Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Orthod Craniofac Res. Author manuscript; available in PMC 2010 May 1.
Published in final edited form as:
PMCID: PMC2683378

Modulation of murine bone marrow-derived CFU-F and CFU-OB by in vivo bisphosphonate and fluoride treatments



Bisphosphonates (BPN) have actions on a variety of cell types including: osteoclasts, osteoblasts, osteocytes, and endothelial cells. The objectives of this report are to review the current state of understanding of the effects of BPNs on orthodontic tooth movement and to provide evidence on BPN's in vivo effects on bone marrow derived osteoprogenitor cells.

Material & Methods

Mice from the C3H/HeJ (C3H), C57BL/6J (B6), FVB/NJ (FVB) and BALB/cByJ (BALB) strains were treated for 3-weeks with 0, 3, 30, or 150 mcg/kg/week alendronate (ALN) administered s.c. alone or in combination with 50ppm fluoride (F). Bone marrow cells were harvested and subjected to in vitro CFU-F and CFU-OB assays.


Baseline differences in CFU-F, CFU-OB/ALP+, and CFU-OB/total were observed between the 4 strains. Strain specific responses to ALN and F treatments were observed for CFU-F, CFU-OB/ALP+, and CFU-OB/total. F treatment alone resulted in decreases in CFU-F (p = 0.013), CFU-OB/ALP+ (p = 0.005), and CFU-OB/total (p = 0.003) in the C3H strain. CFU-F (p = 0.036) were decreased by F in the B6 strain. No significant (NS) effects of F were observed for FVB and BALB. ALN treatment resulted in a significant decrease in CFU-F (p = 0.0014) and CFU-OB/total (p = 0.028) in C3H only. ALN treatment had NS effect on CFU-OB/ALP+ in all 4 strains.


Genetic factors appear to play a role in ALN's effects on CFU-F and CFU-OB/total but not on CFU-OB/ALP+.

Keywords: bisphosphonates, alendronate, mesenchymal stem cells, CFU-F, osteoprogenitors

Clinical Relevance

There is a rapidly growing population of patients receiving oral and parenteral bisphosphonates to manage/treat diseases of the bone and in cancer. Concerns have emerged towards unwanted and potentially serious side effects of bisphosphonate therapy. Successful orthodontics relies upon orchestrated function of many of the same cells affected by bisphosphonates. There is great interest to better understand to what extent bisphosphonate therapy may have on orthodontic treatment and outcome as well as any possibility that orthodontic treatment may trigger events capable of leading to serious side effects like osteonecrosis of the jaw.


Nitrogen containing bisphosphonates (BPN) are a major class of bone seeking compounds that are used for the treatment of bone diseases such as Paget's disease of bone, multiple myeloma, bone metastases, osteoporosis (adults), and pediatric bone diseases (i.e. osteogenesis imperfecta). BPNs are stable analogues of naturally occurring inorganic pyrophosphate (PPi) containing two phosphonate groups attached to a single carbon atom, forming a “P-C-P” structure. BPNs have high affinity for hydroxyapatite (HAP) crystals and have multiple direct effects on HAP, including inhibition of calcification, crystal growth, and crystal dissolution. BPNs have highly selective localization and retention in bone preferentially compartmentalizing to bone undergoing resorption or formation and can achieve high local levels (1-3).

Bisphosphonates can inhibit of bone resorption indirectly through impairment of osteoclast function. The non-nitrogenous bisphosphonates can disrupt osteoclast cellular metabolism and induce apoptosis (4,5). In long term bone marrow cultures BPNs inhibit osteoclast differentiation (6,7) and appear to act through osteoblasts to inhibit osteoclast function (7-9). Despite these actions, BPN's greatest effects on osteoclast function come from inhibition of farnesyl pyrophosphate synthase (FPPS) within the melavonate pathway (10-12). FPPS generates isoprenoid lipids during the post translational modification of small GTP-binding proteins (i.e. Rho, Rac, cdc42, and Rab) important for osteoclast formation, function, and survival.

