Assessment of Pulpal Status in Primary Teeth Following Direct Pulp Capping in an Experimental Canine Model

(1) Background: This study aimed to assess the pulpal response of primary teeth by pulse-oximetry (PO) in a canine model, following direct pulp capping (DPC). (2) Methods: Forty-eight primary teeth from eight canine subjects were divided into three treatment groups, based on the DPC material—calcium hydroxide (CH), MTA, BiodentineTM)—and three corresponding control groups. Data from PO pulp testing were correlated with laser Doppler flowmetry (LDF) testing, computer tomographic (CT) densitometry and histological analysis; the experiment lasted 14 days. (3) Results: SpO₂ recordings revealed statistically significant differences (p = 0.002, <0.05) between the treatment and control groups, and no significant differences (p = 0.257, >0.05) were observed between treatment groups. LDF recordings showed significant differences (p = 0.002, <0.05) between the treatment and control groups and identified significant differences between materials (p = 0.001, <0.05). CT densitometry indicated vital pulps in all teeth, with pulpal inflammation detected in 6/8 CH-capped teeth and 2/8 MTA-capped teeth. Histologic evaluation confirmed vital pulp in all specimens, with different degrees of inflammation. (4) Conclusions: Within its limitations, the present study confirms the diagnostic value of PO evaluation of pulpal status in primary teeth with histologic means after pulp-capping procedures in a canine model. However, various degrees of pulpal inflammation elicited by different pulp-capping materials seem not to correlate with the obtained PO values.


Introduction
Dental trauma and deep carious lesions are leading causes of pulp disease in primary teeth. The primary goal of pulp therapy is to maintain the integrity and health of the teeth and their supporting tissues while maintaining the vitality of the pulp [1]. The prognosis of

Materials and Methods
The study was carried out at the Banat University of Agricultural Sciences and Veterinary Medicine "Regele Mihai I al Romaniei" from Timisoara (USAMVBT), in collaboration with the University of Medicine and Pharmacy "Victor Babes" Timisoara (UMFVBT). The experiment was approved by the ethics committees of both universities (approval nr. 133/07/2020 of USAMVBT, approval nr. 51/2020 of UMFVBT) and by the local Sanitary-Veterinary and Food Safety Authority (approval nr. 24876/10.08.2020). No animals were harmed during the procedures.
The study lasted two weeks and included primary teeth from eight canine subjects (aged three months old, belonging to the same litter) and was concluded with the extraction of three primary teeth/subject, which were soon replaced by permanent successors. All in vivo interventions (direct pulp capping, vitality tests and imagistic research) were performed under general anesthesia.

Operative Procedures
The selected teeth-mandibular canines (804), mandibular second premolars (806) and third premolars (807)-were divided into 6 groups, based on the capping material that will be used: the calcium hydroxide group (Dycal ® , Dentsply Sirona, Charlotte, NC, USA)-the 804 s; the MTA group (Bio-MTA Plus, PPH CERKAMED Wojciech Pawłowski, Stalowa Wola, Poland)-the 806 s; the Biodentine TM group (Biodentine TM , Septodont Ltd., France)-the 807 s; and the corresponding control group-704 s, 706 s and 707 s. The direct pulp-capping procedure was performed under general anesthesia. The anesthesia protocol consisted of premedication with xylazine (1 mg/kg b.w., i.v.) and ketamine (5 mg/kg b.w., i.v.) followed by induction with propofol (3 mg/kg b.w., i.v.); general anesthesia was maintained with isoflurane vaporized in oxygen using intermittent positive pressure ventilation; postoperative analgesia was provided with one dose of butorphanol (0.4 mg/kg b.w., s.c.) administered 15 min before recovery. No other medication was administered in the following period during the experiment. Rubber dam was used to isolate the teeth from the oral environment. Before cavity preparation, the teeth were disinfected using 2.5% sodium hypochlorite. The cavities were prepared on the buccal surface of each tooth. A standardized pulp exposure of 1 mm in diameter was performed with a 010 round diamond bur at high speeds under thorough water irrigation. Hemorrhaging was controlled by light pressure with moist saline cotton pellets before placement of the pulp-capping material. Pulp-capping materials were applied in each cavity using Dovgan carriers and round condensers, and the final restoration was completed with conventional glass ionomer cement (GC EQUIA Forte Fill-capsules, GC Europe). At 14 days postintervention, the treated teeth were extracted under general anesthesia, as atraumatic as possible, by a designated veterinary surgeon (C.I.) in the Department of Small Animal Surgery of USAMVBT.

