the 3D scanning analysis pathway or the biological validation (CCK-8) pathway, ensuring independent samples are used for each evaluation method.

2. Tooth extraction procedures

1. Perform all extractions under standard local anesthesia.
2. Method A: Conventional extraction (Control group)
   1. Perform gingival separation around the tooth neck.
   2. Position dental forceps at the cervical area of the tooth crown.
   3. Apply rotational forces until the tooth is loosened. Remove the tooth from the alveolar socket using the forceps.
3. Method B: Minimally invasive extraction (MI group)
   1. Perform gingival separation.
      CAUTION: Handle the periotome carefully to avoid soft tissue injury or instrument breakage.
   2. Insert a periotome into the PDL space as deep as possible, reaching at least two-thirds of the root length.
   3. Circumferentially sever the PDL fibers around the root using the periotome. Loosen the tooth.
   4. Vertically pull the tooth out of the alveolar socket using dental forceps.

3. Sample preparation and staining

1. Immediately upon extraction, rinse the teeth with sterile saline for 10 min to remove blood and debris.
   CAUTION: Toluidine blue is a hazardous chemical. Wear appropriate personal protective equipment (gloves, goggles) when handling.
2. Stain the root surface by immersing the tooth in a 0.1% toluidine blue solution (prepared in phosphate-buffered saline, PBS) for 40 min to visualize the PDL tissue.
3. Rinse with saline for 10 min to remove the dye solution and gently blow-dry the surface with a three-way syringe to remove excess moisture.
   NOTE: The root surface is expected to exhibit a uniform blue-purple coloration, while the exposed cementum should remain unstained, appearing light beige.

4. 3D digital Scanning

1. Calibrate the intraoral digital scanner according to the manufacturer's instructions to ensure an accuracy of 0.02 mm.
2. Secure the tooth crown using hemostatic forceps to avoid obstructing the root surface view.
3. Scan the entire root surface under standard scanning conditions. Perform a complementary scan of the crown to complete the 3D model.
4. Use the scanner's proprietary software to export the digital scan data as a high-resolution 3D object (OBJ) file and associated data package; the texture image is saved in JPG format.
   NOTE: The live scan preview must display a continuous, high-density mesh devoid of any holes or artifacts on the root surface. The texture image should clearly distinguish the blue-stained regions from the unstained root surface.

5. 3D model reconstruction and quantitative analysis

1. Image pre-processing
   1. Threshold calibration
      1. Import the JPG texture map into image editing software. Convert the image to 8-bit grayscale.
      2. Open the Threshold tool (Image > Adjust > Threshold) and inspect the histogram to record the minimum grayscale value capturing all blue-stained PDL pixels. Determine the minimum grayscale threshold using three independent observers.
   2. Import the original JPG texture map into image editing software. Navigate to Select > Color Range. Choose Shadows mode and set Range to 65 and Tolerance to 20% (calibrated from the 5.1.1 threshold data).
      NOTE: Verify that all blue-stained PDL regions are fully selected and that no unstained root surface is included.
   3. Copy the selected, blue-stained regions and paste them onto a new white layer to generate a binary mask (blue for PDL, white for bare root). Save the processed texture image to overwrite the original JPG file.
2. Quantitative measurement
   1. Import the processed 3D model into reverse engineering software.
   2. Use Polygons > Trim to define the Region of Interest (ROI) to exclude gingival and periapical tissues. Set the upper boundary of the ROI at 2 mm below the cementoenamel junction (CEJ). Exclude the apical 3 mm from the analysis.
   3. Select the "enhanced blue regions" within the ROI. After selection, the residual PDL tissue is accentuated in red.
   4. Navigate to Analysis > Compute > Compute Area to calculate the total surface area of the ROI and the surface area of the blue regions (residual PDL).
   5. Calculate the percentage of PDL coverage using Equation 1:
      ​PDL Coverage (%) = (Area of Residual PDL/Total Area of ROI) x 100
3. Visualization
   1. Capture images of the four root surfaces (buccal, lingual, mesial, and distal) perpendicular to the tooth axis for detailed visual assessment.

