J Bone Miner Metab (2002) 20:223–227 © Springer-Verlag 2002 Microstructural properties of bone in rat vertebra after long-term clodronate treatment Antti Koivukangas 1 , Juha Tuukkanen 2 , Petri Lehenkari 1 , Raija Peura 3 , Ritva Hannuniemi 4 , Katriina Kippo 4 , Timo Jämsä 5 , and Pekka Jalovaara 1 1 Division of Orthopedic Surgery, University of Oulu, P.O. Box 5000, FIN-90014 University of Oulu, Finland 2 Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland 3 Institute of Electron Optics, University of Oulu, Oulu, Finland 4 Biomedical Research Center, Leiras Oy, Turku, Finland 5 Department of Medical Technology, University of Oulu, Oulu, Finland treatment of bone disorders associated with increased bone resorption, such as osteoporosis, Paget’s disease, and metastatic bone diseases [2,3]. The accumulation of BPs in bone reaches a plateau only after a very long time. In humans, this means years or even decades of administration [1]. It is also known that the skeletal half-life of BPs is long, ranging between 3 months and 1 year [4]. Short-term treatments with therapeutic doses of BPs are generally well tolerated in both experimental and clinical contexts [4,5], whereas etidronate at high doses has been shown to impair normal mineralization in animals as well as in humans [5,6], and impaired min- eralization has also been reported after pamidronate treatment of Paget’s disease and fibrous dysplasia [7]. There are only a few experimental studies of the long- term effects of BPs on the skeleton. Etidronate has been shown to provoke spontaneous fractures and impair normal mineralization in dogs after 12 months of treat- ment [6]. Beneficial bone effects of 2-year alendronate treatment in rats have been reported [8]. Moreover, 1-year tiludronate treatment administered to grow- ing monkeys turned out to be safe [9], and 1-year pamindronate treatment of dogs increased bone stiff- ness, as calculated sonographically from the dog sterna [10]. In long-term studies of experimental osteoporosis, 2-year treatment of baboons with alendronate [11] and zoledronate treatment of monkeys for 69 weeks pre- vented experimental osteopenia [12] and involved no adverse effects. There are, however, only a few studies concerning the long-term effects of clodronate in grow- ing rats [13,14]. These studies have shown that long- term treatment has a beneficial effect on bone density and strength at the macroscopic level, while a high dose of clodronate decreased the bone growth rate. The ultrastructural properties of bone after 4 months of clodronate treatment were studied by Wink as early as 1986 [15]. This study showed that treatment with clodronate prevented the endosteal bone surface Abstract Bisphosphonates (BPs) are known to increase bone mineral density, but it is not known how this increase manifests at low hierarchic levels of the bone structure. The present study aimed to clarify the effects of the long-term use of clodronate on the microstructure and chemical composition of bone. The second lumbar vertebral body (L2) in growing rats, subjected to 32 weeks’ treatment with clodronate at ei- ther a therapeutic dose of 2 mg/kg, or a high dose of 10 mg/kg, or physiological saline (control group), was studied by scan- ning electron microscopy for morphology, by backscattered electron image (BSE) for density, and by energy dispersive spectrometry for material analysis. BSE images showed that the degree of mineralization in the different areas of trabecu- lar bone of the vertebral body varied in both the control and the study groups, but this variation seemed to be different in the control and study groups. BSE analysis showed that there was more high-density bone (white area) in the low-dose clodronate group than in the controls, but the difference be- tween the high-dose clodronate group and the control group was not significant. The density of the white area (high-density bone) was slightly increased in the low-dose clodronate group. There were no differences in the density of the gray area (low- density bone) between the groups. Neither the distribution of Ca, P, or Mg, nor the total mineral content, was affected by the clodronate treatment. Our results indicate that long-term clodronate treatment at the therapeutic level increases the proportion of high-density bone in the vertebral body in non- osteoporotic rats. Key words bisphosphonate · clodronate · long-term · rat Introduction Bisphosphonates (BPs) decrease bone turnover and, therefore, bone loss [1]. They are widely used in the Offprint requests to: P. Jalovaara Received: October 29, 2001 / Accepted: January 25, 2002