Calcif Tissue Int (1992) 51(Suppl 1):S11-S15
Calcified Tissue International © 1992 Springer-Verlag New York Inc.
Effect of Ipriflavone and Estrogen on the Differentiation and Proliferation of Osteogenic Cells Yoshio Kakai, Toshio Kawase, Tarnotsu Nakano, Yuko Mikuni-Takagaki, and Shigeru Saito Department of Oral Biochemistry, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka-shi, Kanagawa 238, Japan
Summary. The effect of ipriflavone (IP) on the proliferation and differentiation of rat osteoblast-like (ROB) cells and human periodontal ligament fibroblasts (HPLF) was studied in the presence and absence of estrogen. ROB cells were isolated from newborn rat calvaria by sequential collagenase digestion and H P L F from the outgrowth of human periodontal ligament in culture. The alkaline phosphatase (ALP) activity, employed as a marker of bone cell differentiation, was significantly enhanced by IP in both cell types; however, the concentration at which IP had a maximal effect was lower in ROB cells than in H P L F (10 -1° versus 10 -7 M, respectively). Cell proliferation judged by DNA content was either constant (ROB cells) or slightly increased (HPLF) by IP up to 10-10 M, and decreased significantly above that concentration. In addition, the dose-dependent effect of estrogen on the growth and differentiation of each cell type in the presence and absence of IP was also tested. At the concentrations of IP which showed maximum effects in the induction of ALP, I0 -1° M for ROB cells and 10 -7 M for HPLF, IP inhibited DNA increase in an estrogen-independent manner. Estradiol (10-11-10-9 M) itself increased the growth rate of both cell types significantly in a dose-dependent manner. Regardless of the concentrations of estradiol tested, ALP activities of both ROB cells and H P L F were elevated by the addition of IP. The ratio of ALP in the presence and absence of IP was similar over the range of estradiol concentrations tested. Thus, we conclude that IP modulates osteogenic cell differentiation of both ROB cells and H P L F and the effect is estrogen independent.
Key words: Ipriflavone - Estrogen - Osteoblast - Periodontal ligament fibroblast - Differentiation.
The loss of bone mass in osteoporosis results from an imbalance of bone formation and resorption, which is controlled by the interaction between osteoblasts and osteoclasts [1]. Ipriflavone (IP), 7-isopropoxy-3-phenyl-4H-1b e n z o p y r a n - 4 - o n e , an isoflavone derivative, has been reported to prevent bone loss in animal models of experimental osteoporosis [2, 4-6] and in osteoporotic patients [7, 8]. A direct inhibition of 45Ca release by IP was first demonstrated in radii of rat long bone in culture [9]. In ovariectomized rats, IP stimulated estrogen-induced calcitonin release [5], whereas in predonisolone-induced osteoporosis, IP decreased parathyroid hormone (PTH) and increased calcitonin [4]. Further in vitro studies using UMR-106 cells, a
Offprint requests to: S. Saito
clonal cell population of rat osteosarcoma, showed that the cells exposed to IP and its metabolites exhibited the following characteristics: inhibition of cAMP response to PTH, stimulation of collagen biosynthesis, alkaline phosphatase (ALP) activity, and cell growth [10]. These results suggest that IP not only affects resorptive processes but also modulates osteoblastic cell differentiation. In this report, we describe further the direct effect of IP on the growth and osteogenic differentiation of rat osteoblastic-like (ROB) cells and human periodontal ligament fibroblasts (HPLF) (morphologically a typical fibroblast but able to express osteoblastic phenotypes in a particular environment l11, t2]).
Materials and Methods Materials Pregnant Sprague-Dawley rats were obtained from NIPPON SLC (Hamamatsu, Japan). Bacterial collagenase (14-501B) and fetal calf serum (FCS) were purchased from Wako Pure Chemical Co. (Osaka, Japan) and Whittaker (Maryland), respectively. 17-[3estradiol (Sigma E8875) was purchased from Sigma Chemical Co. (Osaka, Japan), and IP was provided from Takeda Chemical Industries, Ltd. (Osaka, Japan).