6. Biological validation (Cell viability assay)

NOTE: This section describes the validation steps performed on a subset of samples (n = 8 per group) to corroborate the digital analysis.

1. Immediately after extraction, place the teeth in a sterile centrifuge tube containing pre-warmed (37 °C) culture medium.
2. Transport samples to the laboratory in an ice box at 4°C within 1 h. Use Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin for sample transport and cell viability testing.
3. Rinse the teeth with sterile phosphate-buffered saline (PBS) in a biosafety cabinet immediately upon arrival.
4. Carefully scrape the PDL tissue from the middle third of the root (ROI is defined by graduated periodontal probe) using a sterile scalpel blade at room temperature within 10 min of arrival to maximize cell viability.
5. Digest the collected tissue with 0.25% trypsin-EDTA for 30 min. Centrifuge the cell suspension at 120 × g for 5 min at room temperature (22–25 °C) to obtain the cell-containing precipitate.
   NOTE: Following centrifugation, a discernible white pellet should form at the bottom of the tube. If no pellet is observed, the sample should be re-centrifuged at 120 × g for an additional 1 min.
6. Resuspend the precipitate in a mixture of 600 µL Cell Counting Kit-8 (CCK-8) solution and medium at a 1:10 ratio (v/v), prepared according to the manufacturer's instructions.
7. Aliquot this suspension at 200 µL per well into a 96-well plate (3 technical replicates per sample). Incubate the plate at 37°C in a humidified incubator with 5% CO₂ for 3 h.
8. Transfer 50 µL of the supernatant from each well to a new 96-well plate.
9. Measure the absorbance (Optical Density, OD) at 450 nm using a microplate reader. Record the mean OD values for statistical comparison.
   CAUTION: (i) Collect Toluidine blue staining solution and rinse waste as hazardous chemical waste and dispose of them according to institutional chemical safety guidelines; (ii) Treat PDL tissue, used scalpels, and cell culture waste as biological hazardous waste (Biosafety Level 1) and autoclave or incinerate them per institutional biosafety protocols; (iii) Discard PPE (gloves, pipette tips, paper towels) in designated biohazard containers.

7. Statistical analysis and methodological validation

1. Data collection: Compile the quantitative data, including the total root surface area (mm²), total stained area (mm²) from the 3D analysis, PDL coverage percentage (%), and the OD values from the biological assay.
2. Normality test: Assess the data distribution for normality using the Shapiro-Wilk test.
3. Group comparison:
   1. Compare the PDL coverage and OD values between the MI and Control groups.
   2. Use an unpaired Student’s t-test for normally distributed data or a Mann-Whitney U test for non-normally distributed data.
   3. Calculate the effect size (Cohen’s d) to determine the magnitude of the difference.
4. Methodological robustness analysis:
   1. Perform a linear regression analysis with "Root Surface Area" as the independent variable and "PDL Coverage" as the dependent variable.
   2. Calculate the Pearson correlation coefficient (r) to assess if the measurement method is biased by tooth size. A non-significant correlation (P > 0.05) indicates robustness.
5. Reliability analysis:
   1. Generate violin plots combined with box plots to visualize the probability density and distribution characteristics of the data.
   2. Compare the shape and concentration of the data distribution between groups.
6. Intra-observer and inter-observer reliability testing:
   1. Randomly select 16 samples from the total dataset (8 per group) for reliability assessment.
   2. For intra-observer repeatability, repeat the full segmentation and measurement procedure on the same 16 samples at one-week intervals for a total of two trials.
   3. For inter-observer agreement, provide standardized training to two additional independent observers on the segmentation protocol.
   4. Have all three observers independently perform ROI definition, color thresholding, and area computation on the same 16 samples.
   5. Calculate Intraclass Correlation Coefficient ICC(A,1) with 95% confidence intervals to quantify absolute agreement for both intra-observer and inter-observer comparisons. Report ICC values >0.90 as excellent, 0.75–0.90 as good, 0.50–0.75 as moderate, and <0.50 as poor.
   6. Perform Bland-Altman analysis for all pairwise comparisons. Calculate the mean difference (bias) and 95% limits of agreement (mean difference +/- 1.96 SD). A non-significant paired t-test result (P > 0.05) indicates negligible systematic bias.
7. Software: Perform all statistical analyses using appropriate software. Consider P < 0.05 as statistically significant.

