Effect of combined application of premixed bioceramic paste and diode laser in vital pulp therapy: an immunohistochemical randomized controlled split-mouth in vivo animal experiment

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Restor Dent Endod. 2025;.rde.2026.51.e4

Publication date (electronic) : 2025 December 8

doi : https://doi.org/10.5395/rde.2026.51.e4

Mo’men A. Salama^1,, Dalia M. Fayyad¹, Mohamed I. Rabie¹, Manar A. A. Selim², Mahmoud F. Ahmed^3,4

¹Department of Endodontics, Faculty of Dentistry, Suez Canal University, Ismailia, Egypt

²Department of Oral Biology, Faculty of Dentistry, Suez Canal University, Ismailia, Egypt

³Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt

⁴Department of Surgery and Theriogenology, Faculty of Veterinary Medicine, King Salman International University, Ras Sudr, Egypt

Citation: Salama MA, Fayyad DM, Rabie MI, Selim MAA, Ahmed MF. Effect of combined application of premixed bioceramic paste and diode laser in vital pulp therapy: an immunohistochemical randomized controlled split-mouth in vivo animal experiment. Restor Dent Endod 2026;51(1):e4.

^*Correspondence to Mo’men A. Salama, PhD Department of Endodontics, Faculty of Dentistry, Suez Canal University, 4.5 Km Ring Road, Ismailia 41522, Egypt Email: momen_salama@dent.suez.edu.eg

Received 2025 August 17; Revised 2025 October 11; Accepted 2025 October 14.

Abstract

INTRODUCTION

Preserving pulp vitality is a core component of modern minimally invasive dentistry, boosted by advancements in biomaterials and an enhanced comprehension of pulp biology [1,2]. These advancements have instigated a paradigm change from traditional root canal treatment to more conservative approaches aimed at conserving pulp tissue where clinically feasible [3,4]. Direct pulp capping (DPC) is a crucial procedure of vital pulp therapies for addressing pulp exposures resulting from trauma or caries, especially when the pulp is healthy or shows only reversible inflammation [5]. The principal objective of DPC is to preserve the vitality and functionality of the dental pulp by efficiently sealing the exposure site and facilitating reparative dentinogenesis, particularly through the development of a protective dentin bridge [6,7].

Emerging evidence from comparative investigations highlights the advantages of premixed bioceramic materials, such as Well-Root PT (Vericom, Chuncheon, Korea), over traditional calcium hydroxide formulations for DPC [8]. The premixed bioceramic material, Well-Root PT, is composed of calcium aluminosilicate, zirconium oxide, tantalum oxide, calcium phosphate monobasic, and various fillers [9]. This material’s unique composition enhances biocompatibility, minimizes cytotoxicity, and provides superior sealing properties upon application [9]. The inherent high alkalinity of Well-Root PT creates a favorable microenvironment conducive to pulp healing while simultaneously inhibiting bacterial ingress. Furthermore, Well-Root PT provides clinical performance analogous to mineral trioxide aggregate (MTA) but improved handling characteristics, positioning it as a dependable alternative for both pulp capping procedures and broader regenerative endodontic applications [8,10].

The incorporation of low-level laser therapy (LLLT) alongside DPC represents a notable progression in minimally invasive endodontics, utilizing photobiomodulation to improve dental pulp tissue regeneration [11]. LLLT typical power range of (10–500 mW) [12]. LLLT primarily acts through the absorption of light by mitochondrial photoreceptors, particularly cytochrome C oxidase in the electron transport chain [13]. This absorption leads to a short-term activation of the respiratory chain, enhancing oxidative phosphorylation and increasing adenosine triphosphate production. This process alters the redox state of mitochondria and the cytoplasm, affecting cellular redox mechanisms. The generation of reactive oxygen species at controlled levels acts as signaling molecules to activate various molecular pathways and transcription factors, which promote cellular proliferation, differentiation, and reduced inflammation [13,14]. It has demonstrated efficacy in pain reduction, enhancement of wound healing, the promotion of bone repair and remodeling, assistance in pulp regeneration, and stimulation of angiogenesis [5,14,15]. It improves reparative dentinogenesis by stimulating odontoblast activity and promoting dental pulp stem cell differentiation [16]. Furthermore, LLLT improves dentin matrix structure and mineralization by upregulating the production of structural proteins, such as dentin sialo phosphoprotein (DSPP) [14]. This process promotes the formation of a thicker, more uniform dentin bridge, which enhances long-term sealing efficiency and reduces the possibility of microleakage [17].