As pointed out above bisphosphonates can have actions on osteoblast function. Bisphosphonates exhibit biphasic effects on osteoblast precursors in vitro—stimulatory at low doses and inhibitory at higher doses (13,14). At a lower dose/exposure, ALN inhibits osteoblast apoptosis (15,16) via connexin 43 (17,18). Several investigators also noted that low dose/exposure of bisphosphonates stimulates osteoblast proliferation and differentiation in vitro (14,19,20). On the other hand, at a higher dose/exposure, bisphosphonates have been shown to exhibit no stimulatory effect on osteoblasts (21,22) and suppress bone formation independently of bone resorption (23). Effects of bisphosphonates, such as ALN, have been observed to be dose dependent and animal model dependent (24). However, no study has investigated the dose-dependent modulation of formation of early and late osteoblastic cell precursors by in vivo ALN treatments with genetic background as a factor. Finally, bisphsophonates have actions on angiogenesis. Clodronate, risedronate, ibandronate, pamidronate, and zoledronic acid have anti-angiogenic actions (25-27). Bisphosphonates can inhibit in vitro proliferation, chemotaxis, circulation, and capillary formation of bone marrow endothelial cells via VEGF and VEFG receptors (28-30). Furthermore bisphosphonates can lead to transient reduction in circulating levels of VEGF, bFGF, and Mmp2 following zolendronate infusion (31).

Bisphosphonates and orthodontic tooth movement

The systemic effects of BPNs on orthodontic tooth movement. Alendronate administered subcutaneously (s.c.) inhibited tooth movement in rats to 40% of the control (32). A single intraperitoneal (i.p.) dose of 1500μg/kg of pamidronate prior to orthodontic tooth movement resulted in impaired osteoclast structure and decreased expression of vacuolar-type H+-ATPase and cathepsin K (33). Systemic pamidronate administered shortly before removal of orthodontic force resulted in decreases the extent of initial relapse of orthodontically moved rat molars (34). Similarly s.c. administration of pamidronate inhibited molar tooth movement in rats (35). The topical application of risedronate, alendronate or clodronate in the subperiosteum of following tooth movement prevented relapse of the moved teeth as well as root resorption (32,36-38). Finally there is in vitro evidence using isolated primary human periodontal ligament (PDL) cells subjected to mechanical induced stress (compression) that clodronate is capable of inhibiting prostaglandin E2 (PGE2), cyclo-oxygenase-2 (COX-2), and receptor activator of NF-kappa B ligand (RANKL) gene expression (39).

Awareness to potential risks of BPN in orthodontics has been raised (40-42). However, there are very few case reports in literature of orthodontic treatment of patients who were taking bisphosphonates, one of which involved only 2 patients (41).


Fluoride is an important micronutrient which, similar to bisphosphonates, preferentially compartmentalizes to bone and accumulates with deposition (43,44). Fluoride is known for its anabolic effects on bone and its use as a therapeutic agent for postmenopausal osteoporosis has been investigated with mixed results (45,46). Fluoride can affect osteoblasts anabolically in vivo (47) and in vitro (43) through an undetermined mechanism and results in increased bone mass (43,48). It has been demonstrated using inbred mouse strains that genetic factors play a role in the effects of fluoride both in dental fluorosis (49) and in variation in bone properties in response to fluoride exposure (50,51). Yan et al. (52) used B6 and C3H inbred strains of mice to show that genetic background influences fluoride's effect on osteoclastogenesis. Since fluoride and bisphosphonate target the same physiological compartment, it is possible that there may be some interaction between these two agents.

Bone marrow derived MSCs and osteogenic potential

In 1970s, Friedenstein et al. reported that marrow stromal cells/mesenchymal stem cells (MSC), from the bone marrow, possess the potential to differentiate along multiple mesenchymal cell lineages, including osteoblast precursors (53-55). A standard liquid culture system was developed to isolate MSC by their adherence to the plastic of tissue-culture plates, where clonal populations expand from single precursors—colony-forming-unit fibroblasts (CFU-F). CFU-F is recognized as the early osteoblastic cell precursors and the CFU-F assay is a useful method to enumerate the number of MSCs in bone marrow (53-56). With the addition of ascorbic acid and dexamethasone, the differentiation of the plastic adherent cells can be modified in vitro at the colony level to give rise to cells capable of forming mineralized nodules—colony-forming-unit osteoblasts (CFU-OB) (56).

The use of CFU-F and CFU-OB assays provides an opportunity for the assessment of the effects of in vivo ALN treatment, alone and with fluoride, on early and late osteoblastic cell precursors from different inbred strains of mice.

