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. 2015 Jul 30;227(4):487–496. doi: 10.1111/joa.12359

Apoptosis and reduced microvascular density of the lamina propria during tooth eruption in rats

José Paulo de Pizzol Júnior 1, Estela Sasso-Cerri 2, Paulo Sérgio Cerri 2,✉
PMCID: PMC4580106  PMID: 26228092

Abstract

During tooth eruption, structural and functional changes must occur in the lamina propria to establish the eruptive pathway. In this study, we evaluate the structural changes that occur during lamina propria degradation and focus these efforts on apoptosis and microvascular density. Fragments of maxilla containing the first molars from 9-, 11-, 13- and 16-day-old rats were fixed, decalcified and embedded in paraffin. The immunohistochemical detection of vascular endothelial growth factor (VEGF), caspase-3 and MAC387 (macrophage marker), and the TUNEL method were applied to the histological molar sections. The numerical density of TUNEL-positive cells and VEGF-positive blood vessel profiles were also obtained. Data were statistically evaluated using a one-way anova with the post-hoc Kruskal–Wallis or Tukey test and a significance level of P ≤ 0.05. Fragments of maxilla were embedded in Araldite for analysis under transmission electron microscopy (TEM). TUNEL-positive structures, fibroblasts with strongly basophilic nuclei and macrophages were observed in the lamina propria at all ages. Using TEM, we identified processes of fibroblasts or macrophages surrounding partially apoptotic cells. We found a high number of apoptotic cells in 11-, 13- and 16-day-old rats. We observed VEGF-positive blood vessel profiles at all ages, but a significant decrease in the numerical density was found in 13- and 16-day-old rats compared with 9-day-old rats. Therefore, the establishment of the eruptive pathway during the mucosal penetration stage depends on cell death by apoptosis, the phagocytic activity of fibroblasts and macrophages, and a decrease in the microvasculature due to vascular cell death. These data point to the importance of vascular rearrangement and vascular neoformation during tooth eruption and the development of oral mucosa.

Keywords: apoptosis, eruptive pathway, lamina propria, microvasculature, tooth eruption

Introduction

Tooth eruption involves a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. The eruptive process is divided into five phases: pre-eruptive movement, intra-osseous eruption, mucosal penetration, and both pre-occlusal and post-occlusal eruption (Marks & Schroeder, 1996; Wang, 2013). The initial movements of the developing tooth germ make room for the tooth according to the growth of the maxillary and jaw processes. The active eruptive process culminates with the movement of the tooth towards the oral cavity and thus requires intense bone resorption in the occlusal portion of the bony crypt (Wise, 2009; Cerri et al. 2010). There is evidence that dental follicles play an important role in this cascade because they produce several factors including tumour necrosis factor-α (TNF-α), transforming growth factor β (TGF-β), interleukin-1 (IL-1), colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor kB ligand (RANKL). TNF-α, CSF-1 and RANKL are released by dental follicle cells and stimulate the migration and differentiation of bone-marrow precursor mononuclear cells into osteoclasts (Wise et al. 2005; Wise & King, 2008; Wise, 2009). Moreover, mast cells in the lamina propria participate in the recruitment of osteoclasts and, consequently, stimulate the resorption of bone overlaying the occlusal portion of the tooth germ (Cerri et al. 2010).

After resorption of the bone interposed between the tooth germ and the oral mucosa, the tooth reaches the lamina propria, which then undergoes structural changes that characterize the eruptive phase of mucosal penetration (Marks & Schroeder, 1996). These changes in the connective tissue may include cell death and alterations in the vascular–nervous network. It is known that the vasculature plays a pivotal role both in the maintenance of homeostasis and in the degradation and remodelling of various tissues and organs (Clément et al. 1999). Vascular endothelial growth factor (VEGF) is a well-characterized angiogenic factor that is highly expressed in the endothelial cells (Neufeld et al. 1999; Ferrara et al. 2003). Although tooth eruption is a rapid process, breakdown of the lamina propria involves a series of precisely controlled cellular mechanisms because the eruptive process occurs in the absence of bleeding or inflammatory reactions (Verma et al. 2005). In this context, cell death by apoptosis may be involved in the lamina propria degradation process that occurs during tooth eruption.