The most prevalent non-collagenous protein present in the dentin matrix is dentin sialoprotein (DSP). It regulates dentinogenesis and biomineralization [18]. Furthermore, DSP regulates transcriptional activity and intracellular signaling cascades. These regulatory roles regulate the differentiation of dental pulp stem/progenitor cells, which support both physiological homeostasis and the reparative/regenerative ability of dental tissues [19].

Although both LLLT and advanced bioceramics such as Well-Root PT have individually demonstrated significant therapeutic potential, their combined effects in DPC remain insufficiently explored. Moreover, the available evidence is limited regarding their comparative influence on dentinogenesis-related markers, particularly DSP.

Therefore, this study hypothesized that Well-Root PT would promote pulpal healing and upregulate dentinogenesis-related markers compared to the gold-standard material, White MTA Angelus, and that adjunctive use of LLLT would further enhance this effect. Accordingly, the study aimed to evaluate the pulpal response to DPC using Well-Root PT, with and without adjunctive LLLT, and to compare its efficacy with Angelus MTA through immunohistochemical analysis of DSP expression as a key marker of odontoblastic activity and dentin matrix formation.

METHODS

Experimental design

The study was performed at the Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Suez Canal University. The protocol was approved by the Research Ethics Committee of the Faculty of Dentistry, Suez Canal University (approval No. 529/2022), in accordance with ethical standards and regulations for animal research, and in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. The study was carried out as a randomized, controlled, experimental trial. Eight mature Mongrel dogs were used in this study with intact dentition. The dogs aged between 19 ± 3 months and weighed (15.4 ± 3.2). Dogs were kept for 2 weeks at the animal house before the study for close observation and acclimatization. Sample size was calculated based on prior research [17,20]. Sample size estimation was performed using G*Power version 3.1.9.2 (Heinrich Heine University Düsseldorf, Düsseldorf, Germany) [21], with a significance level (α) of 0.05 and a power (1–β) of 0.80. Adjustments were made to account for potential specimen loss and the clustered (split-mouth) design, resulting in a final allocation of 96 teeth (32 teeth per material group; 16 per subgroup), thereby ensuring adequate statistical power.

The incisor teeth were randomly evaluated by three independent observers who were blinded to the material and technique used. Only the allocator (M.A.S) was aware of the group assignments, which corresponded to the different capping materials. Using a split-mouth design, the teeth were randomly allocated to one of three groups according to a computer-generated random sequence (Microsoft Excel RAND function) prepared by an investigator independent of the surgical procedures. For each dog, the 12 incisors were allocated so that each group (Well-Root PT, MTA, and control) was represented by four teeth per dog. Within each group, two teeth were assigned to the LLLT subgroup and two to the non-LLLT subgroup. The dogs were randomly assigned to be euthanized at either 2 weeks (dogs 1–4) or 8 weeks (dogs 5–8), ensuring equal numbers for each period (Table 1).

Table 1.

Number of teeth assigned to each experimental subgroup at 2 and 8 weeks

RESULTS

Quantitative analysis was performed using the mean area percentage of DSP expression. The DSP expression was evaluated in the pulp tissue at two time points: 2 weeks and 8 weeks. In both the control and MTA groups without LLLT, relatively low mean area percentages for DSP were observed in different parts of the pulp, such as odontoblasts, fibroblasts, collagen fibers, ground substance, and blood vessel walls after 2 weeks. In contrast, the Well-Root PT subgroup exhibited a higher mean area percentage of DSP at the same time point (Figure 1A, C, and E). After 8 weeks, the control subgroup without LLLT maintained low area percentage values, while the MTA subgroup displayed a moderate increase. The Well-Root PT subgroup demonstrated a more pronounced response, with the highest mean area percentage observed in staining (Figure 2A, C, and E).

Figure 1.

Representative microscopic images showing immunohistochemical staining of dentin sialoprotein in pulp tissue (*) after 2 weeks. (A, B) Showing diffuse expression in the control group, with (A) non-LLLT subgroup demonstrating staining of collagen fibers, and (B) LLLT subgroup showing staining of both collagen fibers and the cytoplasm of odontoblasts. (C, D) Showing expression in the MTA group, with (C) non-LLLT subgroup demonstrating patchy staining in collagen fibers and diffuse cytoplasmic staining in odontoblasts, and (D) LLLT subgroup showing extensive staining in both collagen fibers and odontoblasts. (E, F) Showing expression in the Well-Root PT group, with (E) non-LLLT subgroup demonstrating patchy staining in collagen fibers and diffuse staining in odontoblasts, and (F) LLLT subgroup showing similar expression patterns. MTA, mineral trioxide aggregate; LLLT, low-level laser therapy. Well-Root PT: Vericom, Chuncheon, Korea.

