Osteoporosis is a major health problem, with approximately 100 million individuals at risk worldwide. As bone density decreases with age, usually beginning at menopause in women and at age 55 in men, fracture rates progressively increase, particularly in women. As life expectancy increases, the number of patients at risk for fractures is rising.
Current therapies for the treatment of osteoporosis, including estrogen replacement therapy, selective estrogen receptor modulators, and bisphosphonates, are based primarily on blunting the resorption component of the bone homeostasis. Selective androgen receptor modulators, parathyroid hormone analogs, and oxytocin analogs, all with improved pharmacologic properties in bone, are among the potential approaches to eliciting anabolic effects in the skeleton.
However, despite recent successes with drugs that have positive effects on bone formation, there is a clear need for anabolic agents in individuals who have already suffered substantial bone loss.1"4 Mundy et al1 have shown statins, drugs widely used for lowering serum cholesterol, also enhance new bone formation in vitro in rodents. While many retrospective studies and animal models have suggested statins (3-hydroxy-3-methylglutaryl coenzyme A | HMG-CoA] reductase inhibitors) may increase bone mineral density, no exact in vivo, observational morphologic study has demonstrated their positive effect on bone formation.
This experimental in vivo animal model study examines the effect of statin on bone turnover.
MATERIALS AND METHODS
Twenty-four albino rats approximately 8 to 10 weeks of age and weighing approximately 200 g were used in this study to investigate the biological effects of simvastatin (Zocor; Merck, West Point, Pa) on bone. Because there was no injectable form of the drug, 10-mg tablets were dissolved in 600 pg of dimethyl sulfoxide to prepare the injectant.
Daily effective1 dosages of 5 mg/kg/day of 200-g rats were calculated as 1 mg. Animals were kept in individual cages and fed the same brand of rat food with unlimited access to drinking water. The study was approved by Ankara University's ethics committee.
One milligram of simvastatin was injected daily for 5 days in the distal one third of each rat's right leg. Saline solution was injected in the rat's contralateral leg, which served as the control group. Seven, 14. and 21 days following the procedure, eight rats were sacrificed and both legs harvested for the control and experimental groups.
Figure 1: Photomicrograph of a specimen from the control group shows bone (K) and periosteum (P); new bone formation is not visualized (hematoxylin-eosin, original magnification x10). Figure 2: Photomicrograph of a specimen from the 7-day simvastatin group shows lined up osteoblasts (thick arrows), osteoid tissue (O), and osteocytes (thin arrows) (hematoxylin-eosin, original magnification x 20). Figure 3: Photomicrograph of a specimen from the 14-day simvastatin shows increased formation of new capillaries (arrows), thicker osteoid tissue (0), periosteum (P)1 and bone (K) (hematoxylin-eosin, original magnification x1 0). Figure 4: Photomicrograph of a specimen from the 21 -day simvastatin group shows newly formed bone tissue and chondrocytes (C) (Trichrome-Masson, original magnification x 10). Figure 5: Photomicrograph of a specimen from the 21 -day simvastatin group shows the injected area between the arrows (Trichrome-Masson, original magnification x 10).
Specimens were fixed in formalin for 2 days. After separation of the soft tissues, specimens were treated in a decalcification solution of equal parts of 8% hydrochloric acid and 8% formic acid.5 Decalcification solution was replaced every other day, and tissue decalcification took place for 1 5 days.
Tissues were embedded in paraffin, and after conventional histologic processing, 6-pm slices were cut and stained with hematoxylin-eosin and Trichrome-Masson stains. Histopathologic examination was performed using a Zeiss Axioscope photomicroscope, and microphotographs were taken.
In contrast to the simvastatin-injected legs, no stimulated bone formation was seen in any of the control legs (Figure 1). In the 7-day group on the simvastatin side, osteoblasts were increased under the periosteum close to the bone and were arranged in a line for forming the appositional bone. On the other hand, membranous ossification regions also were present. In this region, osteoid tissue was present. Increased activation of new capillaries was directly proportional to the bone formation (Figure 2).