Apoptosis is a programmed cell death pathway that has an important role in the development, maintenance, and remodelling of tissues and organs (Kerr et al. 1972; Cerri et al. 2000; Danial & Korsmeyer, 2004; Cerri, 2005; Czabotar et al. 2014). This form of cell death involves a series of coordinated molecular events that lead to well-characterized morphological changes such as shrinkage, convoluted cytoplasm, condensed chromatin, and the formation of plasma and nuclear membrane blebs; these cells also frequently undergo fragmentation, which gives rise to apoptotic bodies (Cerri et al. 2000; Cerri, 2005; Faloni et al. 2012). These morphological alterations occur due to the actions of intracellular enzymes such as caspases, a family of cysteine-dependent aspartate-specific proteases (Jänicke et al. 1998; Brentnall et al. 2013). Caspase enzymes are divided into two groups based on the timing of their involvement in the apoptotic processes: initiator caspases (such as caspase-1, -2, -4, -5, -8, -9, -10, -11 and -12) or executor caspases (such as caspase-3, -6 and -7) (Hyman & Yuan, 2012; Parrish et al. 2013). Caspase-3 is activated by a variety of apoptotic signals including initiator caspases (Stennicke et al. 1998) and seems to play a key role in apoptotic cell death; therefore, it is considered to be a marker of apoptosis (Green, 2000; Gown & Willingham, 2002; Caneguim et al. 2011; Faloni et al. 2012). Caspases are responsible for the cleavage of several structural proteins such as cytoplasmic cytoskeletal proteins, the degradation of nuclear proteins, and the activation of nucleases that lead to DNA fragmentation (Shi, 2002; Brentnall et al. 2013). DNA fragmentation, which is an important event in apoptosis, can be detected by the TUNEL method (Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labelling) (Gavrieli et al. 1992). The TUNEL method combined with other methods such as caspase-3 immunohistochemistry and ultrastructural features have been used to identify apoptotic cells (Huppertz et al. 1999; Cerri & Katchburian, 2005; Faloni et al. 2012; Longhini et al. 2013; Abdi et al. 2014; Beltrame et al. 2015).

The eruptive pathway is an useful model for investigating the lamina propria degradation process because tooth eruption is a normal physiological phenomenon. Few studies have examined the structural changes that occur along the eruptive pathway of the lamina propria during tooth eruption (Ten Cate, 1971; Verma et al. 2005). Among these studies, an ultrastructural analysis focusing on the breakdown of the extracellular matrix was carried out in monkeys (Ten Cate, 1971). In addition, the structural components and cell types in the opercula of permanent canines, molars and premolars of humans was reported by Verma et al. (2005). However, the use of human biopsies restricts experimental design, hinders the analysis of distinct periods and thus makes it impossible to observe the entire eruptive process. The mechanisms involved in the cell death and microvascularization alterations that are observed in the lamina propria of the eruptive pathway remain unknown. Because connective tissue degradation is required for movement of the tooth through the maxilla to reach the oral cavity, it is reasonable to suggest that apoptosis may be responsible for the physiological degradation of the lamina propria. In addition, the microvascular plexus may also undergo structural changes that allow the tooth movements to occur in the absence of bleeding. Therefore, in this study we evaluate the structural changes that occur in the eruptive pathway of rat molars and focus on apoptotic cell death and the microvascular density in the lamina propria at the different eruptive phases.

Materials and methods

The treatment of the animals was performed in accordance with Brazilian animal care and national laws on animal use. Our research protocol was authorized by the Ethical Committee for Animal Research of the São Paulo State University, Brazil (Araraquara Dental School-UNESP).

We used 20 Holtzman male rats at 9, 11, 13 and 16 days of age. The rats were obtained from the Animal House of the Dental School of Araraquara (UNESP). The animals were housed in polypropylene cages (40  × 30  × 15 cm) that were filled with a layer of white pine shavings. During the experiment, one mother plus five male pups were housed per cage in a room with controlled temperature (23 ± 2 °C) and humidity (55 ± 10%). Rats were maintained under a 12 : 12 light/dark cycle with light onset at 07:00 h. Standardized chow (Guabi Rat Chow, Paulinia, SP, Brazil) and water were provided ad libitum.

The ages of the rats were chosen based on the eruptive phases we wanted to examine. The first molar germs of 9- and 11-day-old rats were at the intraosseus phase, whereas the first molars of 13- and 16-day-old rats were at the mucosal penetration phase (Cerri et al. 2010). Thus, our study was conducted on four groups of rats according to their age (9-, 11-, 13- and 16-day-old). Five rats were used per group.

