Introduction

Pulp preservation is a primary goal for the treatment of Primary dentition. Therefore, vital-pulp therapy is a critical procedure aimed at maintaining and preserving the vitality of primary tooth pulp until the time for its exfoliation. The American Academy of Pediatric Dentistry (AAPD) recommends three treatment modalities for vital-pulp therapy. These modalities are indirect pulp treatment, direct pulp capping, and pulpotomy [1].

For mechanical and/or carious pulp exposure in primary teeth having reversible pulpitis or normal pulp, the procedure of choice is pulpotomy [1]. Formocresol pulpotomies for primary teeth over 12 months show favorable outcomes according to radiographic and clinical studies [2], although several disadvantages have also been noted. For example, Coll et al. [3] conducted a systematic review and meta-analysis. They concluded that the success rate of most pulpotomy procedures not using mineral trioxide aggregate (MTA) decreases to 70% or less after three years. Another study has reported the early exfoliation of pulpotomized teeth [4]. Based on histopathological studies on formocresol pulpotomies, variable pulp-tissue responses reportedly include mild to moderate or severe inflammation, internal resorption, calcific metamorphosis, fibrosis, and necrosis [5, 6]. On the other hand, evidence-based research indicates that MTA is superior to the most commonly used pulp-capping materials; thus, MTA should be deemed as the gold-standard pulpotomy agent for primary molars [7]. MTA is biocompatible, induces dentin-bridge formation, and promotes pulp-tissue regeneration [8]. Several researchers have also expressed apprehension in using pulpotomy as a treatment modality for extensive lesions in primary teeth given its invasive nature [3, 8]. Indeed, the AAPD acknowledges the urgency of identifying new techniques for vital-pulp therapy in primary teeth by using biocompatible medications [1].

In 1978, Dr. Cvek introduced partial pulpotomy (PP) to preserve pulp vitality in complicated crown fractured permanent teeth owing to trauma [9]. The technique has been subsequently recommended for use in permanent teeth with carious exposed pulp and controlled bleeding [1]. Unlike in pulpotomy, the cell-rich coronal pulp tissue is preserved in PP, so the healing and repair ability rates are higher [10]. A 92% success rate for PP in cariously exposed permanent teeth after two months has been reported in a systematic review and meta-analysis [11]. Previous studies have demonstrated that PP is less invasive and more effective in bleeding control than pulpotomy. The chance of clot formation, which may interfere with pulp healing, is also lower in PP [12]. Bakhtiar et al. [13] explored the pulpal response of permanent teeth to different materials used for PP. They found that in 56% of MTA PP-treated teeth, complete dentin-bridge formation without inflammation occurred after eight weeks. However, few studies have focused on the use of PP in primary teeth despite its success in young permanent teeth. Nematollahi et al. [14] conducted a randomized controlled trial and compared the clinical and radiographic success rates of MTA PP with those of formocresol pulpotomy. No significant differences are found between both procedures throughout a 24-month period. Similarly, Abdelhafez et al. [15] reported comparable success rates between various pulpotomy methods in pediatric dentistry.

There is a dearth of studies discussing the efficacy of partial pulpotomy and full pulpotomy procedures using various materials, including mineral trioxide aggregate (MTA), on human primary teeth. However, these studies often focus on clinical and radiographic evaluations rather than detailed histological assessments [14, 15]. Though human studies are invaluable, they are sometimes limited by ethical concerns, variability in patient compliance, and difficulties in obtaining sufficient sample sizes for histological examination.

The present study aims to address these gaps by using an animal model that allows controlled experimental conditions and detailed histological analysis. Puppies’ primary premolars were utilized due to the similarity of their dental development to human primary teeth, including the process of eruption and exfoliation. It allows the researcher to closely monitor the histopathological pulpal responses to partial and full pulpotomy procedures using MTA as a pulp medicament in a controlled environment, thereby providing insights directly translatable to pediatric dentistry. The null hypothesis was that there would be no significant difference in the histopathological response of the pulp tissue of primary teeth to partial pulpotomy (PP) and full pulpotomy (FP) using mineral trioxide aggregate (MTA).