Figure 2.

Representative microscopic images showing immunohistochemical staining of dentin sialoprotein in pulp tissue (*) after 8 weeks. (A, B) Showing expression in the control group, with (A) non-LLLT subgroup demonstrating patchy expression in collagen fibers and patchy expression in odontoblasts, and (B) LLLT subgroup demonstrating diffuse expression in collagen fibers and in the cytoplasm of odontoblasts. (C, D) Showing expression in the MTA group, with (C) non-LLLT subgroup demonstrating patchy expression in both collagen fibers and odontoblasts, and (D) LLLT subgroup showing patchy expression in collagen fibers, diffuse cytoplasmic expression, and some nuclear expression in odontoblasts. (E, F) Showing expression in the Well-Root PT group, with (E) non-LLLT subgroup demonstrating patchy expression in collagen fibers and diffuse cytoplasmic expression in odontoblasts, and (F) LLLT subgroup demonstrating patchy expression in collagen fibers and diffuse cytoplasmic and nuclear expression in odontoblasts. MTA, mineral trioxide aggregate; LLLT, low-level laser therapy. Well-Root PT: Vericom, Chuncheon, Korea.

In the LLLT control subgroup, a slight increase in the mean area percentage was observed at 2 weeks (Figure 1B), with a more pronounced increase by the 8-week time point (Figure 2B). However, both the MTA and Well-Root PT subgroups with LLLT showed higher mean percentages (Figure 1D and F), with a further increase at 8 weeks (Figure 2D and F).

Statistical analysis of the mean area percentage between the study groups and between the indicated time points within the same group was performed. There were significant variations in DSP expression between the non-LLLT and LLLT subgroups within the three groups (p < 0.001). However, post hoc testing did not reveal any significant differences between the non-LLLT MTA and Well-Root PT subgroups, nor between the MTA and Well-Root PT subgroups after the LLLT, at either the 2-week or 8-week time points (Table 2).

Table 2.

Descriptive statistics of the intragroup comparison of DSP immunoexpression area percentage between 2 and 8 weeks

At the 2-week evaluation, the highest mean area percentage of DSP expression was observed in the LLLT Well-Root PT subgroup, followed by the non-LLLT Well-Root PT, the LLLT MTA, and the non-LLLT MTA subgroups. The control group exhibited the lowest levels. By the 8-week time point, a similar trend persisted; however, the LLLT MTA subgroup showed a slightly higher mean value than the non-LLLT Well-Root PT subgroup. In intragroup comparisons, all subgroups showed a statistically significant increase in DSP mean area percentage from 2 to 8 weeks (p < 0.001), indicating a general time-dependent increase regardless of treatment. However, despite differences in mean values, no significant differences were observed between the LLLT and non-LLLT subgroups at either time point in the same group (Table 3). These findings supported the conclusion that the various capping materials significantly influenced the DSP expression signals.

Table 3.

Descriptive statistics of the intergroup comparison of DSP immunoexpression area percentage

DISCUSSION

In this study, the quality of the dentin bridge formed following DPC was evaluated using immunohistochemical analysis to assess the expression of DSP, a reliable marker of odontoblastic activity within the pulp tissue. The efficacy of Well-Root PT and MTA was compared to determine their respective performance. Additionally, the potential role of LLLT in enhancing DSP expression and promoting pulp tissue regeneration was also investigated.

Dogs were selected as the animal model for this study due to their close anatomical and physiological resemblance to human teeth [26]. Their similar pulp-to-dentin ratio and overall tooth structure make them well-suited for investigating pulp responses and dentin bridge formation [27]. Moreover, the healing patterns and tissue reactions in dogs closely mirror those in humans, enhancing the clinical relevance and translational value of the study’s findings [28]. However, it should be noted that healing in dogs occurs more rapidly than in humans. This limitation should be taken into account when extrapolating the findings to clinical scenarios.

In this study, Well-Root PT was compared with MTA as a contemporary bioactive material for DPC. Well-Root PT offers a shorter setting time, which may help reduce operator variability and improve clinical efficiency. However, despite its favorable handling and physical properties, research on its biocompatibility and bioactivity remains limited [8,29], highlighting the importance of evaluating its performance against the well-established MTA. Meanwhile, LLLT was used for its ability to promote rapid hemostasis, sterilize the pulp surface, and reduce inflammation [30,31]. In addition, it supports tissue regeneration and aids in dentin bridge formation. It is also compatible with common pulp capping materials, potentially enhancing their effectiveness [20].

