a Departamento de Ortopedia, Santa Casa de São Paulo, São Paulo, SP, Brasil
b Serviço de Ortopedia e Traumatologia, Hospital Israelita Albert Einstein, Universidade Federal de São Paulo (Unifesp), São Paulo, SP,Brasil
c Instituto de Ortopedia, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (USP), São Paulo, SP, Brasil
d Escola Paulista de Medicina, Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brasil
Introduction
Postmenopausal osteoporosis is a disease characterized bydeterioration of the microarchitecture and reduction of bonemass as a function of increased reabsorption by osteoclasts,with consequently greater susceptibility to fractures. Thiscondition has very high prevalence and occurs more fre-quently than the sum of cases of myocardial infarct, breastcancer and stroke.1Osteoporosis should be considered to bea public health problem,2because it affects individuals withregard to their social, physical and work functions and, there-fore has a socioeconomic impact.3The main cause of lossof bone mass among women is the estrogen deficiency thatarises at the menopause.
Over the last decade, bisphosphonates have become thecornerstone of osteoporosis treatment.5They are used fortreating and preventing this disease and oncological diseasesthat result in increased bone remodeling.6Bisphosphonateshave a chemical structure that has a strong affinity with cal-cium phosphate, which facilitates bonding to bone. Duringbone reabsorption,7the drug is absorbed by the osteoclastsand causes rupture of the cytoskeleton, loss of the pleatedborder, inhibition of lysosomal enzymes, loss of reabsorptiveactivity and death due to apoptosis.8Thus, there is a diminu-tion of osteoclastic activity, without direct interference withneoformation activity.
Sodium alendronate (ALN) is a second-generation amino-bisphosphonate that is a potent inhibitor of osteoclasticreabsorption.10Use of ALN suppresses bone remodeling11,12and increases bone mineral density (BMD), thereby contribut-ing toward avoiding vertebral and non-vertebral fractures.Through using ALN, bone turnover markers present lowerlevels.12Use of a specific ALN dose for treating osteoporosisresults in significant deceleration of disease progression.
The animal model most used for studying postmenopausalosteoporosis comprises ovariectomy, because over a relativelyshort period after ovariectomy, a state of osteopenia very simi-lar to the human condition is obtained.13,14By using animals, itis also possible to investigate the different forms of treatmentand medications that exist on the market. However, there isno consensus regarding the appropriate dose for tests on ani-mals. The aim of this study was to evaluate the bone tissueresponse in ovariectomized rats, to two different concentra-tions of ALN, which is one of the most widely available drugson the market for osteoporosis treatment.
Materials and methods
All the procedures were approved by the Ethics Committee forAnimal Experimentation of the State University of Maringá(protocol no. 033/2009).
Experimental procedure
Female Wistar rats (Rattus norvegicus) of 60 days ofage were anesthetized with an intramuscular injectionof 2-(2,6 xylidine)-5,6-dihydro-4H-1,3-thiazine hydrochloride(Ronpun®) and ketamine hydrochloride (Ketalar®) in a 1:1
ratio. This association was used at a rate of 1 mL/kg of bodyweight for laparotomy and ovary removal. The procedureswere started only after the interdigital and ocular reflexeshad disappeared. Pulmonary ventilation was maintained in aspontaneous manner. Until recovery from the anesthesia, theanimals were observed with regard to their ocular reflex (bystimulating the upper eyelid using gauze), respiration rate (bydirect observation of the thoracic-abdominal movements) andheart rate (checked using a pediatric stethoscope attached tothe anterior region of the thorax).
The animals were divided into four groups of eight ani-mals each: a control group, in which the animals onlyunderwent laparotomy; an ovariectomized group (OVX); anovariectomized group that was treated with 1 mg/kg of ALN(OVX 1 mg); and an ovariectomized group that was treatedwith 2 mg/kg of ALN (OVX 2 mg). After recovery from the anes-thesia, the animals were kept in groups of four animals percage, at a temperature of 20?C, with dark/light cycles of 12 hand free access to water and food throughout the period ofthe experiment. All the animals were weighed at the start andend of the experiment.
During the week following the ovariectomy and for thesubsequent 90 days, the treated groups were injected intra-muscularly with ALN 1 mg/kg or 2 mg/kg, twice a week, alwaysat the same time of day. After the treatment period, the ani-mals were sacrificed by means of an overdose of ketamine. Theleft femur of each animal was removed and fixed in a solutionof 4% paraformaldehyde for 48 h. The soft tissues were thendissected and, following this, the bone was demineralized informic acid for 21 days. The samples were then washed inrunning water for 6 h and processed for embedding in paraf-fin. Semi-serial sections of thickness 7 m were cut along themajor axis of the femur. These sections were stained withhematoxylin and eosin (H&E) for analysis.
To determine the area occupied by the organic matrix, twoimages of a standardized area in the proximal diaphysis werecaptured, just below the epiphyseal disk of the left femur(Fig. 1A and B). The images were obtained from three sectionsper slide, on four slides per animal, thus totaling 24 imagesper animal. The image capture system used consisted of anOlympus BX41 microscope (Tokyo, Japan) with an Olympus Q Color 3 RT camera (Tokyo, Japan) coupled to it. The imageswere obtained using a 20× objective lens.
