The effect of estrogen deficiency on rat pulpodentinal complex

Dental pulp, which occupies the center of each tooth, consists of soft connective tissue. Although the composition and structure of pulp differ from those of dentin, the two tissues exist in an intimate embryologic and functional relationship. The dentin and pulp are usually considered together as a functional complex termed the pulpodentin complex1). Osteoporosis is a condition of low bone mass and microarchitectual deterioration of bone tissue, which can result in pain, deformity, or fracture. According to the World Health Organization (WHO), osteoporosis is considered to be present when bone mineral density (BMD) is 2.5 standard deviations (SD) below the young normal BMD2). Bone loss due to osteoporosis has been reported to affect the jaws3), and ovariectomy has been shown to cause significant decreases of trabecular and cortical BMD4). Although BMD has been shown to be related to tooth loss5), and estrogen deficiency affects incisor odontoblasts6), but it has not been determined whether osteoporosis affects the pulpodentinal complex of teeth.

In recent years, a number of morphologic studies have used immunohistochemical techniques to localize matrix proteins in bone. Osteonectin is a glycoprotein of 32 kD, and a homologous protein, SPARC, an anagram for secreted protein, acidic and rich in cysteine7, 8). High levels of immunodetectable osteonectin are found in active osteoblasts and marrow progenitor cells, odontoblasts, periodontal ligaments and gingival cells and some chodrocytes and hypertrophic chondrocytes. Osteonectin can be detected in osteoid, bone matrix proper and dentin. Osteonectin was originally isolated from bone, and high affinity binding of calcium ions to this protein suggested that osteonectin plays an important role in mineralization of bone9).

The purpose of this study was to evaluate the changes in the pulpodentinal complex of teeth in rats by comparing and analyzing immunohistochemical sections in ovariectomized rats.

In radiographs, generally the pulpodentinal complexes of OVX and SHAM rats did not differ. (Figure 1 & 2).

Figure 1
Radiographic view of mandibular sections of SHAM Rat.

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Figure 2
Radiographic view of mandibular sections of OVX Rat.

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Immunohistochemical staining for osteonectin, however, showed significant differences in the two groups of rats. The predentin thickness in OVX rats was greater than in SHAM rats. Paired t-test was used and the level of significance for overall differences was set at p < 0.05. (Table 1).

Table 1
The mean and standard deviation for predentin thickness of the OVX rats and SHAM rats are shown (p < 0.05)

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Furthermore, in SHAM rats, osteonectin was more specifically stained in predentin areas than in OVX rats. In SHAM rats, the mineralized compartment of intratubular dentin was not immunoreactive, whereas the unmineralized predentin and adjoining immunoreactive polarized odontoblasts were stained heavily with antibodies to osteonectin antibodies. In OVX rats, however, the predentin layers were weakly stained with osteonectin antibodies. The osteonectin staining pattern in fibrous dental pulp cells of OVX rats was similar to that of SHAM rats. Generally, weakly osteonectin labeling is present in polarized odontoblasts, but absent from the cells of the central pulp and dentin (Figure 3, 4, 5, 6).

Figure 3
Immunohistochemical staining of SHAM Rat (original magnification × 400). Osteonectin (Arrow-brown) in the cytoplasm of active, matrix-producing odontoblasts in predentin areas.

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Figure 4
Immunohistochemical staining of SHAM Rat (original magnification × 400). Adjoining immunoreactive polarized odontoblasts (Arrow) were stained heavily with osteonectin antibodies.

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Figure 5
Immunohistochemical staining of OVX Rat (original magnification × 400). Absence of staining of quiescent odotoblasts in the predentin areas.

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Figure 6
Immunohistochemical staining of OVX Rat (original magnification × 400). Weakly osteonectin labeling (Arrow) is present in polarized odontoblasts, but absent from the cells of the central pulp and dentin.

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These results indicate that estrogen deficiency increased unmineralized predentin areas and decreased osteonectin contents of predentin areas.

