Pulp Chamber

Based on the oral findings of the examined participants, the risk of potential EP infections with oral origin was evaluated based on the presence of treatment need and/or oral foci, respectively. Therefore, three risk classes were defined: (A) The low-risk group reflected no dental or periodontal treatment need. (B) The moderate risk group included patients with dental and/or periodontal treatment need, but no potential oral foci. (C) The high-risk group (patients “at-risk” for EP infection with oral origin) consisted of patients with a potential oral focus for EP infection, including caries, touching the pulp chamber, severe periodontal treatment need (e.g., suppuration, endo-perio-lesion), apical radiolucencies (sign of chronic infection/inflammation), (partly) retained teeth with pericoronal inflammation, inflammation in jawbone or additional inflammatory findings. These findings were reported as potential oral foci in various patient groups before and were adopted for the current study [25 (link),26 (link),27 (link),28 (link)]. Those patients were referred to their family dentist/a special clinic for dental treatment, which was mandatory perquisite for EP insertion in those patients (Table 1). If the dental treatment was not possible until the time point two weeks prior to EP surgery, the EP insertion was deferred accordingly.
Within the cohort of the current study, the occurrence of early infectious complications in the first 3 months after EP insertion was recorded.

Two trained pediatric dentists (K.M., K.G.) participated in the data collection process. The calibration exercise was carried out by an oral and maxillofacial radiologist (K.C.) who regularly conducts hands-on workshops on CBCT assessment and interpretation. Prior to the start of the study, the program consisted of theoretical discussions followed by practical sessions on the evaluation of CBCT images. To check the diagnostic reproducibility of the inter-reliability of the investigators, 10 CBCT images were examined independently by the two aforementioned pediatric dentists. To ensure consistency in measurements, inter-examiner variability was assessed prior to and at the end of the data collection period. The weighted Cohen’s kappa value was 0.893 at baseline and 0.931 at the end of the study, which reflected a high degree of conformity in the examination. Any disagreement between the examiners was arbitrated by the subject expert (K.C.) to reach a consensus.
To ensure image standardization, all CBCT images were chosen from a single machine (Planmeca ProMax
^® 3D Mid) with a standard field of view = 80 mm × 80 mm; voxel size of 0.40 mm; 90 kV and 12 mA; exposure time of 12 s; and slice thickness of 0.4 mm. CBCT images were analyzed with the built-in Romexis
^® digital imaging software, version 3.5.2 (Planmeca, Helsinki, Finland), on a 15.6-inch Samsung LCD screen with an Intel
^® Core
^TM i3 2.4 GHz processor, and 500 GB of memory at a resolution of 1280×1024 pixels in a dark room. The observers evaluated the teeth using the Planmeca Romexis
^® toolbar, by carefully scrolling down through the images from the floor of the pulp chamber in all three orthogonal reconstructions (axial, coronal, and sagittal). The measurement tool was used to determine the total length of the crown of the primary second molar, measured from the tip of the mesiobuccal cusp to the cemento-enamel junction. Based on this length, the crown portion was divided into three levels: coronal, middle and apical thirds. Next, the shapes of the contact areas between the maxillary and mandibular primary molars were examined at various levels, coronal, middle, and apical, and were scored in all three sections (axial, coronal, and sagittal) according to the criteria shown in
Figure 1. Depending on the maximum score among the three levels (the coronal, middle, and apical thirds), the overall score for a particular tooth was assigned. For example, if the scoring of the right maxillary contact between two primary molars was 2 at the coronal third (I shape), 1 at the middle third (X shape), and 0 at the apical third (O shape), then the overall score of this tooth would be the maximum number (that is, 2).

