Mast cells and MMP-9 in the lamina propria during eruption of rat molars: quantitative and immunohistochemical evaluation

Abstract

During the active tooth eruption process, structural changes in the lamina propria are necessary to provide extracellular matrix remodelling and for the establishment of the eruptive pathway. A large number of resident cells, recruited cells and proteases have been demonstrated in the eruptive process, but the participation of MMP-9 and mast cells has not yet been demonstrated. In this study, we set out to evaluate the intensity of MMP-9 immunoexpression, the frequency of mast cells and the correlation between the incidence of mast cells and bone resorption in different phases of tooth eruption. Fragments of maxilla containing first molars, obtained from 9-, 11-, 13- and 16-day-old rats, were fixed in 4% formaldehyde, decalcified and embedded in paraffin. Sagittal sections were stained with Masson's trichrome or submitted to the tartrate-resistant acid phosphatase method for quantification of osteoclasts. Sections stained by 1% toluidine blue were used for quantification of metachromatic mast cells mm(-2) of lamina propria. The expression of MMP-9 in the lamina propria was evaluated by immunohistochemistry. In the 9-day-old rats, the lamina propria contained few mast cells and occasional osteoclasts were found in the bone surface overlying the occlusal portion of the tooth germs. Otherwise, a significant increase in the number of mast cells was observed in the intra-osseous phase of tooth eruption (11-day-old rats), period in which numerous TRAP-positive osteoclasts were found in the bone surface. MMP-9 immunolabelling was detected in fibroblasts, mast cells and macrophage-like cells of the lamina propria in all ages studied. However, an enhanced immunolabelling was evident in the advanced phase of tooth eruption (16-day-old rats). During the intra-osseous phase, the parallel between the high frequency of both mast cells and osteoclasts suggests that mast cells could exert a paracrine function on the osteoclasts and then stimulate bone resorption. The immunoexpression of MMP-9 in different cells of lamina propria, including mast cells, indicates that this enzyme participates in the degradation of ECM, mainly during late phase of mucosal penetration. Thus mast cells and MMP-9 are involved in the complex process of degradation of the eruptive pathway extracellular matrix.

Fig. 1

Light micrograph of a sagittal section of the first molar tooth germ of 9-day-old rat. Developing tooth germ containing dental papilla (DP), dentine (D) and enamel (E) is located inside bony crypt (B). In the cervical region, the initial stage of root dentine formation (white arrows) is observed. Ob, odontoblasts; A, ameloblasts; LP, lamina propria; OE, oral epithelium. Masson’s trichrome. Bar: 200 μm.

Fig. 2

Light micrographs of portions of first molar tooth germs, in different stages of eruption, of 9- (A), 11- (B), 13- (C) and 16-day-old (D) rats, stained with Masson's trichrome. (2A) A continuous layer of bone trabeculae (B) is interposed between the tooth germ (GE) and the lamina propria (LP). OE, oral epithelium; E, enamel. Bar: 100 μm. (2B) Cusp tips of the tooth molar germ in close proximity to lamina propria (LP); bone trabeculae (B) partially covers the occlusal portion of the tooth germ. E, enamel; D, dentine; OE, oral epithelium. Bar: 100 μm. (2C) The cusp tips of the tooth germ penetrate in the lamina propria (LP) which is restricted to a thin layer. OE, oral epithelium; E, enamel; ES, enamel space; D, dentine; RE, reduced enamel epithelium. Bar: 100 μm. (2D) The molar germ passing through the oral epithelium (OE). Note that the lamina propria (LP) is restricted to small regions. DP, dental papilla; D, dentine; ES, enamel space; RE, reduced enamel epithelium. Bar: 100 μm.

Fig. 3

Light micrographs of portions of the eruptive pathway of the first molar stained with toluidine blue (A and B) and, submitted to the TRAP-method and counterstained with toluidine blue (C). (3A) Several mast cells (arrows) in the lamina propria (LP) of an 11-day-old-rat. Note the metachromasia (purple colour) in the mast cell (inset). D, dentine; ES, enamel space; RE, reduced enamel epithelium; B, bone; OE, oral epithelium. Bar: 60 μm; inset bar: 5 μm. (3B) Portion of erupting tooth germ of a 13-day-old-rat. Scarce mast cells (arrows) are seen in the lamina propria (LP). ES, enamel space; D, dentine; RE, reduced enamel epithelium; OE, oral epithelium. Bar: 60 μm. (3C) Several TRAP-positive osteoclasts (OC) on the bone surface (B) and mast cells (arrows) are seen in the lamina propria (LP) of an 11-day-old rat. OE, oral epithelium. Bar: 10 μm.

Fig. 4

Number of TRAP-positive osteoclasts mm⁻¹ of alveolar bone surface and mast cells mm⁻² of lamina propria in rats aged 9 and 11 days. The number of mast cells and osteoclasts increased around 30% from 9- to 11-day-old rats. *Statistically significant (P ≤ 0.05).

Fig. 5

(A–F) Light micrographs of portions of oral mucosa of the eruptive pathway of first molars submitted to the immunohistochemistry for detection of MMP-9 (brown colour) and counterstained with haematoxylin. (5A) Fibroblasts (arrows) exhibit weak or negative MMP-9 immunolabelling in the lamina propria (LP) of a 9-old-day rat. MMP-9 immunolabelled osteoclasts (OC) are observed in the bone surface (B). OE, oral epithelium. Bar: 25 μm. 5B (outlined area of 5A) and 5C show, respectively, a fibroblast (arrows) and a mast cell (MC) strongly MMP-9 immunolabelled in the lamina propria (LP) of 9-day-old rats. Bar: 5 μm. (5D) Scarce cells with immunopositive cytoplasm (arrows) are observed in the lamina propria (LP) of 11-day-old rat. Multinucleated osteoclasts (OC) adjacent to bone surface (B) are positive; note a conspicuous immunostaining in the osteoclast cytoplasm (OC, inset). OE, oral epithelium. Bar: 30 μm; inset bar: 5 μm. In 5E (outlined area of 5D) and 5F, MMP-9-positive immunolabelling is observed in the fibroblasts (arrows) and mast cells (MC) of the lamina propria (LP) of 11-day-old rats. Bar: 5 μm.

Fig. 6

(A–G) Light micrographs of portions of oral mucosa of the eruptive pathway of first molars submitted to immunohistochemistry for detection of MMP-9 and counterstained with haematoxylin. (6A) Positive immunoreaction is observed in some cells (arrows) of the lamina propria (LP) of a 13-old-day rat. OE, oral epithelium. Bar: 25 μm. In 6B (outlined area of 6A), 6C and 6D, fibroblasts (arrows), mast cells (MC) and monocyte/macrophage-like cell (M) show cytoplasm strongly immunolabelled in the lamina propria (LP) of 13-day-old rats. (6B and 6C) Bar: 10 μm; (6D) bar: 5 μm. (6E) Strong positive immunoreaction is observed in numerous cells (arrows) of the lamina propria (LP) of a 16-day-old rat. OE, oral epithelium; RE, reduced enamel epithelium; ES, enamel space. Bar: 30 μm. In 6F (outlined area of 6E) and 6G, strong immunostaining is observed in the cytoplasm of fibroblasts (arrows), monocyte/macrophage-like cells (M) and mast cell (MC). Note that scarce components of extracellular matrix (asterisks) are observed in the lamina propria (LP). Bar: 5 μm.