Introduction

Pulpotomy consists of removing the infected and inflamed coronal pulp and maintaining the vital radicular pulp, aiming at preserving the primary tooth until the ideal period of exfoliation [1, 2]. Pulp capping materials recover the remaining pulp to stimulate dentinal barrier formation [2]. The ideal capping material is biocompatible and can be associated with alternative therapies, such as photobiomodulation (PBM) application [3].

The use of PBM in pulp treatment has gained space in pediatric dentistry due to its anti-inflammatory effects on pulp tissue through increasing vascularization, while decreasing edema and pain [3, 4]. Pulp responses can be evaluated clinically, radiographically, histologically, as well as, through immunohistochemistry by the expression of markers related to the repair of the remaining pulp [1, 5].

Cluster of differentiation 31 (CD31) is a protein that regulates cellular adhesion signaling, leukocyte migration, activation of platelets and T cells, and angiogenesis [6, 7]. This marker is expressed in the endothelial cells of blood vessels of the pulp tissue and is related to the inflammatory response because it participates in the repair of cell diapedesis towards the inflamed area. Additionally, CD31 is linked to angiogenesis and vasculogenesis contributing to the regenerative functions of the pulp [8, 9].

This study aimed to evaluate the effects of photobiomodulation on CD31 expression in pulp tissue repair of human primary teeth.

Material and methods

Ethical considerations

All the parents or legal guardians of the children participating in the research signed the informed consent form at pretreatment screening period, after approval by the Institutional Review Board of Bauru School of Dentistry, University of Sao Paulo regarding ethical aspects (Protocol no. #058/2011) and in accordance with the Helsinki Declaration.

Sample selection

Inclusion criteria for tooth selection were as follows: primary mandibular first or second molars compromised by deep caries with the possibility of proper restoration, vital pulp with no fistula or abscess, and absence of internal or external root resorption at radiographic examination. Exclusion criteria were related to the presence of systemic pathology and history of allergic reaction to local anesthetics or some of the constituents of the dressing materials.

Clinical procedures

Fifteen molars were divided into the study groups: GI—calcium hydroxide (CH), GII—photobiomodulation therapy followed by calcium hydroxide (PBM + CH), and GIII—photobiomodulation therapy followed by zinc oxide and eugenol (PBM + ZOE).

Two previously calibrated pediatric dentists performed pulpotomy procedures, at one single session as described previously [4]. In CH group, the remaining pulp tissue was dressed with calcium hydroxide alone after hemostasis (Biodinâmica Química e Farmacêtica Ltda., Ibiporã, PR, Brazil). In the PBM groups, an indium–gallium–aluminum phosphide (InGaAlP) diode laser (Twin Flex Evolution, MMOptics®—São Carlos/São Paulo, Brazil) was used for the irradiation through 320-μm diameter optical fiber in contact with pulp tissue [3]. The red laser diode parameters were set at 660-nm wavelength, 10-mW power output, 2.5-J/cm2 energy density, 50–60-Hz frequency, output beam area of 0.04 cm2, and irradiation time of 10 s in continuous mode as previously described [3, 4]. Prior to laser application, the output power was checked by a radiometer (Laser Check, MMOptics®—São Carlos/São Paulo, Brazil). After laser irradiation in PBM + CH and PBM + ZOE groups, powdered calcium hydroxide and zinc oxide and eugenol paste covered the pulp remaining, respectively. All teeth were restored with reinforced zinc oxide-eugenol (IRM, Dentsply, Petropolis, RJ, Brazil) followed by resin-modified glass ionomer cement (Vitremer, 3M ESPE, São Paulo, SP, Brazil). Then, the patients were dismissed and recalled at periodic follow-ups at 3-month intervals, until the treated teeth achieved the regular exfoliation period to be extracted for histological and immunohistochemistry analyses [1, 3].

Histological analysis

After extraction, the teeth were immediately fixed in 10% neutral formalin solution for 24 h, and decalcified in 4% EDTA solution for 45–60 days. Then, 5-μm thick serial sections were prepared to be routinely assessed after hematoxylin and eosin staining and submitted to immunohistochemical procedures [1].