Materials and methods


Male mice of the C57BL/6J (B6), BALB/cByJ (BALB), C3H/HeJ (C3H) and FVB/NJ (FVB) inbred strains were obtained from The Jackson Laboratory (Bar Harbor, ME) at 5 weeks of age. Food and water were provided ad libitum. A laboratory rodent diet LabDiet® 5001 (PMI® Nutrition International) was provided and contained 0.95% calcium, 0.67% phosphorous, 4.5IU/gm vitamin D3, and an average [F] of 6.56 ± 0.28 μg/gm. Mice from each strain were caged in trios and housed in the Division of Lab Animal Medicine facility within the Dental Research Center, a fully AAALAC accredited unit, at an ambient temperature of 21°C and maintained on a 12:12 hr light/dark cycle. All experimental procedures were approved by the IACUC at the University of North Carolina at Chapel Hill.

ALN and F treatments

A total of 64 mice per strain were used for this study. After 1 week of acclimation, 8 mice per strain were randomly assigned to one of 8 treatment groups (Group 1 = ALN 3 μg/kg/week; F 0 ppm; Group 2 = ALN 3 μg/kg/week; F 50 ppm; Group 3 = ALN 30 μg/kg/week; F 0 ppm; Group 4 = ALN 30 μg/kg/week; F 50 ppm; Group 5 = ALN 150 μg/kg/week; F 0 ppm; Group 6 = ALN 150 μg/kg/week; F 50 ppm; Group 7= ALN 0 μg/kg/week; F 0 ppm; and Group 8 = ALN 0 μg/kg/week; F 50 ppm).

Fluoride ion (0 or 50ppm) was provided as NaF (Sigma-Aldrich, CAS 7681-49-4) in the drinking water. Alendronate (ALN) (alendronate sodium, a gift from Merck Research Laboratories, Rahway, NJ) was prepared in 0.9% w/v NaCl and administered s.c. The 3 and 30μg/kg/week was administered as a single dose and the 150μg/kg/week was administered as a split dose twice a week. The 3μg/kg/week dose has been previously shown to significantly increased the number of CFU-F colonies in the bone marrow from young and old animals and better permit assessment of bone-forming effects of low dose ALN in osteoporosis (14). The 30μg/kg/week dose was shown to stop bone loss in ovariectomized rats (57). The 150μg/kg/week was considered an equivalent dose for mice to have a maximal effect on osteoclasts without toxic effects. After 3-weeks treatment mice were euthanized. Bone marrow cells were flushed and collected from the tibia and femur from one hind leg of each animal.

CFU-F assays

Bone marrow cells were flushed from the femurs and tibiae of mice and plated in triplicate cultures (six-well plates) at 2 different densities (0.5 × 106 cells/well or 1.0 × 106 cells/well) using complete media prepared with MesenCult™ Basal Medium and Mesenchymal stem cell Stimulatory Supplements (StemCell Technologies; Vancouver, BC, Canada). The formation of CFU-F was evaluated after 14 days of culture in a humidified 5% C02/37°C environment. Cultures were washed with calcium and magnesium free Dulbecco's phosphate buffered saline (PBS) twice and then fixed with cold ethanol. CFU-Fs were stained with Giemsa stain and colonies with >50 cells counted using light microscopy.

CFU-OB assays

The bone marrow cells obtained from the mice were plated in triplicate cultures (six-well plates) at 2 different densities (0.5 × 106 cells/well or 1.0 × 106 cells/well) as described above. The complete media used will be prepared from MesenCult™ Basal Medium and Mesenchymal stem cell Stimulatory Supplements (StemCell Technologies; Vancouver, BC, Canada) plus 50μg/mL of ascorbic acid and 10-8M dexamethasone. After 14 days, the cultures were terminated by washing with PBS twice and then fixed with cold ethanol. Formation of osteoblast progenitors was detected using an alkaline phosphatase assay (86-R, Sigma-Aldrich). Alkaline phosphatase positive colonies were counted. Afterwards the plates were washed with borate buffer and stained with borate buffer containing 1% w/v methylene blue for total colonies.


Results of the CFU-F and CFU-OB assays were reported as mean ± SD of triplicate cultures. For each variable, effects across treatment groups were compared using one-way ANOVA. For comparison of treatment groups in pairs, Student's t-test was used. Adjusted p-values ≤ 0.05 were considered significant.


