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Inorganic Polyphosphate: a Possible Stimulant of Bone Formation

¹ Department of Oral and Maxillofacial Surgery,
² Institute for Oral Science,
³ Department of Oral Microbiology, and
⁴ Department of Orthodontics, Matsumoto Dental University School of Dentistry, 1780 Gobara Hirooka, Shiojiri, Nagano 399-0874, Japan; and
⁵ Regenetiss Inc., 1-5-17, Akabane, Okaya, Nagano 394-0002, Japan

^* corresponding author, uematsu{at}po.mdu.ac.jp

	ABSTRACT

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Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

KEY WORDS: inorganic polyphosphate • osteoblast • osteoclast • bone formation

	INTRODUCTION

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Inorganic polyphosphates [Poly(P)] are orthophosphate polymers found in mammalian nuclei, mitochondria, lysosomes, and plasma membranes (Kumble and Kornberg, 1995). They are also present in human mandibular-derived osteoblast-like cells (Leyhausen et al., 1998). It has been suggested that mammalian Poly(P) functions as a regulator of calcification, decalcification (Fleisch et al., 1966), and bone formation through osteoblast proliferation, particularly by modulating the mineralization process (Leyhausen et al., 1998). Recently, it has been discovered that Poly(P) affects the expression of many genes by controlling rpoS expression (Shiba et al., 2000). Moreover, Poly(P) stabilizes fibroblast growth factor (FGF), which is an important regulator of mitotic activity in fibroblasts (Shiba et al., 2003). Basic FGF stimulates Pi transport (Suzuki et al., 2000), and Poly(P) enhances osteoblastic cell differentiation and cell calcification in osteoblast-like cells (Kawazoe et al., 2004). These findings suggest that Poly(P) stimulates osteoblasts and facilitates periodontal regeneration. However, its precise biological function in periodontal regeneration and its involvement in bone formation remain unknown. In this study, we tested the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation, using an osteoblast-like cell line (MC 3T3-E1), osteoclasts derived from co-cultured mouse calvarian osteoblasts and tibia bone marrow macrophages, and in a rat alveolar bone regeneration model.

	MATERIALS & METHODS

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Materials
Types 75 and 35 sodium phosphate glass [Poly(P); average chain length of 75 and 35 phosphate residues, respectively] and p-nitrophenyl phosphate (p-NPP) were purchased from Sigma (St. Louis, MO, USA). Type 100⁺ glass, with a chain length of more than 100 phosphate residues, was prepared as previously described (Itoh et al. , 2006). As a control, we adjusted Na-PO₄ buffer to pH 7.0 by mixing equal concentrations of Na₂HPO₄ and NaH₂PO₄ solutions. The mouse osteoblast-like cell line MC3T3-E1 was purchased from the Riken Cell Bank (Tsukuba, Japan). Anti-bone alkaline phosphatase polyclonal antibody was purchased from Biogenesis (Poole, England). Fifteen healthy male Wistar rats (age, 8 wks; weight, between 200 g and 220 g) were purchased from Japan SLC, Inc. (Shizuoka, Japan).

Cell Culture
MC3T3-E1 cells were plated at a density of 1.5 x 10⁶ cells/60-mm dish (Falcon, Nippon Becton Dickinson, Tokyo, Japan) in -minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). After 48 hrs, the cells were subcultured in the same medium supplemented with 0.5% FBS. Twenty-four hrs later, cells were treated with the various reagents or with an equal volume of vehicle. Culture medium was changed every 4 days.

