http://informahealthcare.com/grf ISSN: 0897-7194 (print), 1029-2292 (electronic) Growth Factors, Early Online: 1–10 ! 2015 Informa UK Ltd. DOI: 10.3109/08977194.2015.1011270

RESEARCH PAPER

The effect of simvastatin treatment on bone repair of femoral fracture in animal model Joa˜o Paulo Mardegan Issa1, Conrado Ingraci de Lucia1, Bruna Gabriela dos Santos Kotake1, Miliane Gonc¸alves Gonzaga1, Fellipe Augusto Tocchini de Figueiredo1, Daniela Mizusaki Iyomasa1, Ana Paula Macedo1, and Edilson Ervolino2 School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil and 2School of Dentistry of Arac¸atuba, Paulista State University – Arac¸atuba, Sao Paulo, Brazil

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Abstract

Keywords

The aim of this research was to evaluate the fracture healing area in osteoporotic femur of female rats restrained by stainless steel wire by statin administration in two different doses (5 mg and 20 mg). Ninety female rats were divided into six groups (n ¼ 15): SH, SH-5 mg, SH-20 mg, OVX, OVX-5 mg, and OVX-20 mg. The surgery consisted of the fracture of the left femur bone and stabilization by K-wire and the administration was restricted and weekly controlled in the drinking water. The euthanasia was conducted at three different moments, five animals per period: 7 d, 14 d, and 28 d. Densitometry, zymography, and histological analyses showed a significant difference between some groups. According to these findings, simvastatin promoted a positive action for bone repair, especially in the osteometabolic group treated with 20 mg of the drug.

Fracture healing, MMP and ovariectomy, statins, simvastatin

Introduction Osteoporosis is a skeletal disease characterized by reduction and change in mass and bone microarchitecture, increasing susceptibility to fracture. Experimental studies investigate the hypothesis that osteoporosis may affect bone repair (Dai & Hao, 2007). Although there are conflicting results, it is assumed that this disease interferes with mechanical and biological determinants in the process of bone healing (Histing et al., 2012; Kubo et al., 1999; Meyer et al., 2001; Namkung-Matthai et al., 2001; Pohlemann & Menger, 2012; Walsh et al., 1997; Xu et al., 2003). To induce osteoporosis in rats, ovariectomy is the most commonly used procedure, promoting interruption in estrogen synthesis because this hormone can affect bone metabolism (Hernandes et al., 2012; Salazar et al., 2011; Tasci et al., 2011; Wang et al., 2007). Several drugs may be used for the prevention and treatment of metabolic bone disease; however, drugs are still required which can stimulate new formation or prevent bone resorption (Wang et al., 2007). Currently the statins, drugs used to treat hypercholesterolemia, are being investigated for having beneficial effects on bone remodeling in the treatment of bone metabolic disorders (Chan et al., 2000; Sugiyama et al., 2000). The effects of these drugs have been shown to decrease bone resorption, increasing bone mineral density (BMD) and bone formation (Ayukawa et al., 2009; Behonick et al., 2007; Correspondence: Prof. Dr. Joa˜o Paulo Mardegan Issa, School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, CEP 14040904, Av. Do cafe S/N, Sao Paulo, Brazil. Tel: +55 16 33150283. E-mail: [email protected]

History Received 19 January 2015 Accepted 20 January 2015 Published online 23 March 2015

Chan et al., 2000; Funkhouser et al., 2002; Kaji et al., 2008). An explanation for these results is that statins decrease the level of metalloproteinases (MMPs), which are extracellular matrix proteases involved in the inflammatory response and cell differentiation at the fracture site, acting on cartilage, bone, and during the stages of angiogenesis soft callus, remodeling, and repair (Behonick et al., 2007). To measure this parameter, zymography analysis was performed to quantify MMPs, as well as immunohistochemistry. Another method used was densitometric analysis by dual energy densitometry X-ray absorptiometry (DXA) that is currently the most widely used method for diagnosis of osteoporosis, demonstrating to be an accurate and costeffective method. Thus, the objective of the study was to analyze the effects of simvastatin on bone repair through radiographic, biochemical, and histological methods in the SH and OVX groups of rats.