Material and methods

Experimental outcomes measure

Herein, the present study considered the study outcome as the pulp-tissue response to the treatment procedures. A trained oral and maxillofacial pathologist blinded to all procedures examined all samples by using the coding system. After evaluating the slides twice at six-day intervals, intra-examiner reliability was determined using the kappa coefficient (range = 0.88–0.96), representing a nearly perfect agreement. The pulp-tissue reaction to treatments was evaluated based on modified criteria of Jabbarifar et al.’s [16] modified criteria (Table 1).

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Table 1. Criteria of assessment of pulpal response to capping materials.

https://doi.org/10.1371/journal.pone.0312076.t001

Results

Vital-pulp treatment was performed on 72 deciduous premolars. A total of 36 MTA-PP and 36 MAT-FP samples were obtained. The pulp-tissue response of MTA-PP was compared with that of MTA-FP (Table 2). Chronic inflammation (score 3) was higher in MTA-FP (66.7%) than in MTA-PP (47.2%), whereas acute and mixed inflammation (scores 1 and 2, respectively) was higher in MTA-PP (13.9%) than in MTA-FP (33.3%). No significant difference existed between the groups (P = 0.085). Inflammation intensity was less in MTA-PP, with only 2.8% demonstrating severe inflammation (score 3) compared with the 16.7% in MTA-P. The difference was statistically significant (P = 0.004). No significant differences existed between the test and the control groups among all hyperemia types (mild, moderate, and severe). Calcific-bridge presence was higher in MTA-PP (72.2%), but the difference was not statistically significant. Localized and diffuse necrosis were significantly higher (27.8% and 11.1%, respectively; P = 0.014) in the control than in the test group. A nearly equal distribution of fibrosis (scores 1 and 2) was observed between groups (P = 0.290). MTA-PP had higher pulp calcification (33.3%) than MTA-FP (16.7%), but the difference was not statistically significant. MTA-FP had greater pathological internal-root resorption (63.9%) than MTA-PP (19.4%) (P <0.0001).

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Table 2. Comparison of pulp tissue response between MTA partial pulpotomy versus pulpotomy procedures.

https://doi.org/10.1371/journal.pone.0312076.t002

Favorable pulp-tissue response was found 2 months after MTA-PP and MTA-FP procedures, as revealed by histopathological evaluation (Figs 1 and 2). Normal dentinal tubules appeared all over the dentin thickness. The odontoblast layer was preserved, and pulp was normal with no an apparent inflammation. Calcified-bridge presence was noted in MTA-PP (Fig 1E and 1F).

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Fig 1.

Pulp tissue response to MTA-PP (test group) after two months, showing Fig 1A & 1B: MTA material in direct contact with normal pulp tissue (H&E Fig 1. A x10 and 1. B X40). Fig 1C: Note the apparent normal thick odontoblastic layer (black arrows) (H&E × 40). Fig 1D: Evidence of normal dentinal tubules with multiple free pulp calcifications (H&E X20). Fig 1E: A noticeable regenerative calcified bridge (black arrows) adjacent to pulp calcifications (blue arrows) (H&E X10). Fig 1F: High power of the marked calcified bridge formation (H&E X40).

https://doi.org/10.1371/journal.pone.0312076.g001

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Fig 2.

Pulp tissue response to MTA-FP (control group) after two months showing Fig 2A: apparently normal pulpal tissue with continuous odontoblastic layer (arrows) (H&E X10). Fig 2B: Note the preserved thick odontoblastic layer and abundant cell population of fibroblasts (arrows) (H&E X40).

https://doi.org/10.1371/journal.pone.0312076.g002

Meanwhile, different inflammation degrees (i.e., mild to severe) were observed in specimens with unfavorable pulp-tissue response. Both groups demonstrated blood-vessel congestion and dilatation, and the infiltration of acute and/or chronic inflammatory cells and abscess formation was significant (Fig 3). Remarkable fibrosis, pathological root resorption, and necrosis were also detected in a few pulpotomy-treated teeth (Fig 4).