Real-time PCR
Total RNA of the cultured cells was extracted by the acid guanidinium isothiocyanate-phenol-chloroform method. First-strand cDNA was then synthesized with random primers (Takara Shuzo, Osaka, Japan) and Superscript II reverse transcriptase (Gibco BRL, Rockville, MD, USA). Relative levels of osteopontin (OPN), osteocalcin (OCN), alpha-1 chain of type I collagen (Col I), and osteoprotegerin (OPG) mRNA were measured by real-time polymerase chain-reaction (PCR) and normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels, which were used as an internal control. The primer sequences for quantitative PCR are as follows: Fwd-5'-GAACAGACTCCGGCGCTA-3' and Rev-5'-AGGGAGGATCAAGTCCCG-3' for OCN; Fwd-5'-TGATGCCACAGATGAGGACCT-3' and Rev-5'-CAGAGGGCATGCTCAGAAGC-3' for OPN; Fwd-5'-GCGAAGGCAACAGTCGCT-3' and Rev-5'-CTTGGTGGTTTTGTATTCGATGAC-3' for Col I; Fwd-5'-AGCTGCTGAAGCTGTGGAA-3' and Rev-5'-TGTTCGAGTGGCCGAGAT-3' for OPG; and Fwd-5'-ACCACAGTCCATGCCATCAC-3' and Rev-5'-TCCACCACCCTGTTGCTGTA-3' for GAPDH. Real-time PCR was performed with the 7500 real-time PCR system (Applied Biosystems, Weiterstadt, Germany) with Power SYBER^® Green PCR Master Mix (Applied Biosystems) for 40 cycles of 95°C for 15 sec and 60°C for 34 sec, as described in the manufacturer’s protocol.

Alkaline Phosphatase Activity and Mineralization Assay
To assess alkaline phosphatase (ALP) activity, we incubated cells with normal medium (-MEM containing 0.5% FBS) or differentiation medium (normal medium supplemented with 1 µM dexamethazone and 50 µg/mL ascorbic acid) with or without 1 mM Poly(P). Alkaline phosphatase activity was assayed as described previously (Noguchi and Yamashita, 1987). Specific calcification detection was performed with von Kossa staining (30 min, 5% silver nitrate).

Pit Formation Assay
To analyze the influence of Poly(P) on bone resorption, we undertook an osteoclastic study using co-cultured mouse calvarian osteoblasts and tibia bone marrow macrophages, as previously described (Yang et al., 2005). Osteoclasts were determined by tartrate-resistant acid phosphatase (TRAP) staining.

Rat Alveolar Bone Regeneration Model
This study was approved by the Ethical Committee of the Matsumoto Dental University. Fifteen healthy male Wistar rats (age, 7 wks; weight, from 200 g to 220 g) were used. Procedures were performed with the rats under general anesthesia achieved by ketamine HCl (200 mg/kg) administered intraperitoneally, and local anesthesia with 2% lidocaine hydrochloride containing epinephrine (1:80,000). Mucoperiosteal flaps were made on both sides of the mandible, and buccal osseous dehiscences were created by the removal of approximately 3 x 3 mm of bone covering the roots of the bilateral mandibular first molars. A reference notch was marked in the root surface at the levels of the surgically reduced bone crest, by means of a round bur (No. : Morita, Tokyo, Japan). The buccal mucoperiosteal flaps were re-adapted. A 1-mM carboxymethylcellulose (CMC) solution containing 1 mM Poly(P)75 was applied to the right sulcus 5 days a wk after surgery, without systemic antibiotics. On the left side, CMC alone was applied as a control. Rats were killed at 1, 2, or 3 wks following surgery. The mandibles, including the first molars, were dissected and further fixed by immersion in 10% neutral buffered formalin for 48 hrs at 4°C. Specimens were washed and decalcified in 10% neutral buffered EDTA at 4°C for 1 mo. Serial sections (6 µm in thickness) were cut with a cryostat in a bucco-lingual direction, stained with hematoxylin and eosin, and immunostained with anti-bone alkaline phosphatase polyclonal antibody, with the VECTASTAIN^® ABC kit (VECTOR Laboratories, Burlingame, CA, USA). Statistical analysis of the new-bone area was performed with a microcomputer-based image analysis system (Shimazu Corporation, Kyoto, Japan).

Statistical Analysis
Results are presented as means ± SD. Significant differences between two values were determined by the Student’s t test.

	RESULTS

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Expression of Osteoblastic Markers in MC 3T3-E1 Cells
To clarify the influence of Poly(P) on osteoblastic differentiation, we first analyzed the expression of OPN, OCN, Col I, and OPG by real-time PCR and determined ALP activity. After cultivation of MC 3T3-E1 cells with 1 mM Poly (P) 75, OPN gene expression exhibited 2 peaks on days 15 and 30 under these culture conditions (Fig. 1A). OCN gene expression peaked on day 30, corresponding with the second peak of OPN gene expression (Fig. 1B). Col I peaked on day 20 and decreased on day 25 (Fig. 1C). OPG was expressed on day 20 and intensively expressed on day 30 (Fig. 1D). ALP activity also increased gradually (Fig. 1E). In contrast, after cultivation of MC 3T3-E1 cells without 1 mM Poly(P)75, increased OPN, OCN, Col I, and OPG gene expression was not seen, nor was increased ALP activity. These results indicated that MC 3T3-E1 cells resembled mature osteoblasts after cultivation with Poly(P)75 for 30 days.