Material and methods Animals and experimental design The study was conducted according to the protocol approved by the Ethics in Animal Research Committee of the University of Sa˜o Paulo, Campus of Ribeira˜o Preto (Process no. 11.1.1154.53.5). Ninety female Wistar rats (Rattus novergicus albinus), aged 10 weeks, with 220 ± 10 g of body weight were used. The animals were housed in groups of five rats per cage, receiving food and water ad libitum, under climate-controlled conditions: room temperature at 23 ± 1  C and 12 h of dark– light cycle. Ovariectomy or sham-ovariectomy was performed

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on all animals when they were 70 d old. The animals were randomly divided into six groups (n ¼ 15) and, after 90 d of ovariectomy, the animals were subjected to fracture surgery on the left femur (LF) and fracture restrain with K-wire. (1) SH: ovariectomy surgery simulation (SHAM), fracture surgery, and treatment with saline solution (placebo – (PB)). (2) SH-5 mg: SHAM, fracture surgery, and simvastatin treatment (5 mg/kg/d). (3) SH-20 mg: SHAM, fracture surgery, and simvastatin treatment (20 mg/kg/d). (4) OVX: bilateral ovariectomy surgery (OVX), fracture surgery, and PB treatment. (5) OVX-5 mg: OVX, fracture surgery, and simvastatin treatment (5 mg/kg/d). (6) OVX-20 mg: OVX, fracture surgery, and simvastatin treatment (20 mg/kg/d). Euthanasia was conducted at three different moments, five animals per period: 7 d, 14 d, and 28 d. In each euthanasia moment, both femurs were collected. Ovariectomy surgery On each animal from the OVX groups, the osteoporosis induction was conducted by hormonal deprivation due to ovariectomy surgery, with bilateral removal of ovaries with a dorsal incision (OVX). Prior to surgery, the animals were anesthetized with an i.p. injection of Ketamine solution (75–100 mg/kg) and Xylazine (5–10 mg/kg). After the procedure, the animals were medicated with anti-inflammatory and antibiotic (0.1 ml/100 g) Pentabiotic veterinary – ‘‘Fodge Dodge’’ (Agroberto, Laranjal Paulista, Sao Paulo, Brazil). The SHAM animals had their ovaries exposed and reinserted inside the abdominal cavity and received the same post-treatment as others. Bone osteotomy After the animals’ anesthesia, as previously described in OVX, the external measurement of the femur extension was made using a caliper rule and then this measurement was equally divided into three parts. First, an incision was made followed by divulsion of soft tissue (Figure 1A) and bone exposition (Figure 1B). To stabilize the fracture, a little incision was made near the knee, and the patella was laterally displaced to expose the femoral patella surface, where a little hole was made using a 27 G sterile hypodermic needle (Figure 1C). Then a sterile stainless steel wire was inserted through the hole (K-wire, 1.0 mm in diameter) that ran the length of the medullary canal to the fracture site, being slightly recessed from the final third of the proximal end of the femur (Figure 1D). The fracture procedure was conducted using a sagittal section microsaw (Dentscler Ribeirao Preto, Sao Paulo, Brazil) coupled with a low rotation machine for dental use (Dentscler) 1.2 mm thick (Figure 1E). This fracture occurred as an oblique way between the middle third and the distal part of the bone (Figure 1F), subsequently, steel wire was fully inserted into the medulla (Figure 1G and H). Each animal was medicated with antibiotic (Pentabiotic 0.1 ml/100 g of body weight) and post-operatory analgesia with banamine (1 mg/kg) during the first 3 d after the surgery procedure. The animals were able to ambulate after surgery and showed no infections.

Figure 1. Bone fracture: (A) surgical access, (B) femur bone exposure, (C) production of the opening for the intramedullary fixation, (D) positioning the intramedullary fixation, (E) making the fracture, (F) fractured femur bone, (G) fracture reduced with positioned fixation, (H) radiographic image of the intramedullary rod in place.

Administration of the formulation The PB and simvastatin formulations were administrated daily via the collective water troughs (Fassbender et al., 2001). To determine the amount of water to be placed with the drug, before administering the first dose, the measurement of ingested water per rat was performed during 24 h, determining the approximate amount of 12.5 ml/d of consumption per rat. Thus, drug dilution in the drinking water of the rats was performed according to the daily requirement, in this way, in accordance with the weight of each animal X mg were diluted according to the variance in each group. The administration started on the every day of the surgical fracture and lasted the entire experimental period until the euthanasia of the animals from each group (treatment with simvastatin lasted 7, 14, and 28 d). The SH and OVX groups received saline solution (0.9% NaCl) in distilled water (PB). The SH-5 mg and OVX-5 mg groups received simvastatin solution diluted in distilled water in a concentration of 5 mg/kg. And the SH-20 mg and OVX-20 mg groups received