The rats were killed by an overdose of ketamine hydrochloride and xylazine hydrochloride, decapitated and the maxilla removed. Using a dissecting microscope, fragments of maxilla that contained the first molars were removed and placed in the fixative solution. The fragments of the right maxilla were fixed and processed for light microscopy and the fragments of the left maxilla were fixed and processed for transmission electron microscopy. This study was carried out in accordance with the US National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80-23, 1996), and every effort was made to reduce the number of animals used.

Light microscopy

The fragments of the maxilla that contained the first molar germs were fixed for 48 h at room temperature in 4% formaldehyde (freshly prepared from paraformaldehyde) and buffered at pH 7.2 with 0.1 m sodium phosphate. After decalcification for 10 days in a 7% solution of EDTA (ethylenediaminetetraacetic acid) containing 0.5% formaldehyde (Cerri et al. 2010), buffered at pH 7.2 with 0.1 m sodium phosphate (PBS), the fragments of maxilla were dehydrated in graded concentrations of ethanol and embedded in paraffin. Sagittal sections were stained with Masson’s trichrome and subjected to the TUNEL method and immunohistochemical detection of caspase-3, macrophages and VEGF.

TUNEL method

The TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labelling) method for detecting DNA breaks (Gavrieli et al. 1992) was performed as previously described (Cerri et al. 2000) and according to the Apop-Tag Plus Kit (Millipore; Temecula, CA, USA). Sections of involuting mammary gland provided by the manufacturer of the kit were used as positive controls. Negative controls were incubated in a TdT free-enzyme solution.

Numerical density of TUNEL-positive cells in the lamina propria

Images were captured using a DP-71 Olympus camera attached to an Olympus BX-51 light microscope. Quantitative analyses were performed using an Olympus Image Pro-Express 6.0.

The number of TUNEL-positive cells in the lamina propria was quantified in five rats per group for a total of 20 rats. Three non-serial sections per animal were used, and in each section, four to six fields of lamina propria were captured, for a total standardized area of 0.4 mm2 of lamina propria per animal. In each field, the number of TUNEL-positive cells was computed by one blinded and calibrated examiner at 1750×. Quantification was performed twice at intervals of at least 8 weeks to obtain the number of TUNEL-positive cells per mm2 of lamina propria per animal.

Immunohistochemistry for cleaved caspase-3

To unmask the antigenic sites, deparaffinized sections were immersed in 10 mm sodium citrate buffer pH 6.0, and placed into a microwave oven at 90–94 °C for 30 min. After the inactivation of endogenous peroxidase in 3% hydrogen peroxide for 20 min, the sections were washed in 0.1 m PBS pH 7.2 and then incubated overnight in a humidified chamber at 4 °C with rabbit anti-caspase-3 antibody (active form) (Millipore) diluted 1 : 100. The sections were washed with PBS and the immunoreaction detected by the Labelled StreptAvidin-Biotin system (Labelled StreptAvidin-Biotin system – LSAB-plus Kit; DAKO Corporation, Carpinteria, CA, USA). The immunoreaction was revealed by Betazoid DAB (DAB – Biocare Medical, Concord, CA, USA), and the nuclei were counterstained with Carazzi’s haematoxylin. For negative controls, the sections were incubated in non-immune serum instead of primary antibody.

Immunohistochemistry for detection of macrophages

For antigen retrieval, deparaffinized sections were immersed in 0.1% CaCl2 containing 0.1% trypsin for 1 h at 37 °C. After washings, the endogenous peroxidase was inactivated in 3% hydrogen peroxide for 20 min, and the sections were incubated overnight in a humidified chamber at 4 °C with mouse anti-macrophage monoclonal antibody (MAC387 – Santa Cruz Biotechnology®, USA) diluted 1 : 100. After washing in PBS, the immunoreaction was detected by using a Vectastain Kit (Vector Laboratories, Inc., Burlingame, CA, USA). Sections were incubated in biotinylated anti-mouse IgG for 40 min at room temperature and then in an avidin–biotin–peroxidase complex for 40 min. Peroxidase activity was revealed by 0.06% 3,3′-diaminobenzidine (Sigma-Aldrich Chemie, Germany), and the sections were counterstained with Carazzi’s haematoxylin. For negative controls, the immunohistochemical reaction was performed replacing the primary antibody with non-immune serum.