Samples were prepared and extracted in the paleogenetics clean room at the Institute for Archaeological Sciences, University of Tübingen (INA). The surface of the dedicated sampling hood was cleaned with HPLC water and UV irradiated by an internal light source between uses. Any calculus was removed from the surfaces of the teeth using dental scalers that had been rinsed with bleach and HPLC water and UV irradiated for 10 minutes between uses. Large calculus samples were pulverized with a tube pestle. Teeth were then sectioned horizontally at the cementoenamel junction and dentin was drilled from the pulp chamber using a dental drill. For calculus samples weighing over 20 mg, half the pulverized material was carried over for extraction. For dentin samples over 70 mg, aliquots of approximately 50 mg were taken for extraction. Dentin and calculus samples were extracted using a previously described silica-based method^{75 (link)}. In brief, samples were submerged in a digestion buffer with final concentrations of 0.45 M EDTA and 0.25 mg/mL proteinase K (Qiagen, the Netherlands) and rotated at 37 °C until decalcified. After incubation, samples were centrifuged and the supernatant was purified using a 5 M guanidine-hydrochloride binding buffer with High Pure Viral Nucleic Acid Large Volume kits (Roche, Switzerland). The extracts were eluted in 100 μl of a 10 mM tris-hydrochloride, 1 mM EDTA (pH 8.0), and 0.005% tween-20 buffer (TET). One extraction blank was prepared for every ten samples, and one positive control of cave bear bone powder was processed alongside each extraction batch to ensure efficiency. The extracts were quantified using a Qubit High Sensitivity fluorometer (Life Technologies) (Supplementary Table S1).

The micro-CT data sets were then transferred to the Mimics 21.0 (Materialise, Leuven, Belgium) software to perform 3D reconstruction of the teeth and root canal systems. The root canal configurations in the mandibular incisors were examined and described by the Vertucci’s classification [4 (link)]: Type I: a single canal is present from the pulp chamber to the apex (type 1–1). Type II: two separate canals leave the pulp chamber but join to form one canal short of the apex (type 2–1). Type III: one canal leaves the pulp chamber and divides into two within the root, but they merge again to exit as one canal (type 1–2-1). Type IV: two separate and distinct canals are present from the pulp chamber to the apex (type 2–2). Type V: one canal leaves the pulp chamber which divides into two separate and distinct canals with separate apical foramina (type 1–2). Type VI: two separate canals join within the root to form one canal, which divides into two distinct canals again short of the apex (type 2–1-2). Type VII: one canal leaves the pulp chamber, divides and rejoins within the root body, and finally redivides into two distinct canals short of the apex (type 1–2-1–2). Type VIII: three separate and distinct canals are present from the pulp chamber to the apex (type 3–3).
The type and number of the accessory canals were also determined.
The calibration was performed by an expert endodontist (Yongchun Gu) and an observer (Ying Tang). In the pilot study, the observer was trained and calibrated to read the micro-CT images with a sample size of 20 (10 single- and 10 double-canaled incisors) that did not belong to the study sample. The observer evaluated the micro-CT images using sagittal, axial, and coronal views and digital 3D tooth models to identify the root canal morphology, and each tooth received a single score. Disagreements were discussed, until a consensus was reached after adequate deliberation.
The inter- and intra-observer errors was evaluated according to Cohen's kappa test. Each observer evaluated the same 20 teeth twice independently with an interval of two weeks. Substantial Kappa values were obtained (the intra-observer kappa value was 1.0 for both observers [Yongchun Gu and Ying Tang], and the inter-observer kappa value was 0.9, all p = 0.000), suggesting the inter- and intra-observer agreement were both excellent.

