Raloxifene supports early fracture healing more than estrogen in ovariectomized rats

Summary

Objectives: Most people suffering from osteoporosis are undiagnosed: the first osteopenic fracture strikes an untreated organism. Therefore implant fixation often fails and bone healing is disturbed. In this basic research project, we explore possibilities to improve the quality of the osteopenic bone immediately after a fracture to avoid these complications.

Methods: Thirty-six female rats, which developed osteopenia within ten weeks duration after ovariectomy (OVX), underwent a standardized metaphyseal osteotomy with a bridging T-plate-fixation. After that rats were divided into three groups, which received soyfree food supplemented with raloxifene (R: 2.02 mg/d) or estradiol-17β-benzoate (E: 0.09 mg/d), orally, or soyfree food (SF) only. During fracture healing the rats were subcutaneously injected with fluorescent agents to help label and visualize the process of bone formation. Bones were analyzed using a three-point bending test, histological sections and microradiographs.

Results: Raloxifene and estradiol have exerted anabolic effects on the trabecular bone. Both substances induced fracture healing mainly via endosteal callus formation (R: 2.08 ± 0.66 mm2, E: 2.02 ± 0.75 mm2 vs. SF: 1.78 ± 0.74 mm2). Due to early bridging and advanced fracture healing, less bone occurred in the later stages after application of test substances. The biomechanical features of the callus formations determined by the Yield load of R- (100.3 ± 28.4 N) were at the level of E-treated bone (93.8 ± 29.7 N) being higher in both comparing to the osteopenic bones (SF: 76.4 ± 18.8 N).

Conclusions: Raloxifene and estrogen had supporting effects in the therapy of fractures of osteopenic bone. They improved not only the bone, but also the callus structure. The raloxifene- and estradiol-treatment enhanced the mechanical properties of the osteopenic bone, which probably lead to (micro) fracture risk reduction. Taking into account all the results there is an advantage for raloxifene; additionally its negative side effects detected in human settings, with respect to e. g. breast cancer propagation, are less than that of estrogens.

Zusammenfassung

Einleitung: Die meisten Menschen, die an Osteoporose leiden, sind nicht ausreichend diagnostiziert: Die erste osteoporotische Fraktur trifft somit den unbehandelten Organismus. Deshalb findet sich bei diesen Patienten häufiger eine gestörte Frakturheilung und, in Folge dessen, postoperatives Implantatversagen. In dem vorliegenden experimentellen Projekt werden Möglichkeiten zur Verbesserung der Qualität und Quantität des osteoporotischen Knochens während der akuten Phase der Frakturheilung untersucht.

Methoden: Sechsunddreißig drei Monate alte weibliche Ratten wurden einer Ovarektomie unterzogen. Nach zehn Wochen erfolgte eine standardisierte metaphysäre Tibia – osteotomie mit T-Platten-Osteosynthese, sowie die randomisierte Zuteilung zur Raloxifen- (R: 2,02 mg/d), Estradiol-17β-benzoat-(E: 0.09 mg/d) oder unbehandelten Gruppe mit sojafreiem Futter (SF). Während der fünfwöchigen Frakturheilung erfolgte eine subkutane Fluoreszenzmarkierung der Knochenneubildung. Postmortal wurden die Tibiae biomechanisch, histomorphologisch, histomorphometrisch und mikroradiografisch untersucht.

Ergebnisse: Raloxifen und Estradiol haben eindeutig anabole Effekte auf den trabekulären Knochen. Beide Substanzen induzieren die Frakturheilung primär über den endo-stalen Kallus (R: 2.08 ± 0.66 mm2, E: 2.02 ± 0.75 mm2 vs. SF: 1.78 ± 0.74 mm2). Aufgrund der früheren Fraktur-Überbrückung und der fortgeschrittenen Heilung unter Raloxifen und Estradiol ist die Knochen-Neubildung in der fünften Woche im Vergleich zur osteoporotischen Gruppe aufgrund der fortgeschrittenen Heilung geringer. Die biomechanischen Eigenschaften (yield load) des Kallus unter Raloxifen (100,3 ± 28,4 N) sind äquivalent zu denen unter Estradiol (93,8 ± 29,7 N), wobei beide signifikant besser als die der osteoporotischen Vergleichsgruppe sind (SF: 76,4 ± 18.8 N).

Schlussfolgerung: Die Raloxifen- und Östrogen- Therapie verbessert die Fraktur - heilung und Struktur des osteoporotischen Knochens in der Ratte. Trotz der geringen Kallusdimension unter Östrogen induzieren beide Substanzen sehr gute mechanische Kalluseigenschaften. So besteht die Option, dass das Auftreten von Mikrofrakturen als Basis der makroskopischen osteoporotischen Fraktur signifikant gegenüber dem nicht behandelten Organismus gesenkt wird. Unter Berücksichtigung aller Ergebnisse ergibt sich für Raloxifen ein Vorteil gegenüber Östrogen, auch weil seine Nebenwirkungen in klinischen Studien, z. B. hinsichtlich des Mammakarzinomrisikos, geringer sind.