Immunohistochemistry for VEGF detection and numerical density of blood vessel profiles

To allow antigen recovery, deparaffinized sections were immersed in 10 mm sodium citrate buffer, pH 6.0, and placed in a microwave oven at 90–94 °C for 30 min. After inactivation of endogenous peroxidase in 3% hydrogen peroxide for 20 min, the slides were washed in 50 mm PBS at pH 7.2 and incubated with Sniper (Biocare’s Background Sniper® – Biocare Medical, USA) for 30 min at room temperature. Subsequently, the sections were incubated overnight in a humidified chamber at 4 °C with mouse monoclonal anti-VEGF antibody (Biocare Medical®, USA) that was diluted 1 : 400 in Van Gogh Diluent® (Biocare Medical, USA). After washing in PBS, the VEGF immunoreaction was detected using a MACH 4 Universal HRP-Polymer kit (Biocare Medical). The peroxidase activity was revealed by Betazoid DAB (3,3′-diaminobenzidine – Biocare Medical®), and the nuclei were counterstained with Carazzi’s haematoxylin. For negative controls, the primary antibody was replaced with non-immune serum.

The numerical density of VEGF-immunolabelled blood vessel profiles in the lamina propria was quantified in five rats per group for a total of 20 rats. In each rat, two non-serial sagittal sections containing the eruptive pathway of the first molar were used. The images were captured using a camera (DP-71, Olympus) attached to a light microscope (BX-51, Olympus) at 1750×. The number of VEGF-immunolabelled blood vessel profiles was quantified in a total standardized area of lamina propria (0.08 mm2) per animal by one blinded and calibrated examiner. The quantification was carried out twice, with intervals of at least 8 weeks between the analyses. In each rat, the total number of VEGF-immunolabelled blood vessel profiles was divided by a total standardized area (0.08 mm2) of the lamina propria to obtain the number of blood vessel profiles per mm2.

Transmission electron microscopy

Fragments of the maxilla that contained the lamina propria overlaying the first molar germs were fixed for 16 h in a solution of 4% glutaraldehyde and 4% formaldehyde buffered at pH 7.2 with 0.1 m sodium cacodylate (Cerri, 2005). After decalcification for 10 days in a solution of 7% EDTA buffered at pH 7.2 in 0.1 m sodium cacodylate, the specimens were postfixed in cacodylate-buffered 1% osmium tetroxide at pH 7.2 for 1.5 h. Subsequently, the specimens were washed in distilled water and immersed in 2% aqueous uranyl acetate for 2 h. After washing, the specimens were dehydrated in graded concentrations of ethanol, treated with propylene oxide and then embedded in Araldite.

Semithin sections stained with aqueous solution that contained 1% toluidine blue and 1% sodium borate were examined in a light microscope and suitable regions were carefully selected for trimming the blocks. Ultrathin sections were collected on grids, and stained in alcoholic 2% uranyl acetate and in lead citrate solution and examined using a Philips CM 100 transmission electron microscope.

Statistical analysis

The statistical analyses were performed using Statistical SigmaStat 3.2 software (Jandel Scientific, Sausalito, CA, USA). The differences in numerical density of TUNEL-positive cells and of blood vessel profiles were statistically analyzed among all groups (9-, 11-, 13- and 16-day-old rats). Because the groups are independent samples, a one-way anova was used for the analysis of variance. The post-hoc Kruskal–Wallis test was applied for TUNEL-positive cells, and the post-hoc Tukey test was used to examine the number of blood vessel profiles in the eruptive pathway. The significance level was set at P ≤ 0.05.

Results

Sagittal sections of maxillae revealed first molar tooth germ at different eruptive phases. Continuous bone trabeculae overlaying the occlusal portion of the first molar germ was observed in the 9-day-old rat group (Fig.1A). At 16 days, the cusp tips of the upper first molars were crossing a thin layer of lamina propria between oral epithelium and reduced enamel epithelium; this stage is typical of the mucosal penetration phase of tooth eruption (Fig.1B).

Figure 1.

Figure 1

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Light micrographs of sagittal sections of the first molar tooth germs of 9- (1A) and 16-day-old (1B) rats. (1A) The molar tooth germ is at an advanced stage of crown formation; dentine (D) and enamel (E) are present throughout the crown. The initial stage of root dentine formation (arrows) is observed in the cervical region. Note that the developing molar tooth germ is located inside the bony crypt. A continuous layer of bone trabeculae (B) is observed between the tooth germ and the lamina propria (LP). DP, dental papilla; HRS, Hertwig’s epithelial root sheath;OE, oral epithelium. (1B) The molar germ is at an advanced stage of radicular dentine formation (R). The cusp tips are passing through the oral mucosa. Note the remaining portions of the LP. D, dentine; DP, dental papilla; ES, enamel space; RE, reduced enamel epithelium. Masson’s trichrome stain.