Osteonectin is the major noncollagenous protein in the extracellular matrix of developing bones and teeth. Though the function of this protein is not known, its location in newly deposited osteoid, its high affinity for calcium, hydroxyapatite, and collagen, and its reduction in bone from some animals and humans with osteogenesis imperfecta point up its importance to bone metabolism. The high degree of cross-species immunoreactivity observed using monoclonal antibodies against bone osteonectin indicates that common epitopes have been conserved during evolution10). Although bone is a particularly rich source of osteonectin, this protein and/or its mRNA are also detected in several non-bone tissues11, 12).

Dentin is composed primarily of proteins common to both dentin and bone, including collagen types I, III, and V, bone sialoprotein, osteopontin, dentin matrix protein-1, osteocalcin, and osteonectin13, 14). In the previous study9), periodontal tissues demonstrated different staining intensities for osteonectin: strong staining existed in osteoid and moderate staining was seen in the PDL. The intense staining for osteonectin specifically in the osteoid, but not mineralized bone and cementum leads us to speculate that osteonectin may prevent premature mineralization of the osteoid by inhibiting hydroxyapatite formation. Therefore, immunostaining of osteonectin was effective tool for hard tissue regeneration.

The stronger labeling of osteonectin in pulps of dental germs than in those of erupted teeth suggests that osteonectin is more functionally active in young odontoblasts than in older odontoblasts15). We have observed similar results, in that the mineralized compartment of intratubular dentin in Sham rats was not immunoreactive, whereas the unmineralized predentin and adjoining immunoreactive polarized odontoblasts stained heavily with osteonectin antibodies in these animals.

As like previous study16), the use of conventional radiographs alone to assess bone mineral density of rat mandible as unsatisfactory, and should not be regarded as a substitute for histological examination for the determination of bone mineral loss.

Predentin, which is located adjacent to the pulp tissue, is 2 to 6 µm wide, depending on the activity of the odontoblasts in human. Predentin is the earliest form of dentin and is not mineralized. As the collagen fibers undergo mineralization at the predentin-dentin front, the predentin becomes dentin and a new layer of predentin forms circumpulpally. Thickening of the predentin zone may be due to a cumulative lack of calcification over time or may result from the excessive formation of predentin by odontoblasts. The cause of the thickened predentin zone may be twofold. First, there may be a cumulative lack of calcification of physiologically deposited predentin with time. Second, the thickened predentin layer may, in part, be the result of excessive formation of predentin by odontoblasts17).

Estrogen deficiency causes osteoporosis, which is associated with the enhancement of bone remodeling soon after ovariectomy. In another study, long-term estrogen deficiency in ovariectomized rats decreased the mandibular cortical thickness18). Loza et al.2) examined the relationship between osteoporosis and intraoral bone tissues and reported that the alveolar bone loss in the presence of progressed periodontal disease is more significant in men than with in women, suggesting that hormones and habitual factors such as drinking and smoking affect men more seriously than osteoporosis in women. Postmenopausal osteoporosis progresses immediately after menopause, suggesting it is due primarily to the lack of estrogen production by the ovaries19).

We found that the pulpodentinal complex demonstrated different staining intensities for osteonectin. While intense staining in the osteonectin may indicate active changes in the pulpodentinal complex, weak staining of osteonectin in OVX rats may indicate low content of proteins. These findings suggest that predentin is the metabolic equivalent of osteoid in bone and that increases in predentin thickness may therefore be a reliable indicator of estrogen deficiency. If our result is applicable to human studies, odontoblast is affected by estrogen deficiency.

Little is known, however, about the effects of ovariectomy on odontoblast metabolism, suggesting the need for additional studies to determine the participation of this protein in the metabolism of dental pulp.

We investigated changes in the pulpodentinal complex resulting from ovariectomy. Fifteen 10-week-old female Sprague-Dawley rats were subjected to sham surgery (SHAM), and other rats were ovariectomized bilaterally (OVX). Animals were sacrificed 12 weeks later, and their mandibular molars and associated periodontal supporting tissues were dissected out and fixed in 10% buffered formalin. Each section was dentally radiographed and immunostained for osteonectin. We found that the two groups differed significantly in immunostaining for osteonectin. The predentin thickness in OVX rats was wider than in Sham rats, whereas, in the latter, osteonectin was more specifically stained in predentin areas than in OVX rats. These results indicate that estrogen deficiency increases unmineralized predentin areas and decreases osteonectin contents of the pulpodentinal complex.