DOI: 10.3109/08977194.2015.1011270

20 mg/kg of simvastatin solution in distilled water. The average dose was designed by 10 d of consumption by the animals and monitored daily during replacement of the contents of the troughs. After euthanasia, the right femurs (RF), without fracture, were identified and individually frozen at 20  C for posterior analysis of gelatinases by zymography. The LFs had their metallic fixation elements removed and after they were fixed in 4% formalin solution. Bone densitometry

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After 24 h of bone fixation, the LF was subjected to densitometric analysis by DXA (dual energy of X-ray, DPX-Alpha, LunarÕ , Lunar Corporation, Madison, WI). A specific program for small animals was used to obtain bone density (g/cm2). After capturing the images, a decalcification process of the bone samples was started using 0.5 M EDTA. Quantitative histological analysis Subsequent to the decalcification, the LFs were dehydrated in crescent alcohol series: 70%, 90%, and 95% and absolute alcohol I, II, and III (1 h in each concentration), diaphanized in xylene (three washes of 1 h) and soaked in paraffin. Fivemm-thick semi-serial sections were obtained from each sample. These sections (five sections/animal) were stained with Masson Trichrome and photographs were taken under 50 magnification using an Axio Imager 2 (Zeiss, Go¨ttingen, Germany). The percentage of new trabecular bone in the callus region/total area (BV/TV-%) was determined in photographs using the 4.3 AxioVision program (Zeiss, Oberkochen, Germany). Immunohistochemical analysis After deparaffinization, the bone samples were hydrated with a decreasing series of ethanol (100–100–100–90–70 GL). Antigenic recovery was performed immersing the histological slices in Diva DecloakerÕ buffer (Biocare Medical, Concord, CA), in a pressurized Decloaking ChamberÕ (Biocare Medical, Concord, CA), at 95  C, for 10 min. After washing the histological slices in phosphate-buffered saline (PBS) solution 0.1 M, pH 7.4, they were immersed in 3% hydrogen peroxide for 1 h to block endogenous peroxidase. Subsequently, the histological slices were washed in PBS and treated with 3% bovine serum albumin for 12 h to block unspecific binding sites. Histological slices with samples of all the experimental groups were divided into three lots and each of these were subjected to incubation with one of the following primary antibodies used in this study. The primary antibodies were diluted in PBS plus 0.1% Triton X-100 (PBS-TX), for 24 h, in a humid chamber. In subsequent steps, the Universal Dako Labeled (HRP) Streptavidin-Biotin KitÕ (Dako Laboratories, Carpinteria, CA) was used. After the washes, the histological sections were incubated with secondary biotinylated antibody for 2 h, washed and treated with streptavidin conjugated with horseradish peroxidase for 1 h. After three washes in PBS-TX, reactions were developed using as a chromogen the 3.30 -diaminobenzidine tetrahydrochloride (DAB chromogen KitÕ , Dako Laboratories, Carpinteria, CA). At the end of a series of washes in PBS, the histological sections were counterstained with Harris Hematoxylin