TUNEL-positive cells (brown-yellow colour) were found in the lamina propria at different phases of tooth eruption (Figs.2A–E). However, only a few TUNEL-positive cells were observed in the 9-day-old rat group (Fig.2A), whereas several positive cells (Fig.2B) were found in the lamina propria at the more advanced stage of tooth eruption (16-day-old rat group). In the 16-day-old rats, fibroblasts exhibiting TUNEL-positive nuclei were observed throughout the thin layer of the lamina propria (Fig.2B). TUNEL-positive round or elliptic/ovoid structures of various sizes were also found (Fig.2B–E); some were in close juxtaposition to TUNEL-negative fibroblasts (Fig.2B,C) and round mononuclear cells or macrophage-like cells (Fig.2D,E). Quantitative analysis revealed a significant and gradual increase in the numerical density of TUNEL-positive cells in the lamina propria from 9 to 16 days (P ≤ 0.05). Although significant differences were not found between the 11- and 13-day-old rat groups, it is important to note that a significant increase (P ≤ 0.05) in the number of TUNEL-positive cells was observed in the lamina propria of 16-day-old rats compared with the other groups (Fig.3).

Figure 2.

Figure 2

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Light micrographs of portions of oral mucosa of the eruptive pathway of first molars submitted to the TUNEL method (brown-yellow colour) and counterstained with haematoxylin. (2A) Scarce TUNEL-positive structures (arrows) in the lamina propria (LP) of a 9-day-old rat. The inset shows a strong TUNEL-positive structure (arrow). (2B) Several TUNEL-positive cells in the LP of a 16-day-old rat (arrows). The inset shows a typical fibroblast with a TUNEL-positive nucleus (arrow); a TUNEL-positive nucleus (arrowhead) is juxtaposed to a fibroblast (Fb). (2C) (9-day-old rat) A small and irregular TUNEL-positive structure (arrow) is juxtaposed to fibroblast (Fb) in the LP. (2D) (13-day-old rat) A small TUNEL-positive structure (arrow) seems to be inside the cytoplasm of a round and large mononuclear cell (M) of the LP. (2E) (13-day-old rat) A large mononuclear cell (M) is surrounding a round TUNEL-positive structure (arrow). A large TUNEL-positive structure (arrowhead) is also observed in the LP. B, bone trabeculae; OE, oral epithelium; RE, reduced enamel epithelium.

Figure 3.

Figure 3

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Numerical density of TUNEL-positive cells in the lamina propria of the eruptive pathway of upper first molars from 9-, 11-, 13- and 16-day-old rats. Significant increase in TUNEL-positive cells is observed in 11-, 13- and 16-day-old rat groups compared with 9-day-old rat group. Note that the number of TUNEL-positive cells is significantly increased in the lamina propria of the 16-day-old rat group compared with the other groups. A one-way anova and the post-hoc Kruskal–Wallis test showed a significance of P ≤ 0.05. Statistically significant differences between groups are indicated by asterisks (*).

In the sections submitted to immunohistochemistry for caspase-3 detection, immunolabelled fibroblasts (brown-yellow colour) were observed in the lamina propria of 9-day-old rats. Enhanced immunoexpression was evident in the eruptive pathway of 16-day-old rats (Fig.4A,B). The mammary gland sections (positive controls) exhibited numerous TUNEL-positive nuclei and caspase-3 immunolabelled cells, but the maxilla sections used as negative controls did not have TUNEL-positive cells or caspase-3 immunolabelling (data not shown).

Figure 4.

Figure 4

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(4A,4B) Light micrographs of portions of lamina propria (LP) of the eruptive pathway of first molars submitted to the immunohistochemistry for the detection of cleaved caspase-3 (brown-yellow colour) and counterstained with haematoxylin. Immunolabelled fibroblasts (Fb) are observed in the lamina propria (LP). Note that in (4B) (16-day-old rat), numerous immunolabelled fibroblasts (Fb) are observed compared with (4A) (11-day-old rat). (4C–F) Electron micrographs of portions of the lamina propria of the eruptive pathway of first molars of (4C) 9- and (4D–F) 13-day-old rats. (4C) A fibroblast (Fb) exhibits masses of condensed chromatin filling the entire nucleus (arrows). CF, collagen fibrils. (4D) A cell with masses of condensed chromatin in the nuclear periphery (arrows). (4E) Cytoplasmic projections (P) of a mononuclear cell (M) with an irregular nucleus is partially surrounding a large and ovoid structure (S). This structure (probably a portion of an apoptotic cell) has a nucleus with a compacted block of condensed chromatin (asterisk) and small masses of condensed chromatin (arrowheads). Masses of condensed chromatin (C), small vacuoles (V) and vesicles (Ve) are observed throughout the cytoplasm. (4F) A globular and electron-opaque structure (S) is observed inside a mononuclear cell (M). In this cell, different-shaped lysosomes (L) are observed in the cytoplasm. (4G) Light micrograph of a portion of LP of the eruptive pathway submitted to immunohistochemistry for macrophage detection and counterstained with haematoxylin. Immunolabelling (arrowhead) in the cytoplasm of a round cell (M) is observed in the lamina propria of a 13-day-old rat.