Isolation o f ROB cells cells and H P L F Osteoblastic cells were enriched by using a modified method of Dziak and Brand [13] and Brand [14]. Calvariae (frontal and parietal bones) from 2- to 3-day-old rats were dissected out aseptically, and the soft tissue was carefully removed in isolation medium (IM). The IM consisted of 25 mM HEPES (pH 7.4), 10 mM NaHCO3, 100 mM NaCI, 24 mM KC1, 1 mM CaC12, 5 mg/ml glucose, 2 mg/ml bovine serum albumin (fraction v), 100 U/ml penicillin, and 100 ixg/mlstreptomycin. Pieces of calvaria were placed in a sterile beaker and digested by sterile crude bacterial collagenase (0.75 mg/ml in 12 ml of IM) that was preincubated with 1.67 ~g/ml of N-2-tosyl-lysine chloromethyl ketone (TLCK) for 30 minutes to inhibit clostripain in the enzyme preparation. Incubation of the calvaria-enzyme solution was carried out at 37°C in a shaking water bath. The cells released during the first 30-minute digestion and the second 20-minute digestion were discarded, and the cells from the third 20-minute digestion were filtered through a nylon screen (40-~m pore mesh) and collected by centrifugation. Fresh periodontal ligament (HPL) tissue from healthy human subjects was obtained during the process of extracting the first premolars of patients 10-12 years old who were undergoing orthodontic treatment. Only the HPL attached to the middle one-third of the root was dissected out with a scalpel; the other two-thirds were discarded in order to avoid contamination by gingival and Other fibroblasts. Small pieces of HPL were placed in 2-cm~ wells filled with Dulbecco's modified Eagle's medium (DMEM) under 15-mm Thermanox coverslips. The medium was supplemented with 5% FCS, 50 txg/mlascorbic acid, and penicillin/ streptomycin [11]. Cells were allowed to migrate and proliferate for
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Calcified Tissue International © 1992 Springer-Verlag New York Inc.
Effect of Ipriflavone and Estrogen on the Differentiation and Proliferation of Osteogenic Cells Yoshio Kakai, Toshio Kawase, Tarnotsu Nakano, Yuko Mikuni-Takagaki, and Shigeru Saito Department of Oral Biochemistry, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka-shi, Kanagawa 238, Japan
Summary. The effect of ipriflavone (IP) on the proliferation and differentiation of rat osteoblast-like (ROB) cells and human periodontal ligament fibroblasts (HPLF) was studied in the presence and absence of estrogen. ROB cells were isolated from newborn rat calvaria by sequential collagenase digestion and H P L F from the outgrowth of human periodontal ligament in culture. The alkaline phosphatase (ALP) activity, employed as a marker of bone cell differentiation, was significantly enhanced by IP in both cell types; however, the concentration at which IP had a maximal effect was lower in ROB cells than in H P L F (10 -1° versus 10 -7 M, respectively). Cell proliferation judged by DNA content was either constant (ROB cells) or slightly increased (HPLF) by IP up to 10-10 M, and decreased significantly above that concentration. In addition, the dose-dependent effect of estrogen on the growth and differentiation of each cell type in the presence and absence of IP was also tested. At the concentrations of IP which showed maximum effects in the induction of ALP, I0 -1° M for ROB cells and 10 -7 M for HPLF, IP inhibited DNA increase in an estrogen-independent manner. Estradiol (10-11-10-9 M) itself increased the growth rate of both cell types significantly in a dose-dependent manner. Regardless of the concentrations of estradiol tested, ALP activities of both ROB cells and H P L F were elevated by the addition of IP. The ratio of ALP in the presence and absence of IP was similar over the range of estradiol concentrations tested. Thus, we conclude that IP modulates osteogenic cell differentiation of both ROB cells and H P L F and the effect is estrogen independent.