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(Labvision Corporation, Fremont, CA). As a negative control, the samples were subjected to the procedures previously described suppressing the use of primary antibodies. Zymography steps for MMP-2 and MMP-9 detection Frozen 1 cm samples of the distal end of the RFs were used. They were submerged in liquid nitrogen and crushed in a stainless steel calcified tissue grinder that was specially developed and designed for studies using rat bones. The pistil was manually operated and a mean of 10 cycles was used. After the pistil was pressed against the bottom grinder a 90degree twist to the right was then applied. Thereafter a piece at the bottom of the grinder was taken out, in which the ground bone was deposited. This crushed bone was then weighted and one part (100 mg) of it was used to make a solution with three parts of the following gelatinase extraction buffer: Tris-HCl 50 mM, pH 7.4, CaCl2 10 mM, containing the proteinase inhibitors phenanthroline, phenylmethylsulthonylfluoride, and N-ethylmaleimide (all these inhibitors were used at 1 mM). This sample was then incubated in ice under shaking for 16 h in the refrigerator. After that, the samples were centrifuged (4  C, 10,000 RPM, for 10 min). The supernatant was collected and stored frozen (20  C) for short times (normally just overnight), until protein quantification was carried out, and the samples were then applied to the gelatin-containing sodium-dodecyl-sulphate polyacrylamide gels, and run. After the run, the gels were washed for two 30 min intervals with 2% Triton X-100 solution, and were then incubated overnight with Tris-HCl 50 mM, pH 7.4, CaCl2 10 mM containing 1 mM ZnCl2. On the following day, the gels were stained with Coomassie Brilliant Blue and destained with 30% methanol (v/v) and 10% acetic acid (v/v), being thereafter photographed on a ChemiDoc MP Image System (BioRad Laboratories, Hercules, CA). The exact sequence of staining and destaining times (which is critical to obtain the same background) is described by Kwak et al. (2000). Gelatinolytic bands were observed as white bands against a dark blue gelatin-stained background. Molecular masses were determined according to the migration of the bands relative to the pre-stained protein ladder (PageRuler Plus Prestained Protein Ladder, Fermentas, Burlington, Ontario, Canada), which was used in all the gels. This ladder has pre-stained blue protein bands, except for the 70 kDa and the 25 kDa bands, which are pre-stained in orange color, and the 10 kDa green band. Gelatinolytic activities between different gels were normalized against an internal standard (1 ml of fetal bovine serum, Figure 2), which was also included into every zymogramic test. Gelatinolytic activity was determined by densitometry of the white bands using the free ImageJ software (National Institutes of Health, NIH, Bethesda, MD). Results are expressed as relative fluorescence units. Details on these analyses are also described by Nascimento et al., (Luckman et al., 1998). In Figure 2, there is an example of Gel of a bone sample exposed to simvastatin 20 mg/kg/d for 28 d post-surgery, which illustrates the analyses of gelatinolytic activity; blank bands demonstrate activity of matrix metalloproteinases MMP-9 at 97 kDa (*) and 84 kDa (**) as well as MMP-2 at 72 kDa (***) and 64 kDa (****). For the zymograph analysis, 50 mg of the

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SHAM At all concentrations after 14 d, the percentage of trabecular bone was statistically higher than the 7 d and 28 d periods (0 mg – p50.001; 5 mg – p50.05, and 20 mg – p50.001) (Table 2). Figure 2. Zymographic gel with fetal bovine serum and samples 1–7 of the ovariectomized rats’ group that received the simvastatin drug for 28 d at 20 mg/kg/d. Blank bands demonstrate activity of matrix metalloproteinases MMP-9 at 97 kDa (*) and 84 kDa (**) as well as MMP-2 at 72 kDa (***) and 64 kDa (****).

sample was inserted per lane aiming to obtain visible and distinct bands in the gel (Figure 2 – numbers shown on the gel represent the sample from 1 to 7).

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Statistical analysis The statistical analysis was made using the SPSS17.0 program (SPSS Inc., Chicago, IL). The ANOVA and Tukey tests were performed. Most data presented adherence to normal distribution, but a minority was subjected to parametric tests since non-adherence is certainly due to type 1 error in statistics.

Results Quantitative histology Comparison of the variation of the trabecular bone according to the dose of simvastatin for the same period OVX 7 d – The different concentrations of simvastatin showed no change in the trabecular bone callus region. 14 d – The OVX-20 mg group demonstrated a significant increase in the trabecular bone, differing from the OVX-0 mg (p50.05) and OVX-5 mg (p50.001) groups. 28 d – The OVX-20 mg group demonstrated a significant increase in the trabecular bone, showing statistical differences in the OVX-0 mg (p50.01) and OVX-5 mg (p50.001) groups (Table 1). SHAM Different concentrations of simvastatin showed no change in the trabecular bone callus region regardless of the dose of simvastatin over time (Table 2). Comparison of the variation of trabecular bone according to the days of treatment for the same drug concentration OVX 0 mg – At this concentration and time of 14 d, there was a significant increase in trabecular bone compared with the periods of 7 d and 28 d (p50.001). 5 mg – At this concentration and time of 7 d, there was no statistical difference in trabecular bone compared with 14 d and 28 d; however, in the 14–28 d, there was a significant reduction in trabecular bone with p50.05. 20 mg – At this concentration, all results for the trabecular bone differed significantly between periods. The time of 7 d showed a lower value than 14 d (p50.001) and 28 d (p50.001). For the time of 14 d, BV/TV was greater than 28 d (p50.05) (Table 1).

