approximately 1.66 million fractures due to osteoporosis are estimated to occur worldwide every year , and this number is expected to quadruple by the year 2050 [ 13 ] . because its insidious and silent evolution , fractures are one of the most frequent symptoms .
these fractures result from a loss of bone mass and a fragile microscopic architecture of the bone tissue .
osteoporotic fractures are associated with a direct or indirect force on weakened bone tissue , which induces continuity failures in the trabeculae [ 46 ] .
some medications , such as bisphosphonates , can stop and even reverse the degenerative disease process in the bone .
bisphosphonates are derived from pyrophosphate and are used in diseases with high rates of bone remodeling , such as paget 's disease , bone tumors , malignant hypercalcaemia , and osteoporosis [ 5 , 79 ] . given their high affinity for bone calcium , these substances act specifically on skeletal tissue and reduce its reabsorption by inhibiting osteoclasts .
zoledronic acid ( za ) , a new bisphosphonate , has been shown to be the most potent inhibitor of bone reabsorption .
studies have shown that za is up to 100 times more effective than alendronate and up to 10,000 times more effective than etidronate , besides its once a year posology [ 1013 ] .
several existing bisphosphonates work on the bone tissue increasing its mass ; however , there are not enough studies that explain how those drugs act on bone mechanical performance .
the aim of the present study was to investigate the effects of za on the biomechanical properties of femoral bone tissue in osteoporotic female rats .
eighty 30-day - old female wistar rats ( rattus norvergicus albinus ) were used . after being clinically evaluated and weighted , they were housed in polypropylene cages with a controlled temperature and light - dark cycle environment , with a rodent diet ( labina , nestl purina petcare company ) and water ad libitum . upon reaching a reproductive age ( i.e. , at 60 days of age , which was 30 days after their arrival in the laboratory ) , the rats were equally and randomly assigned in two groups : group o ( oophorectomy ) ( n = 40 ) and group s ( sham surgery ) ( n = 40 ) .
an oophorectomy was performed after intraperitoneal anesthesia with 30  mg / kg of 3% sodium pentobarbital and a bilateral dorsolateral trichotomy just below the last rib , under sterile conditions . a longitudinal incision approximately 1.5  cm in length was made on the skin between the last rib and the hip joint .
the peritoneal cavity was exposed , and the muscle layer was divulsed for access to the ovary and its surrounding adipose tissue .
the ovary was ligated with a 3.0 cotton thread and resected distally to the ligature ( figure 1 ) .
the muscle and skin were joined with a 30 mono nylon suture , and the same procedures were repeated on the contralateral side to remove the other ovary .
the animals in group s were submitted to the same procedure described above , except for the ligation and resection of the ovaries .
a new sorting was performed at 90 days of age ( 30 days after the surgical procedures ) , and groups o and s were further subdivided into four subgroups according to whether they received 0.1  mg / kg of za ( aclasta , novartis biocincias s.a . ) or distilled water ( dw ) intraperitoneally , which yielded the following groups : oza ( n = 20 ) , odw ( n = 20 ) , sza ( n = 20 ) , and sdw ( n = 20 ) .
ten animals were randomly selected from each group and sacrificed after nine or 12 months of receiving the substances ( moments 1 and 2 ) with a lethal intraperitoneal dose of 3% sodium pentobarbital at 80  mg / kg .
the animals ' right femurs were disarticulated at the proximal ( hip ) and distal ( knee ) areas soaked in a 0.9% saline solution , wrapped in aluminum foil , labeled , and stored in a freezer at a controlled temperature of 20c for 24 hours until the mechanical tests were performed .
axial compression tests were performed to determine the mechanical properties of the femurs using the universal testing machine emic , model dl 10,000 , with an accuracy of (0.018 + f/3700 ) kn , calibrated accordingly to iso 7500 - 1:2004 and abnt nbr 6674:1999 standards .
the machine interfaced with a computer running windows 2000 and the mtest program ( ddl , mn , usa ) , which provided graphical control and was used to record the results .
the femurs were thawed 12 hours before the mechanical tests and kept wrapped in gauze soaked with a 0.9% saline solution .
the distal extremities of the specimens were then fixed ( in a proportion of 2  :  3 ) vertically in 35  ml plastic containers containing 30  ml of auto - polymerizing acrylic resin .
the vertical fixation of the samples was achieved with the aid of a goniometer positioned in two orthogonal planes with respect to the femurs .