The current study was carried out in accordance with the Consolidated Standards of Reporting Trials (CONSORT) group’s standards for clinical trial planning and reporting (Figure 1). The study included children of both genders and ages ranging from 4 to 9 years, who needed pulpectomy in their primary mandibular molars (first “D” and second “E”). A total of 110 patients were enrolled after acquiring informed consent from the accompanying parent. Prior to performing the pulpectomy, a four-point pain scale was employed to quantify the pre-instrumentation pain score [26 (link)]. The following four-point scale was used to assess pain: (1) zero—no pain, (2) one—slight pain, (3) two—moderate pain, and (4) three—severe pain (Figure 2) as reported by Topçuoğlu et al. [27 (link)]. The investigation involved radiographic examinations performed prior to treatment that revealed no interradicular radiolucency or periapical lesions. In addition, only teeth with three root canals following access cavity preparation were included in the trial to standardize the groups.
Acute apical periodontitis, necrotic pulp, more than three root canals, and individuals with abscess were disqualified. Patients who took medications up to six hours before the treatment were also barred from participating in the study. Children with a lack of cooperative capacity, those with a systemic ailment, or those with particular healthcare needs were also excluded from the study. A total of 8 individuals were disqualified because they did not match the inclusion criteria.
The block randomization strategy was employed to divide the individuals n = 31, 34, and 37 for XP-endo Shaper, Kedo-SG Blue, and hand file groups, respectively. A statistician created the randomized sequence, and the allocation was hidden by using opaque envelopes. The treatment technique was kept undisclosed from the patients and their parents, and the evaluator who recorded the instrumentation time was likewise blinded. The operator (B.T.) was delivering the therapy, hence the operator could not have been rendered blind.
The pulpectomy treatment was executed by the same operator (B.T.) and completed in a single visit. Non-pharmacological behavior management approaches were employed to change the child’s attitude and seek cooperation. Local anesthetic (2% lignocaine and 1:200,000 adrenaline; LIGNOX, Indoco Remedies Limited, Mumbai, India) inferior alveolar (I.A.) nerve block that enables a reasonably long-lasting anesthesia was performed, proceeded by rubber dam isolation of the tooth (CricDental Rubber Dam Kit, Mumbai, India). This was continued by caries removal and access cavity creation utilizing a high-speed no.4 round diamond point (Dentsply Maillefer, Tulsa, OK, USA). The root canal orifices were then recognized using the DG-16 explorer (Hu-Friedy, Chicago, IL, USA) only after de-roofing the entire pulp chamber. For standardized groups, teeth with only 3 root canal orifices were included in the current clinical trial. The root canal patency was checked for all the canals located using a size #10 (0.02%) k-file (NiTi flex; Dentsply Sirona, Charlotte, NC, USA) followed by estimation of the working length (WL) as 1 mm short of the apex using an electronic apex locator (ProPex Pixi; Dentsply Sirona, Charlotte, NC, USA) and the root canal instrumentation was initiated.
The randomization procedure was used to select the kind of instrumentation for the specific tooth. The adaptive XP-endo Shaper (tip size #30; FKG Dentaire, La Chaux-de-Fonds, Switzerland) was used for root canal instrumentation in Group 1; for Groups 2 and 3, Kedo-SG Blue pediatric rotary files (E1 tip size #0.30; Reeganz Dental Care Private Limited, Chennai, India) and a hand K-file (Dentsply Maillefer, Tulsa, OK, USA) up to #30 were used, respectively. The rotary files were powered with the aid of X-smart Plus endo-motor (Dentsply Sirona, Charlotte, NC, USA) according to the manufacturer’s instructions. The XP-endo Shaper was operated at 800 rpm and 1 Ncm torque, while the Kedo-S file was used at 250 rpm and 2.2 Ncm torque.

After the cavity filling, the roots of each tooth were removed 3 mm below the enamel–cementum junction with a diamond disc (Disc DS022, Clinique, manufacturer information is not provided), under continuous water cooling.
Then, the pulp tissue was removed using small excavators, and a 17% EDTA solution (Endo-Solution, Cerkamed, Stalowa Wola, Poland) was applied to the pulp chamber for 1 min. Then the pulp chamber was thoroughly rinsed with distilled water.
Each tooth fragment was fixed using a cyanoacrylate adhesive (Loctite, Westlake, OH, USA) to the inner face of the metal cap of a glass container. Through the cap, a 3 mm high syringe needle was inserted into the pulp chamber. The needle was connected to a perfusor coupled to a 100 mL saline solution vial. The vial was fixed at a height of 20 cm to produce a hydrostatic pressure of 20 cm H₂O in the pulp chamber [29 ,30 (link)].