Complementary Research
Pulp vitality was assessed prior to (T0) and after the direct pulp-capping procedure at 24 h (T1), 7 days (T2) and 14 days (T3) by PO and LDF. Inclusion criteria: intact healthy primary teeth (clinically and radiographically) and teeth with adequate size for sensor placement (upper and lower canines, second and third premolars); exclusion criteria: fractured teeth, teeth with calculus accumulation and small teeth not suitable for sensor placement (e.g., incisors and first premolars).
The pulse-oximetry measurements were carried out using a pulse-oximeter designed for veterinary use (SOMO PO100VET, SOMO International CO., Ltd., Hong Kong, China), with a compatible nasal alar sensor (Nasal Alar Fast SpO 2 Sensor, Koninklijke Philips N.V., Amsterdam, the Netherlands) of an appropriate size for dental use, for both anterior and posterior teeth. It uses red (640 nm) and infrared (940 nm) wave lengths to transilluminate the tissue and to detect absorbance peaks due to pulsatile blood circulation, and it calculates the pulse rate and oxygen saturation. The gingival margin was isolated using a liquid rubber dam prior to the placement of the sensor on the tooth (Figure 1). The sensor was hand-stabilized on the tooth with the emitter on the buccal side of the tooth and the receptor on the lingual side. The oxygen saturation (SpO 2 ) of the dental pulp was registered in low-light conditions. The initial measurements were performed in all healthy primary teeth that met the inclusion criteria: upper and lower canines (504, 604, 704 and 804), upper and lower second premolars (506, 606, 706 and 806) and third premolars (507, 607, 707 and 807). Following DPC, SpO 2 measurements were repeated for the treated teeth (804 s, 806 s and 807 s) and the corresponding control groups (704 s, 706 s and 707 s) at 24 h, 7 days and 14 days.
with a compatible nasal alar sensor (Nasal Alar Fast SpO₂ Sensor, Koninklijke N.V., Amsterdam, the Netherlands) of an appropriate size for dental use, for both and posterior teeth. It uses red (640 nm) and infrared (940 nm) wave lengths to t minate the tissue and to detect absorbance peaks due to pulsatile blood circulatio calculates the pulse rate and oxygen saturation. The gingival margin was isolated liquid rubber dam prior to the placement of the sensor on the tooth (Figure 1). Th was hand-stabilized on the tooth with the emitter on the buccal side of the tooth receptor on the lingual side. The oxygen saturation (SpO₂) of the dental pulp w tered in low-light conditions. The initial measurements were performed in all hea mary teeth that met the inclusion criteria: upper and lower canines (504, 604, 704 a upper and lower second premolars (506, 606, 706 and 806) and third premolars ( 707 and 807). Following DPC, SpO₂ measurements were repeated for the treated te s, 806 s and 807 s) and the corresponding control groups (704 s, 706 s and 707 s) a days and 14 days. The LDF recordings of the dental pulp flow were carried out using the Moo ments MoorLab VMS-LDF2 monitor (Moor Instruments Ltd., Axminster, UK). Th ment uses laser radiation generated by a semi-conductor laser diode operating at length of 780 + 10 nm and a maximum accessible power of 1.6 mW. The selecte width for the recorded LD signal was 20 Hz-20 kHz, and the sampling frequenc Hz. Calibration was performed according to the manufacturer's instructions. The reading time was 1 min/tooth. The results were recorded and analyzed using M MoorLab V2.01 software. A double silicone impression (Optosil Comfort Putty a vator Universal Plus, Kulzer GmbH., Hanau, Germany) was fixed perpendicular buccal cervical surface of the teeth for stabilization of the probe (Figure 2). Liqui dam (SDI Gingival Barrier, SDI Ltd., Chicago, IL, USA) was used for the supple isolation of the gingival tissue. The laser Doppler signal acquisition was perfor cording to our previous studies [23][24][25]. The physical parameters assessed were pressed in perfusion units (PU), and DC, which indicates the position of the optic reflecting its mechanic stability at the level of the reading area ( Figure 3). The re were carried out on the treated teeth and their corresponding control groups at T0 The LDF recordings of the dental pulp flow were carried out using the Moor Instruments MoorLab VMS-LDF2 monitor (Moor Instruments Ltd., Axminster, UK). The instrument uses laser radiation generated by a semi-conductor laser diode operating at a wavelength of 780 + 10 nm and a maximum accessible power of 1.6 mW. The selected bandwidth for the recorded LD signal was 20 Hz-20 kHz, and the sampling frequency was 40 Hz. Calibration was performed according to the manufacturer's instructions. The average reading time was 1 min/tooth. The results were recorded and analyzed using MoorSoft MoorLab V2.01 software. A double silicone impression (Optosil Comfort Putty and Activator Universal Plus, Kulzer GmbH., Hanau, Germany) was fixed perpendicularly on the buccal cervical surface of the teeth for stabilization of the probe ( Figure 2). Liquid rubber dam (SDI Gingival Barrier, SDI Ltd., Chicago, IL, USA) was used for the supplementary isolation of the gingival tissue. The laser Doppler signal acquisition was performed according to our previous studies [23][24][25]. The physical parameters assessed were flux, expressed in perfusion units (PU), and DC, which indicates the position of the optical probe, reflecting its mechanic stability at the level of the reading area ( Figure 3). The recordings were carried out on the treated teeth and their corresponding control groups at T0 and T1.  Radiographical examinations were performed at T0 and T3 in the Department of Ra diology of USAMVB. Computer tomography (CT) scans were also carried out at T3, using a Siemens SOMATOM Definition AS 64 scanner (Siemens AG, Erlangen, Germany) and SYNGO Examination integrated software (Siemens AG, Erlangen, Germany) for pulp density measurements. The measurements were performed on axial 0.6 mm-thick slices at the same level, in the DPC teeth and their corresponding control groups.