When the 14-day simvastatin group was compared with the 7 -day simvastatin group, the new osteoid tissue was thicker, the osteoid tissue was well organized, and the formation of new capillaries had spread. The region between the periosteum and the bone was wide (Figure 3).
On examination of the 21 -day simvastatin group, stimulated formation of new bone was increased. In contrast to the 14day simvastatin group, almough the formation of new capillaries was more prevalent, the tissue was stained insufficiendy with blood and chondrocytes were arranged close to the bone (Figure 4). Because of the activation of cells and capillaries, the simvastatin side was enlarged. An enlarged chondral area also was observed close to the bone (Figure 5). The important difference in the 21 -day simvastatin group was the beginning of calcification.
Osteoporosis is a major health problem worldwide, and the incidence and impact of osteoporosis have been studied in terms of cost, morbidity, and quality of life. Substantial bone loss continues throughout old age, with an accompanying exponential increase in fracture risk. Any reduction or arrest of bone loss will result in a concomitant reduction in the incidence of fractures.
The main efficacy criterion for drugs against osteoporosis is protection against fractures. Many resorption-inhibiting agents including estrogens, alendronate, risedronate, raloxifene, calcitonin, and calcium-vitomin D supplements meet mis criterion, but anabolic agents still are urgently required.2,6,7 Therefore, this study investigated the benefit of simvastatin on bone formation in healthy animals.
HMG-CoA reductase inhibitors, or statins, interfere wim the events involved in bone formation independent of their hypolipidemic properties.8 The issue of the efficacy of statins in bone formation continues to be debated, with a few in vivo observational studies yet to confirm the suggested benefit noted in epidemiologic studies. As observed by Mundy et al1 in their in vitro study, the results obtained in Ulis study suggest the effect of statins include acceleration of the proliferation and differentiation of osteoprogenitor cells and advancement of blood supply.
Maritz et al9 investigated the effects of statins on femoral bone mineral density and quantitative bone histomorphometry in rats. Their study compared normal intact female rats mat received different doses of simvastatin, atorvastatin, and pravastatin administered orally for 12 weeks; sham-operated and ovari ectomized rats that received 20 mg/kg/day of simvastatin; and control animals. Results indicated high-dose (20 mg/kg/day) simvastatin increased bone formation and resorption, while low doses (1,5, and 10 mg/kg/day) decreased bone formation and increased bone resorption. The effects of simvastatin on quantitative bone histomorphometry also differed at different dosages. In the present study, the results indicated 5 mg/kg/day of simvastatin administered for 5 days had positive effects on bone formation.
To investigate whether statins may represent new drugs for treating osteoporosis, as Whitfield10 speculated, studies need to be conducted using designer statins that are less liner-oriented and thus better for assessing the optimal doses needed for osteogenicity rather man for lowering cholesterol. Until such studies are undertaken, questions will remain about the effect of statins on bone.
In one recent study, Rejnmark et al" reported an antiresorptive effect based on their data that showed plasma levels of bone turnover markers were lower in statin-treated subjects than in controls. In another study. Das12 reported estrogen, statins, and essential fatty acids, in addition to their other modes of action in the prevention of osteoporosis, have me ability to augment constitutional nitric oxide generation, which is known to be beneficial in osteoporosis.
Mundy et al1 suggested that in bone cells, statins increase the gene expression of bone morphogenetic protein-2 (BMP2), which is an autocrine-paracrine factor for osteoblast differentiation. The development of knowledge about the cellular and molecular mechanisms by which BMPs elicit bone formation over time offer important insights into the mechanisms of response to the treatment. The osteoinductive capacity of BMPs has been demonstrated in preclinical models, and the efficacy of BMPs for the treatment of orthopedic patients is currently being evaluated in clinical trials.13
In this in vivo observational study, simvastatin had a positive effect on bone formation, indicating the use of statins for bone formation might be targeted to the BMP-2. Further clinical and experimental studies are needed to confirm these findings and to investigate their mechanism of action.
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