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Fig 3. Unfavorable pulp tissue response two months after treatment.

Fig 3A: MTA-FP (control group) presented a severe infiltration of acute inflammatory cells in the pulp tissue with neutrophil predominance and abscess formation (H&E X40). Fig 3B: Showed moderate collection of chronic inflammatory cells in the MTA-PP (test group) (H&E X40). Fig 3C & 3D: Note signs of hyperemia with congested blood vessels (black arrows) in the control group (H&E Fig 3C X20, and Fig 3D X40).

https://doi.org/10.1371/journal.pone.0312076.g003

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Fig 4.

Unfavorable pulp tissue response to the MTA-FP (control group) showing Fig 4A. Signs of diffuse tissue necrosis (black arrows) are associated with the focal distribution of MTA material within the pulp tissue (H& E X10). Fig 4B: Note the pathological internal root resorption (black arrows) (H&E X40). Fig 4C & 4D: Showed marked fibrosis (black arrows) along the pulpal side of the dentinal wall (H&E Fig 4C X10, and Fig 4D X40).

https://doi.org/10.1371/journal.pone.0312076.g004

Discussion

PP is considered more conservative than full pulpotomy. [10] PP aims to preserve any remaining vital pulp tissue that is functioning and infection free [1]. A healing environment is likely to emerge once this goal is achieved. The development of mineralized tissues in the exposed area is possible and may trigger a reparative response.

The success rate of vital-pulp therapy in primary teeth is usually determined through radiographic and clinical evaluation. Nevertheless, the gold standard in determining such success is still histopathological examination [18]. To our best knowledge, the present work is the first experimental histologically based study that compared the influence of two MTA vital-pulp therapy techniques (pulpotomy and PP) on the primary tooth pulp of puppies. Given the difference in pulp-tissue responses between both procedures, the null hypothesis was hereby rejected.

The age of the puppies in this study was 6–8 weeks because this age range marks the completion of primary dentition. Considering that the shedding process of puppies’ primary teeth occurs beyond the fourth month of their life, the follow-up period was deemed to be only two months [19]. MTA, a regenerative material with a high application-success rate, is considered to be the gold standard for vital-pulp treatment of primary teeth [20]. The design of the present experimental study was split mouth, i.e., both intervention techniques were applied to comparable sites to eliminate inter-subject variability. Standardized procedures were used in teeth treated in this work.

The pulpal responses to different techniques and medicaments have been extensively researched in experimental animals, but the main focus is on permanent teeth [21–24]. Studies on human teeth have also been performed for the same purpose, and pulpotomy is the most widely evaluated strategy [25]. Accordingly, the results on PP were compared with those obtained from permanent teeth."

After 2 months of follow-up, chronic inflammation (score 3) was noted in MTA-P-treated primary teeth and, to a lesser extent, in MTA-PP-treated ones. Additionally, the pulpotomy group had significantly higher inflammation intensity. The observed inflammation may be associated with the surgical trauma and the irritating effect of the capping material [26], which may also explain the increased inflammation intensity correlated with the higher invasiveness of pulpotomy. The study findings coincided with those of Lee et al. [22], Kang et al. [23], and Agamy et al. [25]. Lee et al. [22] evaluated PP on permanent teeth in experimental animals, whereas Kang et al. [23] experimentally studied pulpotomy techniques for permanent teeth. Notably, inflammation intensity was significantly higher in MTA-P. The success of MTA vital-pulp therapy is known to depend on the presence or absence, as well as the intensity and type, of the inflammatory response and the formation of a calcific barrier [27]. Therefore, the pulp response can be considered to be more favorable in primary teeth treated with partial than full pulpotomies.