a cleaver with a blunt concave edge ( approximately 2  mm diameter ) , fixed to the assay machine , was manually positioned axially to the femur and aligned with its head ( figure 2 ) , until the computer software indicated an axial preload of 0,1n .
once the test was initiated , the cleaver of the testing machine descended axially on the femoral head at a constant speed of 30  mm / min [ 1418 ] ( figure 3 ) .
the m test program interrupted the test at the specimen 's breaking point and automatically provided the results . the maximum load ( ml ) ( newtons - n ) upon rupture and the load - deformation curve were automatically provided by the program ( figure 4 ) . the other variables , including the yield point ( yp ) ( newtons - n ) and stiffness coefficient ( sc ) ( newtons per millimeters - n / mm ) , were calculated by applying johnson 's method to the results provided by the program .
the statistical analysis comprised a completely randomized analysis of variance model and tukey 's multiple comparison tests in the sigmastat version 3.5.1.2 ( systat software , inc .
the variables were assessed using a 2  2  2 analysis of variance factorial design with the following three factors ( each with two levels):surgical procedure ( oophorectomy versus sham surgery);substance administered ( za acid versus dw);moment of euthanasia ( nine versus 12 months ) . surgical procedure ( oophorectomy versus sham surgery ) ; substance administered ( za acid versus dw ) ; moment of euthanasia ( nine versus 12 months ) . both longitudinal and cross - sectional statistical studies were performed , in order to investigate the biomechanical parameters and conclude if ( a ) there was a  time influence , 
( b ) if there was a  drug influence  and ( c ) if there was a  surgery influence .
 a repeated measurement two - way analysis of variance ( anova ) for the longitudinal study design over the biomechanical parameters were used . if f values for a given variable were found to be significant , a paired student 's t - test was employed .
equally , the cross - sectional study design used a two - way anova analysis . when f values were found to be significant , a holm - sidak 's post hoc test was used .
for all the comparisons , differences were deemed to be statistically significant at p < 0.05 .
all the biomechanical measurements , averages , and p values are displayed in tables 1 , 2 , 3 , and 4 .
the analysis of variance revealed a significant increase in the maximum load and yield point and a significant decrease in the stiffness coefficient of the femurs of those animals receiving za ( the oza and sza groups ) , as compared to those that received dw ( the odw and sdw groups ) .
a significant decrease in the maximum load and yield point and a significant increase in the stiffness coefficient were observed after nine months ( moment 1 ) in the animals that were submitted to the oophorectomy procedure and received dw ( group odw ) as compared to the nonoophorectomized group ( group sdw ) .
however , this difference was no longer present at 12 months ( moment 2 ) .
the analysis of variance showed that the nonoophorectomized animals that received dw ( group sdw ) had significantly higher maximum load and lower stiffness coefficients at nine months ( moment 1 ) than at twelve months ( moment 2 ) .
zoledronic acid was selected in this study due to its high rates of positive bone balance with only one annual dose , decreasing patients ' noncompliance with the treatment [ 19 , 20 ] .
the trabecular bone is the main site of action of these drugs , preserving the thickness and density of the bone connections and increasing the resistance of the bone to fractures [ 2123 ] .
patients with severe postmenopausal osteoporosis , that have a high osteoclastic activity , may greatly benefit from this third generation bisphosphonate , simply by keeping their bone mass .
bone tissue has the following three important mechanical properties : bone resistance , which roughly translates to the amount of calcium in the tissue and is defined by the maximum load ; stiffness , which represents the fragility of the material and is defined by the stiffness coefficient ; and yield point , which is the limit at which irreversible structural damage occurs without the possibility of restoring the original shape .
these properties can only be studied experimentally by observing bone behavior when subjected to loading forces .
the equipment used in this study , which provided highly accurate results , was developed for testing isotropic materials ( e.g. , metals ) ; however , biological materials are viscoelastic in nature .
the results obtained were not absolute , given the limitations of the measurement techniques . for determination of biomechanical properties , the assay machine applied to the femurs an axial load under the speed of 30  mm / min , considered by other authors [ 1418 ] as the ideal average speed for viscoelastic materials ( bone ) .
it was also taken into consideration the capacity and type of the used assay machine , which does not allow high speed or impact testing . since the scope of this study was to evaluate only fractures around the hip , the femurs were distally fixated in the acrylic resin in a proportion of 2  :  3 ;
in others studies that considered fractures of the femurs at lower sites ( diaphysis and lower diaphysis fractures ) , femurs were fixated in a proportion of 1  :  3 [ 14 , 15 ] .