View larger version (14K): [in this window] [in a new window]   Figure 1. Expression levels of osteoblast-related genes by real-time polymerase chain-reaction and alkaline phosphatase activity in MC 3T3-E1 cells. Cells were cultured in differentiation medium with 1 mM Poly(P) or without 1 mM Poly(P) (Control) for 35 days. Relative mRNA levels of OPN (A), OCN (B), Col I(C), OPG (D), and ALP activity (E)—without treatment (open circles) and following treatment with 1 mM Poly(P) (closed circles)—are shown. Error bars represent the mean ± SD of three independent analyses.

View larger version (73K): [in this window] [in a new window]   Figure 2. Influence of Poly(P) on calcification and decalcification with in vitro analysis. MC 3T3-E1 cells were cultured with 1 mM Poly(P)75 or 1 mM NaPO4 for 25 days and stained by the von Kossa method. Arrow represents nodule formation (A). After co-culture of mouse calvarial osteoblasts and bone marrow macrophages, a pit formation assay in the cultivation with the dentin slice was performed. Dentin decalcified by osteoclasts was stained by Mayer’s hematoxylin (B) and counted microscopically (C). Bar: 5 µm. Error bars represent the mean ± SD of three independent analyses.

Alveolar Bone Regeneration
Areas of newly formed bone that, at 2 wks after surgery, had regenerated over the reference notch marked on the root surface are shown in Fig. 3A. In Poly(P)75-treated rats, intensive ALP immunostaining was observed in osteoblastic cells on the bone surface and in fibroblastic cells in the periodontal ligament, particularly near the cementum, 2 wks after surgery. While the apical migration of the junctional epithelium was more often observed microscopically in rats treated with CMC alone, as compared with rats treated with Poly(P)75 plus CMC, significant differences in the epithelial cell migration could not be found. The mean areas of observed alveolar bone repair in rats with Poly(P)-, CMC-, and no treatment were 3.2 ± 0.6 mm², 0.9 ± 0.2 mm², and 0.7 ± 0.3 mm², respectively (Fig. 3B). New bone formation in rats treated with Poly(P)75 and CMC was 3.6-fold higher than in CMC-treated rats (p < 0.01) 2 wks after surgery. Thus, Poly(P)75 facilitated bone regeneration and resulted in a statistically significant beneficial effect on bone repair when compared with controls in the early stages of rat periodontal regeneration.

View larger version (53K): [in this window] [in a new window]   Figure 3. Histological and immunohistochemical analyses of periodontal tissue generation. (A) HE staining and immunostaining for ALP in the tissue samples given 1 mM CMC or 1 mM CMC + 1 mM Poly(P)75, with the periodontal regeneration mice model, 2 wks following surgery. Arrow: reference notch. Arrowhead: ALP-positive osteoblasts. Bar: 50 µm. (B) Comparison of bone regeneration area 1–3 wks after surgery. CMC: Carboxymethylcellulose. Error bars represent the mean ± SD of five independent analyses.

	DISCUSSION

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Alkaline phosphatase and OPN in the maturation and organization of bone extracellular matrix and OCN in the mineralization phase are characteristic of osteoblast function and activity. They are shown to be indicative characteristics of the MC3T3-E1 cell line, which is used extensively for the study of these sequential events (Balint et al., 2001). Osteocalcin mRNA is a late-stage marker of differentiation and is associated with mineralization (Choi et al., 1996). The von Kossa staining showed the presence of mineralized nodules, indicating induction of calcium phosphate by extracellular matrix mineralization. Thus, Poly(P) induced cell maturation and appeared to be involved in proliferation, matrix development, and matrix mineralization during bone formation. Osteoblasts are very similar to fibroblasts in terms of gene expression and are sometimes viewed as sophisticated fibroblasts (Ducy et al., 2000). Periodontal ligament cells in vivo have the capacity to differentiate into osteoblasts or cementoblasts, through unidentified mechanisms, and to form alveolar bone or cementum (Roberts and Chase, 1981). Therefore, having expression not only in osteoblasts, but also in the functionally oriented periodontal ligament fibroblasts and cementoblasts on the root surface, may lead to new periodontal tissue complex formation. This implies that Poly(P) may contribute to periodontium formation and acute wound healing with homeostasis in periodontal structures, as suggested by the expression of numerous genes affected by Poly(P) via regulation of rpoS expression and the idea that Poly(P) may be related to the expression of other stress-inducible genes (Shiba et al., 2000).