Key words: Ipriflavone - Estrogen - Osteoblast - Periodontal ligament fibroblast - Differentiation.
The loss of bone mass in osteoporosis results from an imbalance of bone formation and resorption, which is controlled by the interaction between osteoblasts and osteoclasts [1]. Ipriflavone (IP), 7-isopropoxy-3-phenyl-4H-1b e n z o p y r a n - 4 - o n e , an isoflavone derivative, has been reported to prevent bone loss in animal models of experimental osteoporosis [2, 4-6] and in osteoporotic patients [7, 8]. A direct inhibition of 45Ca release by IP was first demonstrated in radii of rat long bone in culture [9]. In ovariectomized rats, IP stimulated estrogen-induced calcitonin release [5], whereas in predonisolone-induced osteoporosis, IP decreased parathyroid hormone (PTH) and increased calcitonin [4]. Further in vitro studies using UMR-106 cells, a
Offprint requests to: S. Saito
clonal cell population of rat osteosarcoma, showed that the cells exposed to IP and its metabolites exhibited the following characteristics: inhibition of cAMP response to PTH, stimulation of collagen biosynthesis, alkaline phosphatase (ALP) activity, and cell growth [10]. These results suggest that IP not only affects resorptive processes but also modulates osteoblastic cell differentiation. In this report, we describe further the direct effect of IP on the growth and osteogenic differentiation of rat osteoblastic-like (ROB) cells and human periodontal ligament fibroblasts (HPLF) (morphologically a typical fibroblast but able to express osteoblastic phenotypes in a particular environment l11, t2]).
Materials and Methods Materials Pregnant Sprague-Dawley rats were obtained from NIPPON SLC (Hamamatsu, Japan). Bacterial collagenase (14-501B) and fetal calf serum (FCS) were purchased from Wako Pure Chemical Co. (Osaka, Japan) and Whittaker (Maryland), respectively. 17-[3estradiol (Sigma E8875) was purchased from Sigma Chemical Co. (Osaka, Japan), and IP was provided from Takeda Chemical Industries, Ltd. (Osaka, Japan).
Isolation o f ROB cells cells and H P L F Osteoblastic cells were enriched by using a modified method of Dziak and Brand [13] and Brand [14]. Calvariae (frontal and parietal bones) from 2- to 3-day-old rats were dissected out aseptically, and the soft tissue was carefully removed in isolation medium (IM). The IM consisted of 25 mM HEPES (pH 7.4), 10 mM NaHCO3, 100 mM NaCI, 24 mM KC1, 1 mM CaC12, 5 mg/ml glucose, 2 mg/ml bovine serum albumin (fraction v), 100 U/ml penicillin, and 100 ixg/mlstreptomycin. Pieces of calvaria were placed in a sterile beaker and digested by sterile crude bacterial collagenase (0.75 mg/ml in 12 ml of IM) that was preincubated with 1.67 ~g/ml of N-2-tosyl-lysine chloromethyl ketone (TLCK) for 30 minutes to inhibit clostripain in the enzyme preparation. Incubation of the calvaria-enzyme solution was carried out at 37°C in a shaking water bath. The cells released during the first 30-minute digestion and the second 20-minute digestion were discarded, and the cells from the third 20-minute digestion were filtered through a nylon screen (40-~m pore mesh) and collected by centrifugation. Fresh periodontal ligament (HPL) tissue from healthy human subjects was obtained during the process of extracting the first premolars of patients 10-12 years old who were undergoing orthodontic treatment. Only the HPL attached to the middle one-third of the root was dissected out with a scalpel; the other two-thirds were discarded in order to avoid contamination by gingival and Other fibroblasts. Small pieces of HPL were placed in 2-cm~ wells filled with Dulbecco's modified Eagle's medium (DMEM) under 15-mm Thermanox coverslips. The medium was supplemented with 5% FCS, 50 txg/mlascorbic acid, and penicillin/ streptomycin [11]. Cells were allowed to migrate and proliferate for
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