Histologic Examination
The histologic examination was performed according to a modified protoco (Nowicka et al., 2013). The samples were demineralized using Shandon TBD-2 decalcify ing solution (Fisher Scientific, Gothenburg, Sweden) and embedded in paraffin. Five-mi cron-thick serial sections were cut in the bucco-lingual plane of the teeth, stained with hematoxylin-eosin (HE) and then assessed by an experienced oral pathologist (M.M.) us ing the optical microscope Olympus BX46 (Olympus Co., Tokyo, Japan), with an inte grated camera system for image acquisition-at 200×, 100× and 40× magnification. Th samples were divided into three histomorphological sample batches (CH, MTA, Biodenti ne TM ) and analyzed using the following criteria: pulp inflammation (type, intensity and extension), the amount of hard tissue formation at the interface of the capping materia (continuity, morphology and thickness) and the odontoblast cell layer. Each section wa scored using a 1-4 numeric scale, with 1 representing the most desired result and 4 repre  Radiographical examinations were performed at T0 and T3 in the Department of Radiology of USAMVB. Computer tomography (CT) scans were also carried out at T3, using a Siemens SOMATOM Definition AS 64 scanner (Siemens AG, Erlangen, Germany) and SYNGO Examination integrated software (Siemens AG, Erlangen, Germany) for pulp density measurements. The measurements were performed on axial 0.6 mm-thick slices, at the same level, in the DPC teeth and their corresponding control groups.

Histologic Examination
The histologic examination was performed according to a modified protocol (Nowicka et al., 2013). The samples were demineralized using Shandon TBD-2 decalcifying solution (Fisher Scientific, Gothenburg, Sweden) and embedded in paraffin. Five-micron-thick serial sections were cut in the bucco-lingual plane of the teeth, stained with hematoxylin-eosin (HE) and then assessed by an experienced oral pathologist (M.M.) using the optical microscope Olympus BX46 (Olympus Co., Tokyo, Japan), with an integrated camera system for image acquisition-at 200×, 100× and 40× magnification. The samples were divided into three histomorphological sample batches (CH, MTA, Biodentine TM ) and analyzed using the following criteria: pulp inflammation (type, intensity and extension), the amount of hard tissue formation at the interface of the capping material (continuity, morphology and thickness) and the odontoblast cell layer. Each section was scored using a 1-4 numeric scale, with 1 representing the most desired result and 4 repre- Radiographical examinations were performed at T0 and T3 in the Department of Radiology of USAMVB. Computer tomography (CT) scans were also carried out at T3, using a Siemens SOMATOM Definition AS 64 scanner (Siemens AG, Erlangen, Germany) and SYNGO Examination integrated software (Siemens AG, Erlangen, Germany) for pulp density measurements. The measurements were performed on axial 0.6 mm-thick slices, at the same level, in the DPC teeth and their corresponding control groups.