To perform the immunohistochemical analysis, 5-μm thick serial tissue sections were digested with Proteinase K (Dako North America, Carpinteria, CA, USA) for 25 min, and the endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution in methanol (0.01 M) (Easy Path (EP) 12-205222, São Paulo, SP, Brazil) for 10 min. Sections were incubated with polyclonal goat anti-human CD31 antibody (Santa Cruz—(M-20) sc—1506, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:100 dilution for 30 min, and then sections were rinsed with phosphate-buffered saline and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Advance, DAKO North America Inc., Carpinteria, CA, USA) for 30 min. Further staining was performed with 3,3′-diaminobenzidine tetra hydrochloride (DAB) solution and Lillie-Mayer’s hematoxylin was used to counterstained the sections for 60 s.

The histological evaluation was based on the scores previously described for inflammation [3]: 0 (none), 1 (mild), 2 (moderate), 3 (intense), and 4 (necrosis) and vascularization: 0 (absent), 1 (regular amount of blood vessels), and 2 (great amount of blood vessels). For the immunohistochemical evaluation, CD31 expression in the dentin-pulp complex was descriptively analyzed under light microscope. Kruskall-Wallis followed by Dunn test was used to determine statistically significant differences (p < 0.05).

Results

During the evaluation period, all groups demonstrated clinical and radiographic success. Histological analysis of the sections confirmed that the remaining radicular pulp tissue of all teeth remained vital, and the comparison among the groups using the score system revealed no statistically significant difference regarding inflammation and the amount of blood vessels, as shown in Table 1 (p > 0.05).

Table 1 Histological analysis for pulp inflammation and vascularization
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Microscopic descriptive evaluation of GI revealed a variable but mainly mild mononuclear inflammatory infiltrate within dense connective tissue showing regular vascularization. GII presented a dense connective tissue highly vascularized with no inflammatory infiltrate. GIII showed in most of the cases mild to moderate inflammatory infiltration with a loose and highly vascularized connective tissue (Fig. 1a–c).

Fig. 1
figure 1

Representative images of HE staining (a–c) and CD31 immunostaining (d–f) for GI (CH), GII (PBM + CH) and GIII (PBM + ZOE). GI (CH) shows dense connective tissue with regular vascularization and mild mononuclear inflammatory infiltrate (a) in association with CD31-immunostained blood vessels (➔; (d). GII (PBM + CH) shows dense connective tissue with regular vascularization and no inflammatory infiltrate (b), revealing discreet CD31-immunostained blood vessels (➔) scattered by the pulp tissue with none or rare inflammatory cells (e). GIII (PBM + ZOE) shows loose connective tissue highly vascularized with moderate and mainly perivascular inflammatory infiltrate (c), and CD31-immunopositivity in large blood vessels (➔; f). Scale bars indicate 20 μm (d, e), 50 μm (a) and 100 μm (b, c and f); ii means inflammatory infiltrate

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The immunohistochemistry analysis revealed positive CD31 expression in the blood vessels of all the studied groups. Notwithstanding, GI and GIII immunostaining was observed in large blood vessels associated with inflammatory cells, whereas GII showed discreet immunostaining predominantly in small blood vessels scattered by the pulp tissue with none or rare inflammatory cells (Fig. 1d–f).

Discussion

The decision on which material should be placed over the remaining pulp tissue following pulpotomy is controversial because the available capping materials do not present all the requirements for an ideal material. Our study showed that PBM prior to calcium hydroxide assisted in repairing process, decreasing inflammation, and enriching vascularization. The rationale behind this affirmation is that GII sections revealed lack of both inflammatory cells and enlarged blood vessels on histological and immunohistochemistry analyses. Although calcium hydroxide is routinely used as capping material in primary teeth, many studies report different success rates [2, 10, 11]. Further, other histological studies described the variation in chronic inflammatory response to calcium hydroxide [2, 3, 12,13,14,15,16]. Thus, long-term efficacy of calcium hydroxide is considered controversial [13]. Moreover, the pulp inflammatory condition is relevant to reach success after employing calcium hydroxide as capping material in the pulpotomy of primary teeth [17].

Inflammation is part of the repair process that involves the migration of leukocytes, cell differentiation, and proliferation [18,19,20]. Some studies evaluated the expression of markers related to the process of tooth tissue formation after pulp treatment with different materials in animals [21, 22] and human teeth [1]. Lourenço Neto et al. [5] evaluated DMP-1 expression, which acts on hard tissue formation, after the use of PBM over the pulp tissue following pulpotomy and found that PBM associated with calcium hydroxide helped in repairing. As far as we are concerned, this is the first evaluation of CD31 expression after PBM on pulp tissue of pulpotomized primary teeth.