Zymography for MMP-2 Comparison of the variation of the MMP-2 according to the dose of simvastatin for the same period The different concentrations of simvastatin showed no change in MMP-2 levels in the bone callus region for OVX and SHAM groups (Tables 1 and 2). Comparison of the variation of MMP-2 according to the days of treatment at the same concentration. OVX 0 mg: At this concentration, no statistical difference over time was observed. 5 mg: At this concentration, there was a significant increase of MMP-2 in the period of 14 d differing statistically from 7 d and 28 d (p50.05). 20 mg: At this concentration, there was a significant increase of MMP-2 in the 14 d period differing statistically from 7 d and 28 d (p50.05) (Table 1). SHAM 0 mg: The period of 28 d showed that there is a significant decrease in MMP-2 levels, differing from 7 d (p50.05). 5 mg: At this concentration, no statistical difference over time was observed. 20 mg: At this concentration, no statistical difference over time was observed (Table 2). Zymography for MMP-9 Comparison of the variation of MMP-9 according to the dose of simvastatin for the period OVX 7 d: The different concentrations of simvastatin showed no change in MMP-9 levels in the bone callus region. 14 d: The different concentrations of simvastatin showed no change in MMP-9 levels in the bone callus region. 28 d: The OVX-5 mg group differed from the OVX-20 mg group (p50.01), in which reduced levels of MMP-9 were found (Table 1). SHAM The different concentrations of simvastatin showed no change in MMP-9 levels in the bone callus region (Table 2). Comparison of the variation of MMP-9 according to the days of treatment for the same concentration OVX 0 mg: At this concentration, it was observed that there was a significant enzymatic increase after 28 d compared with 7 d and 14 d (p50.01).

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5 mg: The period of 28 d showed higher MMP-9 levels compared with 7 d (p50.001) and 14 d (p50.01). 20 mg: At this concentration, no statistical differences were observed in MMP-9 levels during the periods of 7 d, 14 d, and 28 d (Table 1).

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SHAM 0 mg: At this concentration, in the period of 28 d, the MMP-9 level was statistically higher than 7 d (p50.001) and 14 d (p50.01). 5 mg: At this concentration, in the period of 7 d, the MMP-9 level was statistically lower than 28 d (p50.01). In the period of 14 d, the enzyme level was similar to 7 d and 28 d. 20 mg: At this concentration, in the period of 28 d, the MMP9 levels were higher and statistically different to 7 d (p50.01) and 14 d (p50.001) (Table 2). Bone mineral densitometry Comparison of the variation of BMD according to the dose of simvastatin for the same period OVX 7 d: The concentration of 20 mg was statistically reduced compared to the 0 mg concentration (p50.05), and the dose of 5 mg was similar to 0 mg and 20 mg for BMD. 14 d: The different concentrations of simvastatin showed no change for BMD. 28 d: BMD in the concentration of 0 mg was statistically similar to BMD for 5 and 20 mg. A reduced concentration was shown for 5 mg when compared with the BMD of 20 mg (p50.05) (Table 1).

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Table 1. Mean values (standard deviation) of BMD (g/cm2), of trabecular bone (BV/TV), of MMP-2, and MMP-9 and the comparison between the groups and experimental times. OVX Days

0

5

20

Simvastatin (mg) BV/TV 7 14 28 MMP-2 7 14 28 MMP-9 7 14 28 BMD 7 14 28

8.94 (3.9)A 26.2 (7.7)Ba 10.3 (5.9)Aa 293 (41.7) 194.2 (80.4) 137(68.3)

10.6 (2.8)AB 20 (7.7)Aa 7.42 (2.1)Ba

8.66 (3.5)A 37.9 (1.2)Bb 25.1 (6.6)Cb

140.8 (38.1)A 358.2 (207)B 158.4 (68)A

129.4 (49.8)A 348.6 (91.3)B 133.8 (36.5)A

443.9 (61.8)A 430.7 (72.1)A 770 (84.1)Bab

467 (67.3)A 674 (260)A 941 (224)Ba

513 (62.2) 601 (140) 592 (146)b

0.22 (0.02)Aa 0.21 (0)AB 0.18 (0.02)Bab

0.20 (0.03)ab 0.18 (0) 0.18 (0.01)a

0.18 (0)b 0.19 (0) 0.22 (0.01)b

Different capital letters represent statistical differences between lines of the same column (p50.05) and different Greek letters represent statistical differences when comparing the lines of different columns (p50.05).