Ultrastructural analysis of the eruptive pathway of the first molars revealed shrunken cells that exhibited nuclei with electron-opaque masses of chromatin, which is a feature typical of apoptosis, at all ages analyzed (Fig.4C,D). Some fibroblasts showed conspicuous and tortuous masses of condensed chromatin filling almost all of the nuclei (Fig.4C). Round cells with peripheral condensed chromatin masses were also observed (Fig.4D). Structures exhibiting condensed blocks of chromatin in the nucleus and electron-opaque masses in the cytoplasm were observed next to small vacuoles and vesicles. These structures were usually observed in close juxtaposition to fibroblasts or macrophages, and mononuclear cells occasionally partially surrounded these dense round/ovoid structures (Fig.4E). Small and dense round/ovoid bodies in the cytoplasm of macrophages exhibiting irregular nuclei were also observed (Fig.4F). The immunohistochemistry, in which the macrophage marker (anti-MAC387) was used, revealed immunolabelled macrophages in the lamina propria (Fig.4G).

In some collapsed blood vessels, the endothelial cells showed irregular nuclei with condensed peripheral chromatin (Fig.5A) and with a TUNEL-positive nucleus (Fig.5B) or caspase-3 cytoplasmic immunolabelling (Fig.5C–E). The distribution of blood vessels in the lamina propria during tooth eruption was observed in the sections submitted to VEGF immunohistochemistry. In addition to endothelial cells, positive immunolabelling was observed in osteoblasts and fibroblasts (Fig.6A). Quantitative analysis revealed a significant decrease (P ≤ 0.05) in the numerical density of VEGF-immunolabelled blood vessel profiles in the 13- and 16-day-old rat groups compared with the 9-day-old rat group (Fig.6B).

Figure 5.

Figure 5

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(5A,5F) Light micrographs of portions of lamina propria of the eruptive pathway of (5B–D) 11-, (5E) 13- and (5A) 16-day-old rats. (5A) A portion of a semithin section is stained with toluidine blue. An arteriole has endothelial cells (Ec) and smooth muscle cell (MC) with condensed and tortuous peripheral chromatin. (5B) A portion of a section submitted to the TUNEL method (brown-yellow) and counterstained with haematoxylin. A blood capillary profile (Ca) shows a TUNEL-positive endothelial cell (arrow). (5C–E) Sections submitted to immunohistochemistry for caspase-3 detection and counterstained with haematoxylin. Caspase-3 immunolabelled endothelial cells (arrows) are observed in the blood capillaries (Ca) and venule (Ve). Note the collapsed blood vessels with evident immunolabelling in the endothelial cytoplasm (5C,5D).

Figure 6.

Figure 6

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(6A) Light micrograph of a portion of the lamina propria of the eruptive pathway of a 9-day-old rat. A portion of a section of lamina propria (LP) submitted to immunohistochemistry for the detection of VEGF (brown-yellow colour) and counterstained with haematoxylin. Blood vessel profiles (BV) have VEGF immunolabelled cells; note a conspicuous immunolabelling in the endothelial cells (inset). Fibroblasts (arrows) in the LP and osteoblasts (Ob) in bone (B) surface are immunolabelled for VEGF. Oral epithelium (OE). (6B) Numerical density of blood vessel profiles in the lamina propria of the eruptive pathway of the maxillary first molar in 9-, 11-, 13- and 16-day-old rats. Note a gradual and significant reduction in the number of blood vessel profiles in the 13- and 16-day-old rats compared with the 9-day-old rats. A one-way anova and post-hoc Tukey test showed a significance of P ≤ 0.05. Statistically significant differences between groups are indicated by asterisks (*).