Strain dependent differences in CFU-F, CFU-OB/ALP+, and CFU-OB/total were observed at baseline (Table 1). BALB mice demonstrated significantly higher mean CFU-F compared to B6 (p < 0.0001), FVB (p < 0.0001), and C3H (p < 0.001). FVB had the lowest mean CFU-F and this difference was significant when compared to C3H (p = 0.002) and BALB (p<0.0001) but not B6. BALB also displayed a significantly higher number of ALP+ CFU-OB than B6 (p < 0.0001), C3H (p < 0.001) and FVB (p < 0.001). When the total numbers of CFU-OB were counted, a greater difference between the strains was noted. BALB had significantly higher number of CFU-OB/total compared to B6 (p < 0.0001) and FVB (p < 0.001); C3H also displayed significantly higher number of CFU-OB/total compared to B6 (p = 0.003) and FVB (p = 0.042). These strain dependent differences in bone marrow derived CFUs were consistent with reports from others (58,59). Intrinsic differences were again observed in the ratios of the total CFU-OB that were ALP+ at baseline for each individual strain. BALB and FVB showed higher ALP+/total CFU-OB ratios compared to B6 (BALB, p = 0.004; FVB, p = 0.006) and C3H (BALB, p = 0.011; FVB, p = 0.017).

Table 1
Colony forming units (CFUs) fibroblast and osteoblast following alendronate and fluoride treatment

Fluoride alone did not significantly alter the frequency of CFU-F, CFU-OB/ALP+, or CFU-OB/total for BALB and FVB mice. Significant decreases in mean CFU-F were found in B6 (p = 0.036) and C3H mice (p = 0.013) treated with 50ppm [F]. Only the C3H strain demonstrated significant reductions in CFU-OB/ALP+ (p = 0.005) and CFU-OB/total (p = 0.003).

Systemic alendronate had no significant effect on the frequencies of CFU-F, CFU-OB/ALP+, or CFU-OB/total for the BALB, B6, and FVB strains. Alendronate treatment significantly reduced CFU-F (p = 0.0014) and CFU-OB/total (p = 0.028) in the C3H strain without affecting the frequency of CFU-OB/ALP+.


In our review of the literature we found that little is known regarding bisphosphonates' action during orthodontic tooth movement beyond reductions in movement magnitude and reducing relapse predominantly in animal models (32,34-38). Bisphosphonates have actions on a variety of cell types including those coordinating angiogenesis. Whereas the majority of studies have focused upon bisphosphonates' actions on osteoclasts and osteoblasts, we chose to investigate potential effects of the nitrogen containing bisphosphonate, alendronate in the context of another bone seeking agent fluoride, on the bone marrow mesenchymal stem cell (MSC) pool. This study investigated the inter-strain responses to ALN treatment (+/- fluoride). Four strains of mice, C57BL/6J (B6), BALB/cByJ (BALB), C3H/HeJ (C3H), and FVB/NJ (FVB) were selected based on differences in genetics, bone biology and wound healing characteristics (49-52,60,61). This study tested the hypothesis that systemic ALN and fluoride would interact and affect the bone marrow pool of MSCs capable of forming early (CFU-F) or late (CFU-OB) osteoprogenitor colonies in a strain-specific manner. While the four strains differed in baseline frequencies of CFU-F, CFU-OB/ALP+, and CFU-OB/total their responses to fluoride or alendronate were limited. Fluoride treatment of 50ppm in the drinking water would raise the serum [F] to a physiologically relevant level of approximately 6-10μM/L (52,62) and had the most pronounced effect on the C3H strain by reducing CFU-F, CFU-OB/total and CFU-OB/ALP+ frequency. B6 was the only other strain that responded to F with a reduction in CFU-F. Alendronate across a wide range of doses resulted in only a modest reduction in CFU-F and CFU-OB/total in the C3H strain. Given the relatively small number of strains investigated it would appear that systemic alendronate has minimal effects on bone marrow MSC pool and no effect on the frequency of ALP+ osteoblast precursor potential.