Histologic Examination
The histologic examination was performed according to a modified protocol (Nowicka et al., 2013). The samples were demineralized using Shandon TBD-2 decalcifying solution (Fisher Scientific, Gothenburg, Sweden) and embedded in paraffin. Five-micron-thick serial sections were cut in the bucco-lingual plane of the teeth, stained with hematoxylin-eosin (HE) and then assessed by an experienced oral pathologist (M.M.) using the optical microscope Olympus BX46 (Olympus Co., Tokyo, Japan), with an integrated camera system for image acquisition-at 200×, 100× and 40× magnification. The samples were divided into three histomorphological sample batches (CH, MTA, Biodentine TM ) and analyzed using the following criteria: pulp inflammation (type, intensity and extension), the amount of hard tissue formation at the interface of the capping material (continuity, morphology and thickness) and the odontoblast cell layer. Each section was scored using a 1-4 numeric scale, Diagnostics 2022, 12, 2022 6 of 14 with 1 representing the most desired result and 4 representing the least desired result, as follows: for the type of pulp inflammation: 1 = no inflammation, 2 = chronic inflammation, 3 = acute and chronic inflammation and 4 = acute inflammation; for the intensity of pulp inflammation: 1 = absent or very few inflammatory cells, 2 = mild, defined as an average of <10 inflammatory cells, 3 = moderate, defined as an average of 10-25 inflammatory cells and 4= severe, defined as an average >25 inflammatory cells; for the extension of pulp inflammation: 1 = absent, 2 = mild, defined as inflammatory cells only next to dentin bridge or area of pulp exposure, 3 = moderate, defined as inflammatory cells observed in part of coronal pulp (in one-third or more of the coronal pulp or in the mid pulp) and 4 = severe, defined as all coronal pulp is infiltrated or necrotic; for the odontoblastic layer: 1 = palisade pattern of cells, 2 = presence of odontoblast cells and odontoblast-like cells, 3 = presence of only odontoblast-like cells and 4 = absent; for the continuity of the dentinal bridge: 1 = complete dentin bridge formation, 2 = partial/incomplete dentin bridge formation extending to more than one-half of the exposure site but not completely closing the exposure site, 3 = initial dentin bridge formation extending to not more than one-half of the exposure site and 4 = no dentin bridge formation; for the morphology of the dentinal bridge: 1 = dentin or dentin associated with irregular hard tissue, 2 = only irregular hard tissue deposition, 3 = only a thin layer of hard tissue deposition and 4 = no hard tissue deposition [26].

Statistical Analysis
The data obtained from the histopathological evaluation and the pulp vitality recordings were processed with the statistical software R Version 4.1.2 2021, RStudio 2022.02.3 + 492 "Prairie Trillium" and JAMOVI Version 1.8.3.0. For statistical analyses, both parametric and nonparametric tests were applied, according to the results of the Shapiro-Wilk test of normality. Parametric tests including Independent Samples t-Test and ANOVA and non-parametric tests including Kruskal-Wallis and Mann-Whitney I were used for the comparative evaluation of pulp-testing results before and after DPC and for comparative histological analysis. A p value < 0.05 was considered statistically significant. In some situations, we also considered the effect size as a qualitative assessment of the effects (values > 0.5 can be considered as a medium to large effect).

