The Role of the Osteoclast During Endochondral Ossification in a Rat Fracture Model

Bisphosphonates (BPs) are the most common treatment for osteoporosis, due to their powerful ability to inhibit osteoclastic bone resorption. They are also being investigated to augment callus production during fracture healing, however, concerns exist as to the effects of BPs during both initial fracture union and hard callus remodelling. Endochondral ossification during fracture repair is a critical process leading to initial union, and is assumed to be dependent on osteoclast function. Hard callus remodelling, known to be dependent on osteoclast function, is important to the completion of bone repair.

The role of osteoclasts during initial endochondral fracture union was investigated using the BP zoledronic acid (ZA) and in a genetic model of osteoclast inactivity, the incisor absent (ia/ia) rat. In addition, the effect of differing ZA treatment regimes on hard callus remodelling was investigated using both Bolus and Weekly ZA dosing. A Bolus of 0.1mg/kg ZA or 5 Weekly doses of 0.02mg/kg ZA or Saline were administered commencing 1 week post surgery in a rat femoral fracture model. Femoral fractures were also produced in ia/ia rats. Examinations were performed up to initial union and throughout callus remodelling.

ZA treatment did not alter the rate of endochondral fracture union. All fractures united by 6 weeks, with no difference in the percentage of cartilaginous callus between treatment groups at any time point. Fracture union was achieved by 3 weeks in both ia/ia and control rats, again with no difference in the percentage of cartilaginous callus. In contrast, marked differences in hard callus were evident in the ZA treated groups. ZA increased callus bone mineral content, volume and importantly increased callus strength. Bolus ZA treatment did not delay the commencement of hard callus remodelling at 4 weeks post fracture, whereas this was delayed in the Weekly ZA group. By 12 and 26 weeks Bolus ZA had the same callus content of remodelled neocortical bone as Saline, however Weekly ZA had significantly less than saline at these times. These extensive delays in hard callus remodelling with Weekly ZA dosing produced a fracture callus of inferior material properties.

In conclusion, neither ZA treatment nor the absence of active osteoclasts in ia/ia rats delayed endochondral fracture union. Thus, this study confirms the redundancy of osteoclasts in this process. Bolus ZA treatment was superior to Weekly ZA dosing; hard callus remodelling proceeded, producing a strong fracture callus with improved material properties. This study supports the use of less frequent ZA doses during fracture repair.