Supported by a grant from the National Institute of Craniofacial and Dental Research DE014853


1. Russell RG. Bisphosphonates: from the laboratory to the clinic and back again. Bone. 1999;25:97–106. [PubMed]
2. Russell RG. Bisphosphonates: from bench to bedside. Ann N Y Acad Sci. 2006;1068:367–401. [PubMed]
3. Russell RG. Bisphosphonates: mode of action and pharmacology. Pediatrics. 2007;119 2:S150–62. [PubMed]
4. Frith JC, Monkkonen J, Blackburn GM, Russell RG, Rogers MJ. Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5′-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro. J Bone Miner Res. 1997;12:1358–67. [PubMed]
5. Frith JC, Monkkonen J, Auriola S, Monkkonen H, Rogers MJ. The molecular mechanism of action of the antiresorptive and anti-inflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis. Arthritis Rheum. 2001;44:2201–10. [PubMed]
6. Hughes DE, MacDonald BR, Russell RG, Gowen M. Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow. J Clin Invest. 1989;83:1930–5. [PMC free article] [PubMed]
7. Nishikawa M, Akatsu T, Katayama Y, Yasutomo Y, Kado S, Kugal N, et al. Bisphosphonates act on osteoblastic cells and inhibit osteoclast formation in mouse marrow cultures. Bone. 1996;18:9–14. [PubMed]
8. Vitte C, Fleisch H, Guenther HL. Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption. Endocrinology. 1996;137:2324–33. [PubMed]
9. Yu X, Scholler J, Foged NT. Interaction between effects of parathyroid hormone and bisphosphonate on regulation of osteoclast activity by the osteoblast-like cell line UMR-106. Bone. 1996;19:339–45. [PubMed]
10. Fisher JE, Rodan GA, Reszka AA. In vivo effects of bisphosphonates on the osteoclast mevalonate pathway. Endocrinology. 2000;141:4793–6. [PubMed]
11. van Beek E, Pieterman E, Cohen L, Lowik C, Papapoulos S. Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo. Biochem Biophys Res Commun. 1999;255:491–4. [PubMed]
12. Luckman SP, Hughes DE, Coxon FP, Graham R, Russell G, Rogers MJ. Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J Bone Miner Res. 1998;13:581–9. [PubMed]
13. Still K, Phipps RJ, Scutt A. Effects of risedronate, alendronate, and etidronate on the viability and activity of rat bone marrow stromal cells in vitro. Calcif Tissue Int. 2003;72:143–50. [PubMed]
14. Giuliani N, Pedrazzoni M, Negri G, Passeri G, Impicciatore M, Girasole G. Bisphosphonates stimulate formation of osteoblast precursors and mineralized nodules in murine and human bone marrow cultures in vitro and promote early osteoblastogenesis in young and aged mice in vivo. Bone. 1998;22:455–61. [PubMed]
15. Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest. 1999;104:1363–74. [PMC free article] [PubMed]
16. Plotkin LI, Manolagas SC, Bellido T. Dissociation of the pro-apoptotic effects of bisphosphonates on osteoclasts from their anti-apoptotic effects on osteoblasts/osteocytes with novel analogs. Bone. 2006;39:443–52. [PubMed]
17. Plotkin LI, Bellido T. Bisphosphonate-induced, hemichannel-mediated, anti-apoptosis through the Src/ERK pathway: a gap junction-independent action of connexin43. Cell Commun Adhes. 2001;8:377–82. [PubMed]
18. Plotkin LI, Lezcano V, Thostenson J, Weinstein RS, Manolagas SC, Bellido T. Connexin 43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts in vivo. J Bone Miner Res. 2008 Jul 2; Epub ahead of print. [PMC free article] [PubMed]
19. von Knoch F, Jaquiery C, Kowalsky M, Schaeren S, Alabre C, Martin I, et al. Effects of bisphosphonates on proliferation and osteoblast differentiation of human bone marrow stromal cells. Biomaterials. 2005;26:6941–9. [PubMed]
20. Im GI, Qureshi SA, Kenney J, Rubash HE, Shanbhag AS. Osteoblast proliferation and maturation by bisphosphonates. Biomaterials. 2004;25:4105–15. [PubMed]
21. Yaffe A, Kollerman R, Bahar H, Binderman I. The influence of alendronate on bone formation and resorption in a rat ectopic bone development model. J Periodontol. 2003;74:44–50. [PubMed]