Currently, PBM is used as adjuvant to stimulate the healing process of damaged hard tissues [3, 4, 23, 24]. To reach anti-inflammatory action, the light energy is absorbed by cellular chromophores and transformed into chemical energy, resulting in alterations in cell metabolism [25]. Fibroblasts have many photoreceptor molecules, which release chemical mediators when stimulated, mainly at the beginning of the healing process [26]. The positive biostimulation induced by laser regulates different factors related to healing process: modulation of inflammation, acceleration of epithelialization, increase of collagen synthesis, and stimulation of vascular neoformation [3, 27]. The blood vessel neoformation allows providing enough nutritional supply to fibroblasts during tissue repairing process [20].

The growth of new blood vessels based on preexisting vessels is known as angiogenesis [28, 29], which is regulated by many cytokines and growth factors [28, 30, 31]. Angiogenesis can be quantified by immunohistochemical analysis of micro-vessel density, through identifying proteins expressed on the surface of endothelial cells marked by monoclonal antibodies [28]. CD31, a transmembrane glycoprotein from endothelial cells, is one of the markers involved in this process. CD31 is part of the endothelial intercellular junction and is present on the surface of the inflammatory cells, such as monocytes, macrophages, and neutrophils. Additionally to angiogenesis, endothelial cells play a fundamental role in controlling coagulation, thrombolysis, vascular permeability, and inflammation, favoring leukocyte migration [7].

The literature suggests that CD31 molecule has pro-inflammatory action, promoting inflammation by stimulating leukocyte migration; and anti-inflammatory effects, due to its capacity of recruiting cytoplasmic phosphatases that inhibit leukocyte activation; reduces the production of pro-inflammatory cytokines; and restores the integrity of the vascular barrier through mechanisms not fully understood yet [8]. Pimenta et al. [32] histologically evaluated CD31 expression in inflamed and sound pulp tissue of permanent teeth referred for serial extraction. CD31 expression was greater in the inflamed tissue when associated with the expression of the lymphatic marker VEGF-3. Notwithstanding, CD31 was also identified in sound pulp tissue. Bruno et al. [33] evaluated through immunohistochemistry the inflamed and sound pulp tissue of permanent teeth and found CD31 expression both in sound pulp tissue and in pulp tissue with pulp inflammation. The greatest density of CD31 + blood vessels was seen in the teeth with irreversible pulpitis. Similarly, Sawa et al. [34] analyzed the CD31 immunoexpression in inflamed and sound pulp tissue of third molars and found larger blood vessels in inflamed pulp, although CD31 expression was seen in blood vessels of sound pulp tissue. Nevertheless, CD31 expression was more intense especially in medium-size vessels. Large blood vessels favor the increase of tissue fluid with inflammatory cells, which corroborates this present study showing intense CD31 expression in GI and GIII, with larger blood vessels. On the other hand, in GII, small blood vessels showed discreet CD31 expression. The intense expression of adhesion molecules in vascular tissue is associated to leukocyte migration, a critical step in the beginning of the immunological response to tissue inflammation [35].

Angiogenesis modulation through PBM has been investigated to verify a contribution to repairing process. However, the literature lacks consensus on the ideal laser parameters and expected therapeutic effectiveness. Schindl et al. [36] evaluated in vitro the effects of 670-nm LLL irradiation with different dosimetries, showing that energy densities of 2, 4, and 8 J/cm2 stimulated endothelial cell proliferation. Similarly, Góralczyk et al. [18] found that 635-nm LLL irradiations with similar energy densities stimulated cellular proliferation and positively modulated growth factors and receptors involved in the mechanism of vascular neoformation. Based on this information, the wavelength (660 nm) and energy density (2.5 J/cm2) of the red laser employed in our study probably favored the repair of the pulp tissue.

Our study showed the advantages of PBM associated with calcium hydroxide in pulp tissue repairing of primary teeth undergoing pulpotomy. Laboratorial analysis of pulp tissue repair is essential to judge the pulp capping material effectiveness [37], because although many studies reveal high clinical and radiographic success rates of pulp capping materials, inflammatory alterations of the pulp are seen histologically [2, 3, 16, 38, 39]. Thus, the histological results of this study suggest that associations of standard therapies with new technologies may be indicated aiming at advances in the pulp treatment of primary teeth.

Conclusion

The CD31 expression was greater in the group PBM when associated with calcium hydroxide. Photobiomodulation therapy followed by calcium hydroxide helped in repairing and seemed to regulate angiogenesis and leukocyte migration processes in the remaining pulp tissue after primary teeth pulpotomy.