the maximum load , defined as the maximum load supportable before the breaking point , was automatically determined by the software ; it represents the highest point in the load - deformation curve .
the region of interest ( roi ) chosen in the present study was the proximal femur , which is a region that is greatly affected by osteoporosis due to the high concentration of trabecular bone .
the significantly increased maximum load in the za groups ( oza and sza ) can probably be explained by inhibition of osteoclast activity , which diminished the osteoporotic process even in the oophorectomized group ( oza ) . in similar biomechanical investigations [ 14 , 15 ] , compressive tests conducted on bone regions with lower concentrations of trabecular bone , such as the tibial diaphysis , produced nonsignificant results .
the hormonal deficit caused by a bilateral oophorectomy results in a loss of bone mass and consequent decrease in the maximum load .
the animals in the present study that received za ( the oza group ) , even those subjected to oophorectomy , had maximum loads equivalent to those of the nonoophorectomized group ( the sza group ) .
the yield point is the weight limit that the body safely supports before plastic deformation occurs , ( i.e. , an irreversible deformation or microstructure rearrangement that does not allow the bone to return to its initial shape and dimension ) .
this variable is defined as the last point in the linear phase ( elastic phase ) of the load - deformation curve . as the yield point
is a rarely studied variable , few studies are available for comparison . za increased the maximum yield point values in this study , which increased the safety zone over which the bone microstructure remained intact and within the limits of elastic deformation .
choosing an roi with a high concentration of trabecular bone may have positively influenced these results [ 1315 ] .
the stiffness coefficient is one of the major biomechanical evaluation parameters ; it indicates the stability or rigidity of the material under the maximum load .
the stiffness coefficient is obtained from the relationship between the applied load and body deformation .
a higher stiffness should be interpreted as a greater propensity to break under a fixed deformation , which is typical of brittle materials , such as glass .
the stiffness coefficient is calculated from the linear portion of the elastic phase of the load - deformation curve ( figure 4 ) .
in other words , za reduced femoral stiffness and rendered the bone to be more ductile and less likely to fracture .
in a similar study of human tibiae at different patients ages , burstein et al .   concluded that the resistance ( maximum load ) was similar in young and elderly individuals ; however , the  old 
brittle  and characteristic of  fragile  materials ; thus , it had a greater stiffness .
hormonal deficiency tends to increase the stiffness coefficient , making the bone more fragile or brittle .
similar to previous results [ 8 , 25 , 26 ] , za could maintain and even reduce the bone stiffness coefficients of the oophorectomized animals in the present study ( group oza ) to levels similar to those of the non - oophorectomized animals ( group sza ) .
these results suggest that the drug maintained the absolute ( i.e. , organic and inorganic ) bone integrity , which would explain the decrease in the femoral stiffness coefficient .
zoledronic acid administered intraperitoneally at a single dose of 0.1  mg / kg significantly increased the maximum load and yield point and reduced the stiffness coefficient in the proximal region of the femur of oophorectomized rats after nine and 12 months of observation .
 
 aim . to investigate the biomechanical effects of zoledronic acid ( za ) on femurs of female osteoporotic rats after follow - up periods of 9 and 12 months 
 . methods . 
 eighty female wistar rats were prospectively assessed . at 60 days of age 
 , the animals were randomly divided into two groups : bilateral oophorectomy ( o ) ( n = 40 ) and sham surgery ( s ) ( n = 40 ) . at 90 days of age , groups o and s were randomly subdivided into four groups , according to whether 0.1  mg / kg of za or distilled water ( dw ) was intraperitoneally administered : oza ( n = 20 ) , odw ( n = 20 ) , sza ( n = 20 ) , and sdw ( n = 20 ) . the animals were sacrificed at 9 and 12 months after the administration of the substances , and then their right femurs were removed and analyzed biomechanically . 
 axial compression tests that focused on determining the maximum load ( n ) , yield point ( n ) , and stiffness coefficient ( n / mm ) of the proximal femur were performed in the biomechanical study . results . 
 za significantly increased the maximum load and yield point , reducing the stiffness coefficient concerning the oophorectomy status and follow - up period . conclusion . 
 zoledronic acid , at a dose of 0.1  mg / kg , significantly increased the maximum loads and yield points and reduced the stiffness coefficients in the femurs of female rats with osteoporosis caused by bilateral oophorectomy . 
1. Introduction
2. Materials and Methods
3. Statistical Analysis
4. Results
5. Discussion
6. Conclusions