Discussion

Our combined results obtained from the TUNEL method, caspase-3 immunohistochemistry and ultrastructural images confirm the occurrence of apoptosis in the lamina propria during tooth eruption. The significant increase in the incidence of apoptosis at 16 days indicates that programmed cell death is an important event during degradation of the eruptive pathway. In addition, the occurrence of apoptosis in the vascular cells and the decreased microvascular density suggest that vascular changes are necessary for the passage of the tooth and for the development of gingiva.

TUNEL-positive cells were found in the lamina propria at all ages analyzed, which indicates that cell death occurs at all the different eruptive phases. The TUNEL method is widely used to identify cell death (Cerri, 2005; Faloni et al. 2007; Gonçalves et al. 2008; Cruzoé-Souza et al. 2009; Longhini et al. 2013) because it shows DNA breaks. DNA fragmentation constitutes one of the molecular changes observed in cell death by apoptosis (Gavrieli et al. 1992; Huppertz et al. 1999; Danial & Korsmeyer, 2004). However, it has been reported that the TUNEL method should be combined with other markers to obtain a definitive identification of apoptotic cells (Danial & Korsmeyer, 2004; Cerri, 2005; Cerri & Katchburian, 2005). Thus, caspase-3 immunolabelling and ultrastructural features are used for the identification of apoptotic cell death (Caneguim et al. 2011; Faloni et al. 2012; Longhini et al. 2013; Beltrame et al. 2015).

Strong immunolabelling for caspase-3 was observed in the cells of lamina propria, primarily in the mucosal penetration stage of the eruptive process, i.e., in the 16-day-old rats. Caspases constitute a family of proteases that are involved in the complex and orchestrated cascade of molecular events that control cell death by apoptosis (Danial & Korsmeyer, 2004; Lockshin & Zakeri, 2004; Connolly et al. 2014). Caspase-3 is an executor caspase (Green & Amarante-Mendes, 1998; Demon et al. 2009; Khalil et al. 2014) that is widely used as a marker of apoptosis (Huppertz et al. 1999; Caneguim et al. 2011; Faloni et al. 2012; Longhini et al. 2013). In addition, ultrastructural analysis of the lamina propria revealed cells with irregular nuclei with tortuous and conspicuous masses of condensed chromatin, which is typical of apoptosis (Cerri et al. 2000; Cerri & Katchburian, 2005; Sasso-Cerri & Cerri, 2008; Caneguim et al. 2011; Longhini et al. 2013; Beltrame et al. 2015).

In the lamina propria, TUNEL-positive small structures were observed in close proximity to round mononuclear cells that exhibited typical morphological features of macrophages; these structures occasionally appeared to be within the macrophages. In addition, ultrastructural analysis showed round and/or ovoid bodies with electron-opaque masses inside macrophages. These images are typical of the apoptotic bodies that have been described in other tissues (Cerri et al. 2000; Cerri, 2005; Cerri & Katchburian, 2005). These findings indicate that cells of the lamina propria undergo apoptosis and that the apoptotic bodies derived from these dying cells are engulfed and digested by macrophages. In addition to macrophages, typical fibroblasts were observed in close juxtaposition to TUNEL-positive structures, which suggests that these cells may also recognize and internalize apoptotic bodies. It is known that changes in the cellular surface of apoptotic cells and/or bodies constitute one of the signals to attract and/or activate phagocytic cells (Fadok et al. 1992; Gardai et al. 2006; Elliott et al. 2009; Ravichandran, 2010). Although macrophages are professional phagocytic cells, it has been reported that neighbouring resident cells also play a role in the removal of apoptotic bodies (Cerri et al. 2000; Cerri, 2005; Cerri & Katchburian, 2005; Monks et al. 2008). There is evidence that fibroblasts internalize and digest apoptotic bodies during periodontium development (Cerri et al. 2000). In addition, the presence of TRAP-positive structures that are partially surrounded by periodontal fibroblasts suggests that these cells are able to internalize osteoclast apoptotic bodies (Faloni et al. 2012). Apoptotic structures inside the large vacuoles in osteoclasts (Boabaid et al. 2001; Cerri et al. 2003; Faloni et al. 2007) and osteoblasts (Cerri, 2005) support the idea that, in addition to macrophages, these cells are involved in the removal of apoptotic bodies. The results of the present study indicate that macrophages and fibroblasts are phagocytic cells that may be involved in the recognition and internalization of apoptotic structures during the degradation of the lamina propria of the eruptive pathway.