22. Garcia-Moreno C, Serrano S, Nacher M, Farre M, Diez A, Marinoso ML, et al. Effect of alendronate on cultured normal human osteoblasts. Bone. 1998;22:233–9. [PubMed]
23. Iwata K, Li J, Follet H, Phipps RJ, Burr DB. Bisphosphonates suppress periosteal osteoblast activity independently of resorption in rat femur and tibia. Bone. 2006;39:1053–8. [PubMed]
24. Sama AA, Khan SN, Myers ER, Huang RC, Cammisa FP, Jr, Sandhu HS, et al. High-dose alendronate uncouples osteoclast and osteoblast function: a study in a rat spine pseudarthrosis model. Clin Orthop Relat Res. 2004;425:135–42. [PubMed]
25. Fournier P, Boissier S, Filleur S, Guglielmi J, Cabon F, Colombel M, et al. Bisphosphonates inhibit angiogenesis in vitro and testosterone-stimulated vascular regrowth in the ventral prostate in castrated rats. Cancer Res. 2002;62:6538–44. [PubMed]
26. Santini D, Vincenzi B, Avvisati G, Dicuonzo G, Battistoni F, Gavasci M, et al. Pamidronate induces modifications of circulating angiogenetic factors in cancer patients. Clin Cancer Res. 2002;8:1080–4. [PubMed]
27. Wood J, Bonjean K, Ruetz S, Bellahcene A, Devy L, Foidart JM, et al. Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid. J Pharmacol Exp Ther. 2002;302:1055–61. [PubMed]
28. Allegra A, Oteri G, Nastro E, Alonci A, Bellomo G, Del Fabro V, et al. Patients with bisphosphonates-associated osteonecrosis of the jaw have reduced circulating endothelial cells. Hematol Oncol. 2007;25:164–9. [PubMed]
29. Hasmim M, Bieler G, Ruegg C. Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways. J Thromb Haemost. 2007;5:166–73. [PubMed]
30. Scavelli C, Di Pietro G, Cirulli T, Coluccia M, Boccarelli A, Giannini T, et al. Zoledronic acid affects over-angiogenic phenotype of endothelial cells in patients with multiple myeloma. Mol Cancer Ther. 2007;6:3256–62. [PubMed]
31. Ferretti G, Fabi A, Carlini P, Papaldo P, Cordiali Fei P, Di Cosimo S, et al. Zoledronic-acid-induced circulating level modifications of angiogenic factors, metalloproteinases and proinflammatory cytokines in metastatic breast cancer patients. Oncology. 2005;69:35–43. [PubMed]
32. Igarashi K, Mitani H, Adachi H, Shinoda H. Anchorage and retentive effects of a bisphosphonate (AHBuBP) on tooth movements in rats. Am J Orthod Dentofacial Orthop. 1994;106:279–89. [PubMed]
33. Sato Y, Sakai H, Kobayashi Y, Shibasaki Y, Sasaki T. Bisphosphonate administration alters subcellular localization of vacuolar-type H(+)-ATPase and cathepsin K in osteoclasts during experimental movement of rat molars. Anat Rec. 2000;260:72–80. [PubMed]
34. Kim TW, Yoshida Y, Yokoya K, Sasaki T. An ultrastructural study of the effects of bisphosphonate administration on osteoclastic bone resorption during relapse of experimentally moved rat molars. Am J Orthod Dentofacial Orthop. 1999;115:645–53. [PubMed]
35. Keles A, Grunes B, Difuria C, Gagari E, Srinivasan V, Darendeliler MA, et al. Inhibition of tooth movement by osteoprotegerin vs. pamidronate under conditions of constant orthodontic force. Eur J Oral Sci. 2007;115:131–6. [PubMed]
36. Adachi H, Igarashi K, Mitani H, Shinoda H. Effects of topical administration of a bisphosphonate (risedronate) on orthodontic tooth movements in rats. J Dent Res. 1994;73:1478–86. [PubMed]
37. Igarashi K, Adachi H, Mitani H, Shinoda H. Inhibitory effect of the topical administration of a bisphosphonate (risedronate) on root resorption incident to orthodontic tooth movement in rats. J Dent Res. 1996;75:1644–9. [PubMed]
38. Liu L, Igarashi K, Haruyama N, Saeki S, Shinoda H, Mitani H. Effects of local administration of clodronate on orthodontic tooth movement and root resorption in rats. Eur J Orthod. 2004;26:469–73. [PubMed]
39. Liu L, Igarashi K, Kanzaki H, Chiba M, Shinoda H, Mitani H. Clodronate inhibits PGE(2) production in compressed periodontal ligament cells. J Dent Res. 2006;85:757–60. [PubMed]