Preoperative Research
The preoperative radiographical images revealed the normal development of the primary teeth in relation to the adjacent structures ( Figure 3). The following developmental stages were noted in the selected primary teeth: second and third premolars within the stability stage (II/III), with no evidence of root resorption, and canines within the first phase of resorption stage (III/III) with less than one-third of the root resorbed.
PO recordings were carried out at T0 on all selected healthy primary teeth (upper and lower canines and premolars) to serve as reference data. The mean values recorded for pulpal SpO 2 were as follows: 90.9% ± 2.87 SD for canines (C), 89.7% ± 3.21 SD for second premolars (P2) and 89.1% ± 3.13 SD for third premolars (P3)- Table 1. The data were statistically processed using the ANOVA test, which revealed no significant differences (p = 0.054, >0.05) between the three tooth categories (C, P2 and P3). However, a post hoc analysis of the data using the Tukey HSD test shows a larger difference between C and P3, (p tukey = 0.048) with a Cohen's effect size of 0.600 (if >0.5, the effect can be considered medium to significant). Independent Samples t-Test was applied to compare values of upper vs. lower teeth, and no significant differences were detected (p = 0.747, >0.05).
LDF recordings were carried out at T0 only on lower healthy primary canines and premolars (reduced number of teeth due to lengthy procedure). The data were statistically processed using the ANOVA test, which revealed significant differences (p = 0.029, < 0.05) between the three tooth categories (C, P2 and P3). The Tukey HSD post hoc comparison showed the most significant differences (p tukey = 0.022) between P2 and P3.

Imagistic Research
The radiographical images showed no periapical lesion in any of the treated teeth ( Figure 4) at 14 days after the DPC. However, a slight progression in the physiologic root resorption of the primary canines was noted. The data were statistically processed using the ANOVA test, which revealed no significant differences (p = 0.054, >0.05) between the three tooth categories (C, P2 and P3). However, a post hoc analysis of the data using the Tukey HSD test shows a larger difference between C and P3, (ptukey = 0.048) with a Cohen's effect size of 0.600 (if >0.5, the effect can be considered medium to significant). Independent Samples t-Test was applied to compare values of upper vs. lower teeth, and no significant differences were detected (p = 0.747, >0.05).
LDF recordings were carried out at T0 only on lower healthy primary canines and premolars (reduced number of teeth due to lengthy procedure). The data were statistically processed using the ANOVA test, which revealed significant differences (p = 0.029, < 0.05) between the three tooth categories (C, P2 and P3). The Tukey HSD post hoc comparison showed the most significant differences (ptukey = 0.022) between P2 and P3.

Imagistic research
The radiographical images showed no periapical lesion in any of the treated teeth ( Figure 4) at 14 days after the DPC. However, a slight progression in the physiologic root resorption of the primary canines was noted.

Vitality Tests
Statistical analysis of post-operative SpO₂ recordings revealed that there are significant differences (p = 0.002, <0.05) between the treatment vs. control groups (T1-T3), with an effect size of 0.295 (Mann-Whitney non-parametric test)- Figure 6; there are no significant differences (p = 0.257, >0.05) between the three treatment groups overall (T1-T3) and also no significant differences between the groups; no significant differences were observed at T1 (p = 0.559, >0.05) nor at T3 (p = 0.810, >0.05) (One-Way ANOVA and Kruskal-Wallis non-parametric tests). Statistical analysis of LDF recordings revealed that there are significant differences between materials (p = 0.001, <0.05) and also significant differences (p = 0.002, <0.05) between the treatment and control groups at T1 (Independent Samples t-Test).

Vitality Tests
Statistical analysis of post-operative SpO₂ recordings revealed that there are significant differences (p = 0.002, <0.05) between the treatment vs. control groups (T1-T3), with an effect size of 0.295 (Mann-Whitney non-parametric test)- Figure 6; there are no significant differences (p = 0.257, >0.05) between the three treatment groups overall (T1-T3) and also no significant differences between the groups; no significant differences were observed at T1 (p = 0.559, >0.05) nor at T3 (p = 0.810, >0.05) (One-Way ANOVA and Kruskal-Wallis non-parametric tests). Statistical analysis of LDF recordings revealed that there are significant differences between materials (p = 0.001, <0.05) and also significant differences (p = 0.002, <0.05) between the treatment and control groups at T1 (Independent Samples t-Test). Statistical analysis of LDF recordings revealed that there are significant differences between materials (p = 0.001, <0.05) and also significant differences (p = 0.002, <0.05) between the treatment and control groups at T1 (Independent Samples t-Test).