Because hypoxia seems to promote caspase-3 activation and induction of apoptosis (Zheng et al. 2012), it is possible that the significant reduction in the numerical density of blood vessel profiles observed in the mucosal penetration stage (13- and 16-day-old rats) may be responsible for the high incidence of apoptotic cells at this stage of tooth eruption. Moreover, collapsed blood vessels were often found in the lamina propria at advanced stages of tooth eruption (13- and 16-day-old rats). In these vessels, endothelial and smooth muscle cells have irregular nuclei and condensed chromatin. These results, combined with the presence of TUNEL-positive nuclei and strong caspase-3 cytoplasmic immunolabelling, indicate the occurrence of apoptosis in the blood vessels of the eruptive pathway. The occurrence of vascular apoptosis may be responsible for the significant reduction in numerical density of blood vessel profiles in the lamina propria of 16-day-old rats. During this period, the cusp tips of the developing molar germs cross the oral mucosa, thereby initiating the mucosal penetration stage of the eruptive process (Cerri et al. 2010). Thus, apoptosis of vascular cells is a necessary event such that the degradation process of lamina propria provides a way for tooth movement towards the oral cavity. It is possible that in other regions, blood vessel neoformation (vasculogenesis and/or angiogenesis) may occur and thus allow the microcirculation expansion towards the developing gingiva.

In the present study, VEGF immunoexpression by endothelial cells and fibroblasts of the lamina propria suggests that this vascular factor plays a role in the degradation of the eruptive pathway. VEGF is highly expressed during embryogenesis, wound healing, tissue remodelling and inflammatory diseases, and it acts primarily on endothelial cells by stimulating proliferation, migration, and the induction of various genes involved in tissue remodelling (Pufe et al. 2004; Vempati et al. 2014). In addition to endothelial cells, other cells express VEGF receptors, including monocytes, macrophages (Forstreuter et al. 2002), osteoblasts (Mayr-Wohlfart et al. 2002) and chondrocytes (Pufe et al. 2004). Although VEGF is an important mediator of angiogenesis, some evidence indicates that VEGF induces the production of matrix metalloproteinases (MMPs), a family of zinc-dependent proteinases that digest several components of the extracellular matrix during tissue degradation and remodelling (Cerri et al. 2010; Faloni et al. 2012; Singh et al. 2015). High VEGF expression in osteoarthritis has been associated with cartilage destruction because VEGF is able to regulate the expression of MMP-1 and MMP-13 (Pufe et al. 2004). Thus, it is possible that the matrix degradation of the lamina propria during tooth eruption may be a VEGF-dependent process.

It has been demonstrated that cell–matrix interactions are important modulators of vascular cell proliferation (Schwartz et al. 1999), migration (Hou et al. 2000) and survival (Meredith et al. 1993). Evidence indicates that the interaction of αvβ3 integrin with tenascin-C controls vascular smooth cell apoptosis (Jones et al. 1997), whereas the interaction of vitronectin with αvβ3 and/or αvβ3 integrins regulates endothelial cell survival (Isik et al. 1998). Therefore, it is possible that the extensive matrix degradation observed in the eruptive pathway may be responsible for the induction of apoptosis in the blood vessel cells. Further studies are necessary to confirm this theory.

In conclusion, cell death by apoptosis occurs in the degrading lamina propria of the oral mucosa during tooth eruption. In addition to macrophages, fibroblasts are potential phagocytes of apoptotic cells. Vascular cell apoptosis may be responsible for the gradual reduction of the microvascular network throughout the eruptive process to avoid bleeding during tooth eruption. Based on these data, we can hypothesize that an intense rearrangement of the microvascular plexus may occur because the eruptive pathway is degraded and the gingiva is formed concomitantly to the eruptive process. Further studies are necessary to better understand the process of vascular neoformation in the developing oral mucosa.

Acknowledgments

The authors thank Mr Luis Antônio Potenza and Mr Pedro Sérgio Simões for technical support. This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – no. 2009/16943-6 and n° 2011/23064-9), CAPES, CNPq and ProInter (PROPe UNESP – no. 2015/951).

Author contributions

P.S.C. coordinated the study. J.P.P.Jr. performed the histological processing, TUNEL and immunohistochemical reactions, and morphometric and statistical analyses. E.S.C. and P.S.C. carried out the design of the morphometric analyses. P.S.C. and J.P.P.Jr. prepared the specimens for analysis under transmission electron microscopy. All authors selected the images and participated in the design and writing of the manuscript. All authors approved the final version of manuscript.

Conflict of interest

The authors declare no conflict of interest.

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