40. Keim RG. Bisphosphonates in orthodontics. J Clin Orthod. 2006;40:403–4. [PubMed]
41. Rinchuse DJ, Rinchuse DJ, Sosovicka MF, Robison JM, Pendleton R. Orthodontic treatment of patients using bisphosphonates: a report of 2 cases. Am J Orthod Dentofacial Orthop. 2007;131:321–6. [PubMed]
42. Zahrowski JJ. Bisphosphonate treatment: an orthodontic concern calling for a proactive approach. Am J Orthod Dentofacial Orthop. 2007;131:311–20. [PubMed]
43. Farley JR, Wergedal JE, Baylink DJ. Fluoride directly stimulates proliferation and alkaline phosphatase activity of bone-forming cells. Science. 1983;222:330–2. [PubMed]
44. Palmer C, Wolfe SH. American Dietetic A. Position of the American Dietetic Association: the impact of fluoride on health. J Am Diet Assoc. 2005;105:1620–8. [PubMed]
45. Haguenauer D, Welch V, Shea B, Tugwell P, Adachi JD, Wells G. Fluoride for the treatment of postmenopausal osteoporotic fractures: a meta-analysis. Osteoporos Int. 2000;11:727–38. [PubMed]
46. Cranney A, Guyatt G, Griffith L, Wells G, Tugwell P, Rosen C, et al. Meta-analyses of therapies for postmenopausal osteoporosis. IX: Summary of meta-analyses of therapies for postmenopausal osteoporosis. Endocri Rev. 2002;23:570–8. [PubMed]
47. Turner CH, Garetto LP, Dunipace AJ, Zhang W, Wilson ME, Grynpas MD, et al. Fluoride treatment increased serum IGF-1, bone turnover, and bone mass, but not bone strength, in rabbits. Calcif Tissue Int. 1997;61:77–83. [PubMed]
48. Lau KH, Baylink DJ. Molecular mechanism of action of fluoride on bone cells. J Bone Miner Res. 1998;13:1660–7. [PubMed]
49. Everett ET, McHenry MA, Reynolds N, Eggertsson H, Sullivan J, Kantmann C, et al. Dental fluorosis: variability among different inbred mouse strains. J Dent Res. 2002;81:794–8. [PubMed]
50. Mousny M, Banse X, Wise L, Everett ET, Hancock R, Vieth R, et al. The genetic influence on bone susceptibility to fluoride. Bone. 2006;39:1283–9. [PubMed]
51. Mousny M, Omelon S, Wise L, Everett ET, Dumitriu M, Holmyard DP, et al. Fluoride effects on bone formation and mineralization are influenced by genetics. Bone. 2008;43:1064–74. [PMC free article] [PubMed]
52. Yan D, Gurumurthy A, Wright M, Pfeiler TW, Loboa EG, Everett ET. Genetic background influences fluoride's effects on osteoclastogenesis. Bone. 2007;41:1036–44. [PMC free article] [PubMed]
53. Friedenstein AJ. Precursor cells of mechanocytes. Int Rev Cytol. 1976;47:327–59. [PubMed]
54. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. 1976;4:267–74. [PubMed]
55. Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet. 1987;20:263–72. [PubMed]
56. Owen M, Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp. 1988;136:42–60. [PubMed]
57. Frolik CA, Bryant HU, Black EC, Magee DE, Chandrasekhar S. Time-dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: effects of raloxifene HCl, tamoxifen, estrogen, and alendronate. Bone. 1996;18:621–7. [PubMed]
58. Dimai HP, Linkhart TA, Linkhart SG, Donahue LR, Beamer WG, Rosen CJ, et al. Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice. Bone. 1998;22:211–6. [PubMed]
59. Phinney DG, Kopen G, Isaacson RL, Prockop DJ. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem. 1999;72:570–85. [PubMed]
60. Al-Qawasmi RA, Hartsfield JK, Jr, Everett ET, Weaver MR, Foroud TM, Faust DM, et al. Root resorption associated with orthodontic force in inbred mice: genetic contributions. Eur J Orthod. 2006;28:13–9. [PubMed]
61. Beamer WG, Donahue LR, Rosen CJ. Genetics and bone. Using the mouse to understand man. J Musculoskelet Neuronal Interact. 2002;2:225–31. [PubMed]
62. Everett ET, Yan D, Weaver M, Liu L, Foroud T, Martinez-Mier EA. Detection of dental fluorosis-associated quantitative trait loci on mouse chromosomes 2 and 11. Cells Tissues Organs. 2008;189:212–17. [PMC free article] [PubMed]