Insufficient oestrogen is believed to be one of the major causes of postmenopausal osteoporosis. Osteoporosis is considered as one of the risk factors for periodontal disease and tooth loss. Daniell (1983) suggested that middle-aged women may be more likely to retain their teeth if they avoid smoking and undertake a programme effective in preventing the progression of osteoporosis. Taguchi et al. (1995) demonstrated that a decrease of mandibular bone mass positively correlates with tooth loss in women. Kribbs (1990) reported that a greater percentage of osteoporotic women than normal women were edentulous. Bone of lesser density may be more readily absorbed and this enhances tooth loss. However, a direct relation between osteoporosis and periodontal disease or tooth loss is not clear.

The periodontal ligament is the connective tissue located between the alveolar bone and root surface of the tooth. Cells of this ligament exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. They play an important part in maintaining the integrity of the periodontal tissue. Our purpose now was to investigate the effects of oestradiol on the function of periodontal ligament cells by measuring the production of osteocalcin in vitro.

Cells were obtained from the healthy periodontal ligament of teeth extracted from two males (21 and 22 years old) and two females (27 and 44 years old) for orthodontic reasons. Informed consent was obtained from the patients before the extractions. The middle third of the periodontal ligament on the root surface was minced and cultured in tissue-culture medium. Dulbecco’s modified Eagle medium with 10% fetal bovine serum supplemented with antibiotics was used to maintain the cells. When the outgrown cells from the tissue explants had become confluent, they were transferred and cultured in a tissue-culture flask. Stocks of these cells were prepared and stored in liquid nitrogen for use in the experiments. The cells were used in passages eight to ten.

The periodontal ligament cells cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum supplemented with 10 mM of ?-glycerophosphate and 50 ?g/ml of ascorbic acid for 20 days were stained with alizarin red and observed under a microscope to confirm the presence of mineral-like nodules.

Cells of the periodontal ligament obtained from 44-year-old female were seeded in 6-well multiwell tissue-culture plates at the concentration of 3.0×10⁵ cells/well in Dulbecco’s modified Eagle medium with 10% fetal bovine serum. After 3 hr the cells were washed with phosphate-buffered saline and the medium was changed to ASF-301 (Ajinomoto Co. Ltd., Tokyo, Japan) containing 17-? oestradiol (Sigma, St. Louis, MO, USA) and cultured for 20 days. The concentrations of 17-? oestradiol were 0, 0.2, 2.0 and 20 ng/ml; these concentrations of the sex hormone are within the physiological range. The main components of ASF-301 medium are 10 mg/ml of bovine insulin, 5 mg/ml of human transferrin, 10 mM 2-mercaptoethanol, 10 mM of 2-aminoethanol and 10 nM of sodium selenite.

The amount of osteocalcin in the culture medium was analysed by two-step sandwich enzyme immunoassay (Gla-type Osteocalcin EIA Kit, Takara, Otsu, Japan). In brief, a 96-multiwell plate was coated with monoclonal antibody against bovine osteocalcin by incubating the plate overnight at 4°C. A portion (100 ?l) of the culture medium was added to the wells and incubated for 1 hr at room temperature, followed by incubation with 100 ?l of antibody labelled with peroxidase. Then o-phenylenediamine–HCl was added to the wells and incubated for 10 min. The reaction was stopped by adding 1N H₂SO₄ and the absorbency was measured at 492 nm. The production of osteocalcin by periodontal ligament cells obtained from other donors (both males and females) was also measured with or without 20 ng/ml of oestradiol.

For statistical analysis, the student-t test was used.

The cells obtained from both male and female donors produced mineral-like nodules that were stained by alizarin red; Fig. 1 shows a representative picture of the nodules. As shown in Fig. 2, production of osteocalcin by the periodontal ligament cells was observed after 10 days of culture, and this production increased with the time. The addition of oestradiol (20 ng/ml) to the culture medium enhanced the production significantly. The amount of osteocalcin at day 15 with 20 ng/ml of oestradiol (8.37±1.05 ng/ml) was 10 times more than without oestradiol (0.87±0.17 ng/ml). Oestradiol enhanced the production of osteocalcin in a dose-dependent manner at 20 days of culture. Fig. 4 shows the effects of oestradiol on osteocalcin production by periodontal ligament cells obtained from different donors. Cells obtained from both male and female donors produced osteocalcin, and oestradiol enhanced the production significantly.

Fig. 1. Mineral-like nodules of the peridontal ligament cells obained from a 21-year-old male. Alizarin red, original magnification ×100.

Fig. 2. Time-dependent changes in osteocalcin production by periodontal ligament cells cultured with (solid symbols) or without (open symbols) 20 ng/ml of oestradiol. The values are the mean and SD of six cultures. Cells obtained from 44-year-old female. **Significantly different compared to without oestradiol, p<0.001.

Fig. 3. Effects of different concentrations of oestradiol on osteocalcin production by periodontal ligament cells cultured for 20 days. Cells obtained from a 44-year-old female. The values are the mean and SD of six cultures. Significantly different from 0 ng/ml of oestradiol, *p<0.05, **p<0.001.

Fig. 4. Production of osteocalcin by periodontal ligament cells from different donors. The number of the cells was adjusted to 3.0×10⁵ cells/ml at day 0 and cultured for 20 days with or without 20 ng/ml of oestradiol. The values are the mean and SD of six cultures. Significantly different from 0 ng/ml of oestradiol, **p<0.001.

The cells used in this experiment initiated mineral-like nodules, which is one of the characters of periodontal ligament cells. Many in vitro studies have shown that such cells produce type I collagen, alkaline phosphatase, osteonectin, osteopontin, and osteocalcin. Nojima et al. (1990) reported that periodontal ligament cells produce a protein immunologically cross-reactive with bovine bone gla protein (osteocalcin). The expression of osteocalcin mRNA by periodontal ligament cells was shown by using the reverse transcriptase-polymerase chain reaction and by in situ hybridization. Here we demonstrate the production of osteocalcin by using the two-step sandwich enzyme immunoassay. Osteocalcin is a vitamin K-dependent Ca²⁺-binding protein of bone matrix synthesized by osteoblasts and considered as a marker for bone formation.

The production of osteocalcin and its stimulation by various hormones have been studied extensively. Osteosarcoma cells are known to produce osteocalcin and 1, 25 (OH)₂ D₃ stimulates its production. Incubation of bone marrow cells with dexamethasone and 1, 25(OH)₂ D₃ stimulates the expression of osteocalcin mRNA.

To the best of our knowledge, this is the first report that oestradiol enhances the production of osteocalcin by periodontal ligament cells. In our experiment the cells obtained from both male and female donors were affected by oestradiol. Oestrogens are known to be important in males also because oestrogen-receptor knock-out mice of both sexes showed phenotypic changes in gonads, mammary glands and skeletal tissues. Thus it is possible that the production of osteocalcin in males is also enhanced by oestradiol.