Histologic Analysis
The histologic evaluation of teeth confirmed the presence of vital pulp tissue in all specimens, exhibiting different degrees of inflammation. MTA and Biodentine TM were well tolerated by the pulp tissue, while in CH-capped teeth, a persistent inflammation was noted (Figure 7). There was evidence of moderate and severe acute and chronic pulpal inflammation in eight out of eight specimens in the CH group, mild and moderate acute inflammation in six out of eight specimens in the MTA group and mild acute inflammation in two specimens out of eight specimens from the Biodentine TM group. Odontoblast and odontoblast-like cells were discovered adjacent to the dentinal bridge in most specimens from the MTA and Biodentine TM groups, and in case of the CH group, only two specimens presented odontoblast-like cells. Complete dentinal bridge formation was not observed in any of the specimens from the three groups.

Histologic Analysis
The histologic evaluation of teeth confirmed the presence of vital pulp tissue in all specimens, exhibiting different degrees of inflammation. MTA and Biodentine TM were well tolerated by the pulp tissue, while in CH-capped teeth, a persistent inflammation was noted (Figure 7). There was evidence of moderate and severe acute and chronic pulpal inflammation in eight out of eight specimens in the CH group, mild and moderate acute inflammation in six out of eight specimens in the MTA group and mild acute inflammation in two specimens out of eight specimens from the Biodentine TM group. Odontoblast and odontoblast-like cells were discovered adjacent to the dentinal bridge in most specimens from the MTA and Biodentine TM groups, and in case of the CH group, only two specimens presented odontoblast-like cells. Complete dentinal bridge formation was not observed in any of the specimens from the three groups. Results of the histologic evaluation of all specimens from the three groups, according to the numeric 1-4 scale, are summarized in Table 3. Table 3. Modal values of the different criteria characterizing the histologic features of the three DPC biomaterials, according to the scores (1-4).

Biomaterial Criteria All Criteria Inflammation
Odontoblastic Layer Dentinal Bridges Ca (OH)2  3  4  2  4  MTA  2  1  3  2  Biodentine TM  1  1  3  1 Statistical analysis of the histologic evaluation criteria revealed significant differences between the responses of teeth to the three biomaterials (p < 0.001, <0.05, ANOVA statistical test). Detailed Tukey HSD post hoc comparisons between materials revealed the highest statistically significant differences between the Biodentine TM and CH groups (ptukey < 0.001), significant differences between the MTA and CH groups (ptukey = 0.001) and no significant differences between the Biodentine TM and MTA groups (ptukey = 0.712).

Discussion
This study assesses the pulpal status in primary teeth, aiming to determine whether a small pulse-oximetry nasal alar sensor can be used in a dental setting (and possibly serve as a basis for the development of a customized dental sensor) for monitorization of pulp Results of the histologic evaluation of all specimens from the three groups, according to the numeric 1-4 scale, are summarized in Table 3. Table 3. Modal values of the different criteria characterizing the histologic features of the three DPC biomaterials, according to the scores (1-4).

Biomaterial Criteria All Criteria Inflammation Odontoblastic Layer Dentinal Bridges
Ca (OH) 2  3  4  2  4  MTA  2  1  3  2  Biodentine TM  1  1  3  1 Statistical analysis of the histologic evaluation criteria revealed significant differences between the responses of teeth to the three biomaterials (p < 0.001, <0.05, ANOVA statistical test). Detailed Tukey HSD post hoc comparisons between materials revealed the highest statistically significant differences between the Biodentine TM and CH groups (p tukey < 0.001), significant differences between the MTA and CH groups (p tukey = 0.001) and no significant differences between the Biodentine TM and MTA groups (p tukey = 0.712).