Year	Entry
1998	Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res. October 1998;355(suppl):S7-S21.
2000	Boivin GY, Chavassieux PM, Santora AC, Yates J, Meunier PJ. Alendronate increases bone strength by increasing the mean degree of mineralization of bone tissue in osteoporotic women. Bone. November 2000;27(5):687-694.
2002	Boivin G, Meunier PJ. The degree of mineralization of bone tissue measured by computerized quantitative contact microradiography. Calcif Tiss Int. June 2002;70(6):503-511.
2005	Odvina CV, Zerwekh JE, Rao DS, Maalouf N, Gottschalk FA, Pak CYC. Severely suppressed bone turnover: a potential complication of alendronate therapy. J Clin Endocrinol Metab. March 2005;90(3):1294-1301.
1998	Lacey DL, Timms E, Tan H-L, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian Y-X, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. April 17, 1998;93(2):165-176.
1991	Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, Golub E, Rodan GA. Bisphosphonate action: alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest. December 1991;88(6):2095-2105.
2002	Street J, Bao M, deGuzman L, Bunting S, Peale FV Jr, Ferrara N, Steinmetz H, Hoeffel J, Cleland JL, Daugherty A, van Bruggen N, Redmond HP, Carano RAD, Filvaroff EH. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA. July 23, 2002;99(15):9656-9661.
2007	Allen MR, Burr DB. Mineralization, microdamage, and matrix: how bisphosphonates influence material properties of bone. BoneKEy Osteovision. February 2007;4(2):49-60.
1999	Gerber H-P, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med. June 1999;5(6):623-628.
2004	Knothe Tate ML, Adamson JR, Tami AE, Bauer TW. The osteocyte. Int J Biochem Cell Biol. January 2004;36(1):1-8.
2003	Currey JD. The many adaptations of bone. J Biomech. 2003;36(10):1487-1495.
2002	Everts V, Delaissé JM, Korper W, Jansen DC, Tigchelaar-Gutter W, Saftig P, Beertsen W. The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation. J Bone Miner Res. January 2002;17(1):77-90.
1999	St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. August 15, 1999;13(16):2072-2086.
1984	Bonnarens F, Einhorn TA. Production of a standard closed fracture in laboratory animal bone. J Orthop Res. 1984;2(1):97-101.
2004	Bone HG, Hosking D, Devogelaer J-P, Tucci JR, Emkey RD, Tonino RP, Rodriguez-Portales JA, Downs RW, Gupta J, Santora AC, Liberman UA; Alendronate Phase III Osteoporosis Treatment Study Group. Ten years' experience with alendronate for osteoporosis in postmenopausal women. NEJM. March 18, 2004;350(12):1189-1199.
1999	Kong Y-Y, Yoshida H, Sarosi I, Tan H-L, Timms E, Capparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penninger JM. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature. January 28, 1999;397(6717):315-323.
1999	Ferguson C, Alpern E, Miclau T, Helms JA. Does adult fracture repair recapitulate embryonic skeletal formation? Mech Dev. September 1999;87(1-2):57-66.
2000	Mashiba T, Hirano T, Turner CH, Forwood MR, Johnston CC, Burr DB. Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces some biomechanical properties in dog rib. J Bone Miner Res. April 2000;15(4):613-620.
2003	Kronenberg HM. Developmental regulation of the growth plate. Nature. May 15, 2003;423(6937):332-336.
1993	Turner CH, Burr DB. Basic biomechanical measurements of bone: a tutorial. Bone. 1993;14(4):595-608.
2002	Boskey AL, Spevak L, Paschalis E, Doty SB, McKee MD. Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone. Calcif Tiss Int. August 2002;71(2):145-154.
1996	Black DM, Cummings SR, Karpf DB, Cauley JA, Thompson DE, Nevitt MC, Bauer DC, Genant HK, Haskell WL, Marcus R, Ott SM, Torner JC, Quandt SA, Reiss TF, Ensrud KE. Randomised trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Lancet. December 7, 1996;348(9041):1535-1541.
2005	Thomsen JS, Laib A, Koller B, Prohaska S, Mosekilde L, Gowin W. Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies. J Microsc (Oxford). 2005;218(2):171-179.
2002	Parfitt AM. Targeted and nontargeted bone remodeling: relationship to basic multicellular unit origination and progression. Bone. January 2002;30(1):5-7.
1990	Reinholt FP, Hultenby K, Oldberg A, Heinegård D. Osteopontin: a possible anchor of osteoclasts to bone. Proc Natl Acad Sci USA. June 1990;87(12):4473-4475.
2001	Hausman MR, Schaffler MB, Majeska RJ. Prevention of fracture healing in rats by an inhibitor of angiogenesis. Bone. December 2001;29(6):560-564.
2003	Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. May 15, 2003;423(6937):337-342.
1996	Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabi CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science. August 2, 1996;273(5275):613-622.
2003	Komatsubara S, Mori S, Mashiba T, Ito M, Li J, Kaji Y, Akiyama T, Miyamoto K, Cao Y, Kawanishi J, Norimatsu H. Long‐term treatment of incadronate disodium accumulates microdamage but improves the trabecular bone microarchitecture in dog vertebra. J Bone Miner Res. March 2003;18(3):512-520.
2004	Komatsubara S, Mori S, Mashiba T, Li J, Nonaka K, Kaji Y, Akiyama T, Miyamoto K, Cao Y, Kawanishi J, Norimatsu H. Suppressed bone turnover by long-term bisphosphonate treatment accumulates microdamage but maintains intrinsic material properties in cortical bone of dog rib. J Bone Miner Res. June 2004;19(6):999-1005.
2005	Bouxsein ML, Myers KS, Shultz KL, Donahue LR, Rosen CJ, Beamer WG. Ovariectomy‐induced bone loss varies among inbred strains of mice. J Bone Miner Res. July 2005;20(7):1085-1092.

Year

Entry

1998

Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res. October 1998;355(suppl):S7-S21.

2000

Boivin GY, Chavassieux PM, Santora AC, Yates J, Meunier PJ. Alendronate increases bone strength by increasing the mean degree of mineralization of bone tissue in osteoporotic women. Bone. November 2000;27(5):687-694.

2002

Boivin G, Meunier PJ. The degree of mineralization of bone tissue measured by computerized quantitative contact microradiography. Calcif Tiss Int. June 2002;70(6):503-511.

2005

Odvina CV, Zerwekh JE, Rao DS, Maalouf N, Gottschalk FA, Pak CYC. Severely suppressed bone turnover: a potential complication of alendronate therapy. J Clin Endocrinol Metab. March 2005;90(3):1294-1301.

1998

Lacey DL, Timms E, Tan H-L, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian Y-X, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. April 17, 1998;93(2):165-176.