The area occupied by collagen was assessed using imageanalysis software (Image-Pro Plus®, version 4.5, Media Cyber-netics, Silver Spring, MD, USA), which obtained the area insquare micrometers.
The area evaluated was composed of trabecular bone andto calculate the area occupied by the organic matrix, the fol-lowing methodology was adopted: firstly, the total area of theimage seen using the 20× objective lens was calculated; then,the empty areas on each slide (corresponding to trabeculatedbone areas occupied by bone marrow) were measured andsummed in order to subsequently subtract these areas fromthe total area (Fig. 2). The final result corresponded to the areaoccupied by the organic matrix. All the calculations were per-formed using the Excel software (Microsoft Corporation). The
mean area in m2in each animal was obtained for statisticalcomparisons.
Statistical analysis
The statistical analysis was performed using the GraphPadPrism R 3.1 software. The data obtained were presented as themean ± standard deviation. The variance analysis test usedwas ANOVA. Tukey's test was used as a post-test for com-parisons between the means. The significance level used was5%.
Results
The animals' initial and final weights in grams are shown inFig. 2. Fig. 3 presents the results from the analysis on the area(m2) occupied by the organic bone matrix.
Discussion
Experimental ovariectomy is a study model that makes it pos-sible to evaluate the consequences, in animals, of loss of bonemass in a variety of situations.15The commonest study topic isthe efficacy of medications available on the market for treatingosteoporosis.4,8,10,16In this model, a period of three monthsafter removal of the ovaries is sufficient for a significant lossof bone mass in the femurs of ovariectomized rats to haveoccurred.17
Ovariectomy leads to a significant progressive increase inbody weight.18In the present study, the ovariectomized ratswere treated with two different concentrations of ALN for 90days. There was an increase in body weight in the untreatedovariectomized rats and in those that received ALN at the rateof 1 mg/kg. The animals that received 2 mg/kg did not presentany change in body weight, in comparison with the control group These results demonstrate that the dose of 2 mg/kg wasmore efficient in preventing gains in body mass.Estrogen deficiency may be related to decreased num-bers of leptin receptors in the hypothalamus, thereby causingdiminished satiety,19greater food intake and consequentweight gain. On the other hand, there is also the possibilityof diminished energy expenditure in females with estrogendeficiency, which would facilitate the gain in body mass.
The ovariectomized rats received treatment with 1 or2 mg/kg of ALN twice a week. It is important to emphasizethat the treatment started one day after ovariectomy and,therefore, these female rats did not already present a con-dition of bone loss. Through histomorphometric analysis onthe organic matrix, it was observed that the treatment with2 mg/kg of ALN had a preventive effect on the loss of bonemass. The animals that received 1 mg/kg presented a reduc-tion in bone matrix similar to that of the ovariectomizedanimals.
Bisphosphonates decrease the degree of bone growthbut without interfering with the quality of the mechanicalresistance of the bone.21In other words, ALN enables pre-vention of reabsorption, but the bone maintains its normalstructural and mechanical characteristics.22Bisphosphonatesmake the bone turnover slower and provide more time forbone structural organization, without impairing or altering themechanical properties of the tissue.
In humans, the recommended dose of ALN is 1 mg/kg ofbody weight, once a week. However, rats have a metabolismthat is twice as fast as that of humans and for this reason, thedrug was administered twice a week. The dose of 1 mg/kg waschosen to be similar to that applied to humans. On the otherhand, the dose of 2 mg/kg was chosen to be the test dose.
There is no consensus between different authors regardingthe ALN dose that should be administered to animals. Itoet al.16observed, in a study on ovariectomized rats, that dailyadministration of 0.2 mg and 1 mg/kg, for 12 weeks, caused asignificant increase in the quantity of organic matrix, in rela-tion to the untreated ovariectomized rats; the bone mineraldensity (BMD) of the ovariectomized group was 24.2% lowerthan that of the control group. Allen24evaluated the concen-trations of 0.1, 0.2 and 1 mg/kg/day of ALN during a one-yeartreatment period. It was observed that the best results wereobtained with higher concentrations of the medication.
Several other authors have tested different concentrationsof ALN, at different times, either in or not in association withother substances such as vitamin K,25vitamin D3,16calcium26and estradiol.4The results from these studies demonstratedin a general manner that ALN has an anabolic effect on lossof bone mass in ovariectomized animals.
Our results showed that the dose of 1 mg/kg twice a weekwas insufficient to stimulate an anabolic effect. However,Masaya et al.16and Allen24demonstrated that this same con-centration, given as daily treatment, produced an increase inbone mineral density after 12 weeks and one year, respectively.
Conclusion
At least three parameters need to be taken into consider-ation when ALN is used in animal models: the dose, treatment duration and posology. Thus, for lower concentrations of themedication, the doses should be more frequent and the treat-ment should be for a longer period, in order to achieve thedesired results; whereas for higher concentrations, the fre-quency of administration can be lower.
Conflicts of interest
The authors declare no conflicts of interest.
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