Regarding hyperemia, all scores (mild, moderate, and severe) were almost the same, and no significant differences existed between teeth treated with partial and full pulpotomies. Mild hyperemia (score 1) achieved the highest score, confirming earlier findings about pulp-tissue response to MTA pulpotomies [22–24].

Calcific-bridge presence was higher (72.2%) in MTA-PP than in MTA-FP (52.8%). Although the difference was not statistically significant, it can be considered a sign of success for MTA-PP. Inflammatory cells reportedly play a crucial role in tissue healing and repair. The healing process is supported by macrophages producing various growth factors and cytokines. Proper balance must exist between pro- and anti-inflammatory cytokines for healing promotion and tissue repair post-injury [28]. Decreased infection or inflammation in the pulp enables regenerated odontoblast-like cells to differentiate and create reparative dentin or calcific bridges [29]. This finding was consistent with that of Bakhtiar et al. [13]. They observed complete dentin bridges in 56% of cases treated with MTA PP on permanent teeth scheduled for extractions. The study observation also agreed with that of Khalil et al. [21], but their study involves experimental rats and direct pulp capping.

In the current study, The test group demonstrated significantly less pulp-tissue necrosis than the control group, indicating that the response to treatment was more favorable. When at least one area of coronal pulp tissue demonstrates coagulation or liquefaction necrosis in any treated teeth, it is deemed as a primary indicator of pulp change that is irreversible [30]. The necrosis incidence is higher in the pulpotomy group, which may be due to the increased inflammation intensity. Balance must be maintained between inflammatory cells, which is crucial for healing. A disrupted inflammatory reaction can decrease the healing potential, resulting in a degeneration of the odontoblastic layer and even in the necrosis of pulp tissue. The necrosis (diffused type) score of 2 in MTA-FP was consistent with that found by Jabbarifar et al. [16], who found that necrosis was an unfavorable outcome of the procedure.

PP- and pulpotomy-treated teeth had a nearly identical fibrosis degree. Interestingly, fibrosis was absent in the experimental rats’ permanent-tooth pulp after MTA pulpotomy conducted by Salako et al. [31]. Their findings are the opposite of those observed in this current study, as 26 out of 28 fibrosis cases had scores of 1 and 2. The discrepancy may be explained by the different dentition types and experimental animals used. Rowe [32] showed a significant healing potential for rat pulp.

Although the test group had a higher pulp-calcification percentage than the control (33.3% and 16.7%, respectively), the coronal pulp had free/adherent pulp stones. This presence is considered a deviation from the normal histological features of healed pulp tissue [30]. The percentage observed was much lower than that reported by Jabbarifar et al. [16], in which the calcification rate in permanent teeth was 51.9% three months after MTA pulpotomy. The longer evaluation periods and different dentition types may explain the variation between the findings.

Pathological internal-root resorption was more prevalent in MTA-FP than in MTA-PP, further confirming MTA-PP superiority. Since these teeth were extracted before their physiological resorption time, the applied PP/FP technique might induce a histological response like internal resorption. The likelihood of internal resorption can be increased by certain factors such as reduced radicular pulp tissue, which decreases healing potential. The invasiveness of the procedure is another factor. Moreover, the vascularity of the apical region increases with physiologic resorption, which can attract odontoclasts and thus initiate internal resorption [26].

This study has some limitations. First is the inconsistent direction of sectioned teeth throughout the study. Second, is the absence of caries or inflammation in puppies’ teeth. Third, the decalcification of sectioned samples increases the difficulty and duration of the examination compared with conventionally H&E-stained histological sections. However, these findings can serve as a baseline for future experimental and clinical works. Accordingly, it is suggested that further clinical studies be conducted on human primary teeth to evaluate PP’s effectiveness and determine the suitable biological materials.

Conclusion

Partial MTA pulpotomy procedure showed promising histological results that could be applied to vital pulp therapy in primary teeth.

Supporting information

Acknowledgments

The Researchers would like to thank the Deanship of Graduate Studies and Scientific Research at Qassim University for financial support (QU-APC-2024-9/1).