In this study, DSP expression was evaluated at two time points: 2 weeks, to assess the early healing response, including odontoblast differentiation and initial dentin formation [32]. Furthermore, the 8-week period was used to examine long-term outcomes such as reparative dentin bridge formation, reduced inflammation, and maintained pulp vitality. These time points are appropriate for evaluating the effectiveness of pulp capping materials [17,33]. However, the present study cannot fully confirm the long-term formation of a reparative dentin bridge.

Immunohistochemical analysis conducted at both 2 weeks and 8 weeks revealed that the highest mean values of DSP expression were observed in the subgroups treated with MTA and Well-Root PT, with or without adjunctive LLLT. In contrast, the control subgroups, which received only a Teflon barrier without any bioactive material, exhibited the lowest levels of DSP expression. The increased DSP expression observed with MTA and Well-Root PT is attributed to their bioactive properties, which promote odontoblast differentiation and reparative dentin formation. These materials release calcium ions that form hydroxyapatite-like crystals, fostering a favorable environment for pulp healing [1,34]. They also upregulate growth factors like bone morphogenetic protein-2 and transforming growth factor-beta and create an alkaline pH that reduces inflammation, further enhancing odontoblastic activity and DSP synthesis [35,36]. The findings of this study are in agreement with the results reported by Chae et al. [8], who demonstrated that despite releasing fewer calcium ions compared to MTA, Well-Root PT exhibited comparable bioactivity, as evidenced by hard tissue formation and DSP expression.

The study found no significant difference in DSP expression between the LLLT and non-LLLT groups, suggesting that the diode laser, under the applied parameters, did not enhance DSP expression beyond the effect of the capping materials alone. Similarly, Martín et al. [37] reported that diode laser activation during dentin conditioning resulted in lower expression levels of odontoblast-related markers such as DSPP and DMP-1 compared with EDTA alone, supporting the notion that diode laser irradiation may not significantly enhance DSP expression or odontoblastic differentiation. Additionally, the laser settings used may not have been optimal for influencing odontoblastic activity. On the other hand, Alharbi et al. [17] reported that the application of LLLT prior to DPC enhanced the expression of other markers, such as RUNX2 and osteocalcin. This discrepancy may be attributed to differences in the biomarkers assessed, as DSP is specifically associated with odontoblastic differentiation, whereas RUNX2 and osteocalcin are more broadly related to early osteogenic activity. Additionally, variations in LLLT parameters—such as energy settings, number of applications, and exposure duration—could contribute to the inconsistent outcomes across studies. In contrast, Deng et al. [38] attributed this effect to the coagulative and hemostatic properties of the LLLT, which may reduce tissue moisture and potentially interfere with the optimal setting reaction, as it relies on a moist environment for proper hardening.

A notable observation was that at 8 weeks, the LLLT MTA subgroup showed a slightly higher mean DSP expression than the non-LLLT Well-Root PT subgroup. Although not statistically significant, this finding may indicate that the stimulatory effects of LLLT on odontoblastic activity and dentin matrix protein expression were not transient but persisted over time. Future research with extended follow-up periods and varied laser protocols could help clarify the potential synergistic role of LLLT.

Statistically significant differences in DSP expression were also observed between the two evaluation time points within each subgroup. These findings align with Alharbi et al. [17], who reported significant changes in marker expression between early and late follow-up periods in pulp tissues treated with different capping materials, indicating progressive healing and maturation. This temporal variation likely reflects the dynamic nature of the pulp healing process, which begins with an initial inflammatory response followed by progressive reparative events [39]. The increased DSP expression observed at later stages may indicate ongoing odontoblast differentiation and dentin matrix deposition [40]. Additionally, biological variability among the animals, including differences in immune responses and healing capacities, may have contributed to the observed differences between the time points. Finally, the findings of this study support the bioactive potential of both MTA and Well-Root PT in promoting odontoblastic activity and DSP expression.

The limitations of this study include the indirect evaluation of reparative dentin bridges due to insufficient histological clarity; and the use of only a single LLLT wavelength and protocol, which may not capture its full therapeutic potential. Future studies should employ varied laser parameters, longer follow-up periods, and additional odontoblast-specific markers to better elucidate LLLT’s role in vital pulp therapy.

CONCLUSIONS

Well-Root PT may serve as a viable alternative to MTA for DPC procedures, demonstrating comparable biological performance in promoting DSP expression and pulp healing. While LLLT did not significantly influence the outcomes observed in this study. Therefore, the choice of suitable biocompatible capping material appears to play a more critical role in the success of DPC than the use of LLLT. Further studies are required to explore the optimization of LLLT parameters and their potential synergistic effects with various capping materials.

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