Discussion
This study assesses the pulpal status in primary teeth, aiming to determine whether a small pulse-oximetry nasal alar sensor can be used in a dental setting (and possibly serve as a basis for the development of a customized dental sensor) for monitorization of pulp conditions in both healthy and pulp-capped teeth. LDF, CT, radiographical and histological examinations were used to assess and compare the results. The findings of our study indicate that pulse-oximetry can be a valuable aid in monitoring pulp statuses in primary teeth, having correctly identified vitality in all healthy and treated teeth (accordance between PO, LDF, CT densitometry and histologic data); the first null hypothesis stating that pulse oximetry cannot provide a confirmation of the pulp status (vital/non-vital) in primary teeth following direct pulp capping was, therefore, rejected. The study presents comprehensive histologic data about the pulpal response induced by three DPC materials-CH, MTA and Biodentine TM (regularly used by pediatric dentists in practice). Significant statistical differences between the responses of teeth to the three biomaterials were registered; consequently, the second null hypothesis stating that that there is no statistically significant difference between the pulpal response generated by calcium hydroxide and calcium silicate-based materials was also rejected. While the outcome for the two calcium-silicate cements (MTA and Biodentine TM ) was similar-minimal pulpal inflammation, with no statistically significant difference between the two materials-the outcome for CH was poorly marked acute and chronic inflammation, with statistically significant differences between Biodentine TM and CH and also between the MTA and CH groups.
The study was carried out on primary teeth from dogs, which have a life span of approximately five months, with an eruption at about three weeks and shedding at around six months of age [29]. Dogs have been used in numerous studies as animal models for studying the physiopathology of dental pulp. Canines and the premolars of dogs are anatomically close to those of humans. Similarly, dogs share many biochemical and physiologic characteristics with humans, particularly concerning the immune system [30]. As far as we know, only one study in the literature (Riehl et al. 2016) used PO to detect the blood flow in vital canine teeth [15]. However, this study was carried out in intact permanent canines without investigating the pulpal status after pulp-capping procedures.
The majority of existing PO studies was carried out on anterior teeth [14], due to limitations imposed by the size of the sensor. In our study, we selected, for the pulseoximetry vitality testing, the nasal alar SpO 2 sensor because the small size of the sensor makes it appropriate for dental use, even in the posterior area of the oral cavity, ensuring a satisfactory tooth-fit. The sensor produces a strong, consistent signal, even in patients with poor perfusion [31]. The data we obtained from PO testing were confronted with data from a second vitality test-LDF (with a standardized technique), CT densitometry of the pulp tissue and histologic analysis-which, in case of the dental pulp, is the reference standard for measuring the diagnostic accuracy of a pulp test [9]. All the afore-mentioned methods confirmed vitality in all teeth at 14 days post-DPC. PO testing after DPC registered values of SpO 2 ranging between 82.2% and 86.8% in the treated teeth. According to the literature, average SpO 2 values of healthy teeth are 84.94%-89.29% for central and lateral incisors, 89.20% for canines [14], 86.20% for premolars [32], and 85.09% for molars [33]. Minimum oxygen saturation levels in healthy pulp were identified at around 77.52% [14]. Anusha et al. reported oxygen saturation levels for pulpal diseases: 85.4%-reversible pulpitis 81.6%irreversible pulpitis; 70.7%-pulp necrosis (94.6%-positive control, healthy teeth; 0%negative control, endodontically treated teeth) [34]. All these data were recorded, however, in permanent teeth. In primary teeth, the values of SpO 2 are influenced in some degree by the physiologic root resorption. There are conflicting data in the literature on this matter. Komatsu et al. (2007) found that the pulpal blood flow of primary teeth showed a tendency to decrease with age, due to the morphological changes in the blood vessels in the pulp [35], while Karayilmaz et al. (2011) reported an increase in pulpal blood flow, which is attributed to the progressive apical enlargement caused by physiological root resorption [36]. Given the heterogeneity of the available data on dental SpO 2 , a conclusion regarding the presence of pulp inflammation in the treatment groups from our study cannot be drawn. It can be speculated that another factor with possible implications for PO readings could be the presence of pulp capping and filling materials, which exhibit different light-scattering patterns in comparison to the hard dental tissues.