Keywords

Fracture healing - osteopenia - raloxifene - estrogen - bone

Schlüsselwörter

Frakturheilung - Osteoporose - Raloxifen - Östrogen - Knochen

• References

• 1 Haussler B, Gothe H, Goel D. et al. Epidemiology, treatment and costs of osteoporosis in Germany – the BoneEVA Study. Osteoporos Int 2007; 18: 77-84.
• 2 Lill CA, Gerlach UV, Eckhardt C. et al. Bone changes due to glucocorticoid application in an ovariectomized animal model for fracture treatment in osteoporosis. Osteoporos Int 2002; 13: 407-414.
• 3 Lill CA, Fluegel AK, Schneider E. Effect of ovariectomy, malnutrition and glucocorticoid application on bone properties in sheep: a pilot study. Osteoporos Int 2002; 13: 480-486.
• 4 Harrison JW, Howcroft DW, Warner JG. et al. Internal fixation of proximal humeral fractures. Acta Orthop Belg 2007; 73: 1-11.
• 5 Kim T, Ayturk UM, Haskell A. et al. Fixation of osteoporotic distal fibula fractures: A biomechanical comparison of locking versus conventional plates. J Foot Ankle Surg 2007; 46: 2-6.
• 6 Danielsen CC, Mosekilde L, Svenstrup B. Cortical bone mass, composition, and mechanical properties in female rats in relation to age, long-term ovariectomy, and estrogen substitution. Calc Tiss Intern 1993; 52: 26-33.
• 7 Bain SD, Bailey MC, Celino DL. High dose estrogen inhibits bone resorption and stimulates bone formation in the ovariectomized mouse. J Bone Miner Res 1993; 8: 435-442.
• 8 Clemett D, Spencer CM. Raloxifene: a review of its use in postmenopausal osteoporosis. Drugs 2000; 60: 379-411.
• 9 Reginster JY, Devogelaer JP. Raloxifene reduces fractures in postmenopausal women with osteoporosis. Clin Orthop Relat Res 2006; 443: 48-54.
• 10 Deal C, Omizo M, Schwartz EN. et al. Combination teriparatide and raloxifene therapy for postmenopausal osteoporosis: results from a 6-month double-blind placebo-controlled trial. J Bone Miner Res 2005; 20 (11) 1905-1911.
• 11 Cao Y, Mori S, Mashiba T, et al. Raloxifene, Estrogen and Alendronate affect the processes of fracture repair differently in ovariectomized rats. J Bone Miner Res 2002; 17: 2237-2246.
• 12 Miwa M, Sibonga JD, Sarkar G. et al. Estrogen deficiency causes delayed fracture repair. Trans Orthop Res Soc 1999; 24: 482.
• 13 Thompson DD, Simmons HA, Pirie CM. et al. FDA Guidelines and animal models for osteoporosis. Bone 1885; 17: 125-133.
• 14 Stuermer EK, Sehmisch S, Rack T. et al. Estrogen and raloxifene improve metaphyseal fracture healing in the early phase of osteoporosis. A new fracture-healing model at the tibia in rat. Langenbecks Arch Surg 2010; 395: 163-172.
• 15 Stuermer EK, Seidlova-Wuttke D, Sehmisch S. et al. Standardized bending and breaking test for the normal and osteoporotic metaphyseal tibias of the rat: effect of estradiol, testosterone, and raloxifene. J Bone Miner Res 2006; 21: 89-96.
• 16 Seidlova-Wuttke D, Stuermer KM, Stuermer EK. et al. Contrasting effects of estradiol, testosterone and of a black cohosh extract on density, mechanical properties and expression of several genes in the metaphysis of the tibia and on fat tissue of orchidectomized rats. Maturitas 2006; 55S: 64-74.
• 17 Stuermer EK, Sehmisch S, Rack T, et al. Details of testosterone, raloxifene and estrogen influence on the microstructure and biomechanics of metaphyseal osteoporotic bone in male rats. World J Urol 2009; 27: 547-555.
• 18 Rahn BA. The fluorochrome sequence labeling of the bone. Nova acta Leopold 1976; 44: 249-255.
• 19 Stuermer KM. The microradiography of the bone. Technique, expressiveness and planimetry. Unfallheilk 1980; 148: 247-251.
• 20 Frost HM. Staining of fresh, undecalcified, thin bone section. Stain Technol 1959; 33: 135-146.
• 21 Melton LJ, Atkinson EJ, O'Fallon WM. et al. Long-term fracture risk prediction with bone mineral measurements made at various skeletal sites. J Bone Miner Res 1991; 6 (S1) 136.
• 22 Kolios L, Hoerster AK, Sehmisch S. et al. Do estrogen and alendronate improve metaphyseal fracture healing when applied as osteoporosis prophylaxis?. Calcif Tissue Int 2010; 86: 23-32.
• 23 Black DM, Cummings SR, Karpf DB. et al. Randomised trial of effect of alendronate on risk of fracture in women with existing vertebral fractures: Fracture Intervention Trial Research Group. Lancet 1996; 348: 1535-1541.
• 24 Chavassieux PM, Marlot ME, Reda C. et al. Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodelling in patients with osteoporosis. J Clin Invest 1997; 100: 1475-1480.
• 25 Delmas PD, Ensrud KE, Adachi JD. et al. Efficacy of raloxifene on vertebral fracture risk reduction in postmenopausal women with osteoporosis: Four-year results from a randomized clinical trial. J Clin Endocrinol Metab 2002; 87: 3609-3617.
• 26 Adomaityte J, Farooq M, Qayyum R. Effect of raloxifene therapy on venous thromboembolism in postmenopausal women. A meta-analysis. Thromb Haemost 2008; 99: 338-342.
• 27 Ettinger B, Black DM, Mitlak BH. et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: Results from a 3-year randomized clinical trial. JAMA 1999; 282: 637-645.
• 28 Roussouw JE, Anderson GL, Prentice RL. et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women's Health Initiative randomized controlled trial. JAMA 2002; 288: 321-333.
• 29 Nakamura T, Imai Y, Matsumoto T. et al. Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts. Cell 2007; 130: 811-823.
• 30 Wronski TJ, Dann LM, Horner SL. Time course of vertebral osteopenia in ovariectomized rats. Bone 1989; 10: 295-301.
• 31 Kalu DN. The ovariectomized rat model of postmenopausal bone loss. Bone Miner 1991; 15: 175-191.