Results

The experimental workflow established in this study successfully demonstrated a non-destructive protocol for the quantitative assessment of residual PDL on extracted teeth. As outlined in the experimental design (Figure 1), the protocol integrated clinical extraction procedures with digital acquisition and biological validation. The application of intraoral digital scanning, followed by 3D reverse engineering, enabled precise reconstruction of the root surface (Figure 2<...

Discussion

The success of intentional replantation hinges critically on the preservation of viable PDL cells on the root surface. While MI extraction methods have been clinically advocated to reduce trauma^8,9,10, quantifying their biological benefit on residual PDL tissue has remained a challenge due to the lack of objective evaluation tools. This study established a proof-of-concept, non-destructive 3D digital analysis protocol to quantif...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well plate	Servicbio	CCP-96H	Multi-well plate for cell culture and absorbance measurement
Biosafety cabinet	Thermo Fisher Scientific	1300 Series4 A2	Class II biological safety cabinet for sterile procedures
Cell Counting Kit-8 (CCK-8) solution	MedChemExpress	HY-K0301	Cell viability assay reagent
Centrifuge	Ustc Zonkia Zcientific Instruments	SC-3610	Laboratory centrifuge for cell precipitation
Culture medium	Gibco	DMEM 11965	Basal medium for cell transport and incubation
Dental forceps	Jinzhong Surgical instruments	K0F130/K0F100	Standard dental extraction instrument
Fetal bovine serum	Anhui Kangyuan Biotechnology Co., Ltd	KY-01003S	Fetal bovine serumsupplement for culture medium
Graduated periodontal probe	Jinzhong Surgical instruments	K6F040	Assist in scraping the PDL from the  middle third of the root
Hemostatic forceps	Jinzhong Surgical instruments	J31130	Surgical tool used to secure the tooth during scanning
Humidified incubator	Thermo Fisher Scientific	Forma	Cell culture incubator maintained at 37°C with 5% CO2
Image editing software	Adobe Systems Inc.	Adobe Photoshop CC 2018	Software for color selection
Image editing software	Wayne Rasband, National Institutes of Health	ImageJ2 v2.14.0	Software for color threshold
Intraoral digital scanner	Aidite (Qinhuangdao) Technology Co., Ltd.	Aidite Cameo	Optical scanner for 3D digital model acquisition
Local anesthesia	Produits Dentaires Pierre Rolland	Primacaine Articaine	Dental anesthetic agent (e.g., Articaine or Lidocaine)
Microplate reader	Biotek	Elx808	Spectrophotometer for measuring OD at 450 nm
Penicillin/Streptomycin	Gibco	15070063	Antibiotic supplement for culture medium
Periotome	Carl Martin	LS529PT	Minimally invasive extraction instrument
Reverse engineering software	Geomagic Inc.	Geomagic Studio 2013	3D modeling software for surface area calculation
Sterile centrifuge tube	Servicbio	EP-1500-J	Plastic tube for sample collection and processing
Sterile phosphate-buffered saline (PBS)	Gibco	10010	Washing buffer for biological samples
Sterile scalpel blade	Jiayuan Medical Devices	Sterile Scalpel Blade	Blade for PDL tissue scraping
Sterile sline	Servicbio	G4702-500ML	Saline solution for rinsing extracted teeth
Toluidine blue	General laboratory supplier	Toluidine blue (0.1% solution)	Biological stain for periodontal ligament visualization
Trypsin	Gibco	25200056	Enzymatic reagent for tissue digestion