In the present study, Poly(P) induced osteoblastic differentiation of MC3T3-E1 with increased ALP activity. Alkaline phosphatase generates orthophosphate (Pi) and pyrophosphate (PPi) from Poly(P) in association with exopolyphosphatase and pyrophosphatase (Caswell et al., 1987). In bone tissue, extracellular PPi has been identified as a calcification inhibitor (Lang et al., 1955). It is assumed that such inhibitors must be removed from the site of mineralization before calcification can occur (Fleisch et al., 1966). The increase in such enzyme activities would be expected to result in an increased production of Pi and a decrease in PPi concentration (Meyer and Reddi, 1985); thus, Poly(P) might be locally destroyed by such enzymes derived from activated osteoblasts and serve as a donor of Pi for mineralization.

Osteoblast exopolyphosphatase activity is inhibited by the bisphosphonates, including etidronate (Leyhausen et al., 1998). Etidronate inhibits the degradation of both long and short polyP chains by the exopolyphosphatase, which shows a marked biological and structural similarity between bisphosphonates and Poly(P) (Leyhausen et al., 1998), suggesting that polyP metabolism in bone tissue may be analogous to the metabolism of bisphosphonates.

In view of the homology among PPi, bisphosphonates, and Poly(P), effects of Poly(P) on osteoclasts can be speculated. The first possibility is an inhibition of osteoclast recruitment. Several bisphosphonates inhibit osteoclast differentiation in various culture systems (Hughes et al., 1989). The second possibility is a decreased adhesion of osteoclasts to the mineralized matrix. Poly(P)-induced externalization of membranous phosphatidylserine suggests the modulation of the membranous structure of osteoclasts, which leads to a decreased adhesion ability to the mineralized matrix (Hernandez-Ruiz et al., 2006). The third possibility is a shortening of the lifespan of the osteoclast. It has been proposed that this might be due to a toxic effect or induction of osteoclast programmed cell death (apoptosis) (Hughes et al., 1995). The apoptotic effect occurs in macrophage-like cells, including osteoclasts, and is nitric-oxide-independent (Rogers et al., 1996). The last possibility is an inhibition of osteoclast activity. It was reported that bisphosphonates decrease the proton accumulation and the protein synthesis by osteoclasts in vitro (Carano et al., 1990), and also decrease the extrusion of acid through a sodium-independent mechanism by true osteoclasts (Zimolo et al., 1995). Part of this effect may be due to a decrease in proton transport by the vacuolar-type proton ATPase, which is inhibited by several bisphosphonates (David et al., 1996). Moreover, long-chain Poly(P) analogs, long-chain bisphosphonates, actually increase lactic acid production (Shinoda et al., 1983), which could provoke a cytotoxic effect in osteoclasts. Thus, excessive quantities of certain phosphates derived from Poly(P) might lead to induction of the apoptosis or cytotoxicity of osteclasts. In the U266 myeloma cell line, Poly(P) induced arrest of the cell cycle and an increase in the proportion of cells with apoptotic bodies (Hernandez-Ruiz et al., 2006), suggesting that the decrease in the pit formation on the dentin slices might be caused by the induction of osteoclastic apoptosis (Fleisch, 1998), in addition to reduction of osteoclast formation. However, it should be noted that more than one mechanism is most likely involved in the mode of action of Poly(P) for both osteoblasts and osteoclasts.

In conclusion, the results of the present study suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization, and in the inhibition of osteoclastic bone resorption. Local delivery of Poly(P) may thus have adjunctive benefits in the treatment of periodontal disease.

	ACKNOWLEDGMENTS

 
This study was supported by a Grant-in-Aid for Scientific Research (No. 18390548) and a Grant-in-Aid for Creation of Innovation through the Business-Academic-Public Sector Cooperation (No. 13204) from the Ministry of Education, Science, Culture, and Sports of Japan.

Received August 11, 2006; Last revision January 13, 2007; Accepted April 15, 2007

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