Our statistical analysis of the PO recordings revealed that there were no significant differences between the three capping materials, while with LDF testing, there was a statistically significant difference between materials, confirmed by CT densitometry and histological analysis. As with PO, controversies exist in the literature regarding the use of LDF for pulpal blood flow detection as well. On the other hand, some studies found LDF to be an accurate vitality testing method to assess the pulpal vascularity changes of human primary teeth [35], while others reported the opposite [37]. Moreover, the LDF procedure is a lengthy and more sensitive technique compared to conventional pulp vitality tests [9,38], which makes it improper for routine use in children; however, it is reserved for research purposes and particular clinical situations such as trauma to the anterior teeth [39] or "dens invaginatus" [40]. In our study, LDF testing was limited to T0 and T1 (tests at T2 and T3 are not completed) due to the rapid growth of the canine specimens in the two-week time span of the study, which negatively impacted the adaptation of the stabilizing silicone impression. It was decided, therefore, not to manufacture a new impression in order to refrain from altering the reading parameters by placing the probe in a different position. Using the current technique presented in this article, the LDF is not suitable for vitality monitorization of canine primary teeth for time periods longer than 24 h.
The CT scans performed at T3 provided data about the density of the pulp tissue in the treated teeth and their corresponding control groups. CT allows the precise threedimensional evaluation of anatomic structures and a direct measurement of tissue density expressed in Hounsfield units (HU). HU is the standard numeric value representing the relative density of body tissues according to a calibrated grey-level scale based on values for air (−1000 HU), water (0 HU) and bone density (+1000 HU) [41]. The density of the dental pulp ranges from −461.5 HU to −170 HU in the case of pulp necrosis, 243.5 HU −396 HU in pulp inflammation and 465 HU −775 HU when the pulp is healthy (with an average of 520 HU for coronal pulp and 892 HU for radicular pulp) [27,28]. The values obtained in our study are indicative for pulp inflammation in most of the CH-capped teeth, and all Biodentine TM -capped teeth and all the teeth in the three corresponding control groups are indicative for healthy pulp in most of the MTA-capped teeth. The data are consistent with histologic findings. Although accurate, the routine use of CT for diagnostic purpose of the pulp status is not recommended in children due to radiation concerns [42].
Histological examination is regarded in the literature as the gold standard (the best available method against which the performance of other diagnostic tests is evaluated) for dental pulp [9,43]. The histological analysis in our study was aimed primarily at confirming the pulp status of the teeth from the three treatment groups. The extraction of the treated dental units was performed at two weeks post-DPC to avoid the overlapping of physiological root resorption. The results confirmed the presence of vital pulp in all specimens and revealed further data about the inflammatory status present in some of the specimens. Overall, the least amount of evidence for inflammation was found in the Biodentine TM group, while in the CH group, markers of acute and chronic inflammation were present in all specimens, with moderate and severe extension and intensity. Dentinal bridge formation was initiated in specimens from all three groups. Nevertheless, this finding must be interpreted with care, as it can be a sign of healing or a reaction to irritation [26]. In this case, given that the teeth were extracted only two weeks after pulp capping, it is premature to consider it a sign of healing, especially in case of CH specimens, where pulp inflammation is present. The results of the histological analysis from this study are in accordance with previous studies [44,45].
The limitations of the present study are the low number of primary teeth included in the experiment, the rapid growth of young canine subjects (aged three months at the debut of the study) and the lack of dedicated devices/sensors for dental pulp vitality testing (which may have affected the measurements, due to the imperfect adaptation of the sensor to the teeth, instability of the hand-held sensor and the different optical properties of the tissues and filling/capping materials).

Conclusions
Within its limitations, the present study confirms, with histologic means, the diagnostic value of the PO evaluation of pulpal statuses in primary teeth after pulp-capping procedures in a canine model. However, various degrees of pulpal inflammation elicited by different pulp-capping materials seem not to correlate with the obtained PO values. LDF, in its current version, is not a suitable pulp testing method for the monitorization of vitality in primary canine teeth for a longer period due to technique-related limitations. The results of our study could contribute to the improvement of pulp-testing methods and their applications in pediatric dental practice. Future research should be focused on developing a dedicated PO sensor for dental use and establishing SpO 2 reference parameters for all dental categories, as well as for treated teeth. The influence of different filling materials (with optical properties that differ from those of the hard dental structures) on the SpO 2 readings should be acknowledged in detail. Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committees of the Banat University of Agricultural Sciences and Veterinary Medicine "Regele Mihai I al Romaniei" from Timisoara (approval nr. 133/07/2020) and of the University of Medicine and Pharmacy "Victor Babes" Timisoara (approval nr. 51/2020) and by the local Sanitary-Veterinary and Food Safety Authority (approval nr. 24876/10.08.2020). Ethical review and approval were waived for this study for in vivo interventions performed in animal (canine) subjects.