Table 2. Mean values (standard deviation) of BMD (g/cm2), of trabecular bone (BV/TV), of MMP-2, and MMP-9 and the comparison between the groups and experimental times. SHAM Days

0

5

20

Simvastatin (mg)

SHAM 7 d: At the concentration of 5 mg, the BMD was statistically reduced, differing from 0 mg and 20 mg (p50.001) and these groups, in turn, showed no differences in this parameter. 14 d: At the concentration of 5 mg, the BMD was statistically reduced in relation to the 0 mg dose (p50.05). 28 d: At the concentration of 0 mg, the BMD was lower when compared with 5 mg (p50.05), and for the 20 mg dose, the BMD did not differ from BMD at the doses of 0 mg and 5 mg (Table 2). Comparison of the variation of BMD according to the days of treatment for the same concentration

BV/TV 7 14 28 MMP-2 7 14 28 MMP-9 7 14 28 BMD 7 14 28

OVX At this concentration, the BMD values did not prove to be statistically different at the times of 7, 14, and 28 d for the different doses (Table 1).

Different capital letters represent statistical differences between lines of the same column (p50.05) and different Greek letters represent statistical differences when comparing the lines of different columns (p50.05).

SHAM 0 mg: At this concentration, the BMD values were not shown to be statistically different at the times of 7, 14, and 28 d. 5 mg: BMD was not significantly different from 7 to 14 d, however, at 28 d, the BMD was higher and differed statistically from 7 d (p50.001) and 14 d (p50.001). 20 mg: At this concentration, the BMD values were not shown to be statistically different at the times of 7, 14, and 28 d (Table 2).

6.28 (1.92)A 26.92 (6.6)B 8.38 (1.88)A

7.16 (0.9)A 20.56 (4.7)B 4.62 (0.66)A

9.60 (4.81)A 19.12 (7.3)B 9.86 (2.09)A

432.2 (34.3)A 343.8 (191)AB 157.2 (34)B

265,2(86.5) 321(178) 141.6 (105)

237.8(43) 254.2(180) 96.6 (30.4)

472 (42)A 510 (64)A 801.8 (117)B

482.8 (48)A 575.8 (161)AB 795 (84)B

0.22 (0.03)a 0.20(0.03)a 0.18 (0.02)a

0.14 (0.02)Ab 0.14 (0.02)Ab 0.23 (0.02)Bb

522.2 (52)A 404.2 (64)A 808.8 (146)B 0.21 (0.01)a 0.19 (0.02)ab 0.22(0.01)ab

Qualitative histological analysis On the seventh day of post-surgery, all the experimental groups showed a blood clot on fracture area and connective tissue abundant in fibroblasts, blood vessels, and inflammatory cells. The fracture bone surfaces revealed an area of cartilaginous tissue bordering the fracture. Subjacent to this cartilage, an area of new bone composed of very thin immature trabecular bone was observed (Figure 3).

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On the 14th day of post-surgery, with the exception of the OVX group, all the other experimental groups showed on the fracture area a prevalence of cartilaginous tissue and a remaining blood clot. The OVX group had the prevalence of connective tissue on the fracture area, in addition to Table 3. Immunohistochemical expression for BSP, OCN, VEGF, MMP-2, and MMP-9 in different groups for different experimental times.

cartilaginous and bone tissue. The OVX-20 mg groups showed a higher quantity of new bone tissue in comparison with the other ovariectomized groups (Figure 4). Table 4. Immunohistochemical expression for BSP, OCN, VEGF, MMP2, and MMP-9 in different groups for different experimental times – SHAM groups. Group

Group

Time (d)

BSP

OCN

VEGF

MMP-2

MMP-9

OVX

7 14 28 7 14 28 7 14 28

+ + + + ++ ++ + ++ ++

+ + + + ++ ++ + ++ +++

++ ++ + ++ ++ + ++ ++ +

+ ++ + + ++ + + ++ +

+ + ++ + + + + + +

OVX-5 mg

BSP

OCN

VEGF

MMP-2

MMP-9

SH

7 14 28

+ +++ +++

+ ++ +++

++ ++ +

+ ++ +

+ ++ ++

SH-5 mg

7 14 28

+ ++ +++

+ ++ +++

++ ++ +

+ ++ +

+ + +

SH-20 mg

7 14 28

+ ++ +++

+ ++ +++

++ ++ +

+ ++ +

+ + +

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OVX-20 mg

Time (d)

Figure 3. Histological characteristics of the fracture callus of the femurs using masson trichrome staining at 7 postoperative days, in different experimental groups. Groups: SH (A), OVX (B), SH-5 mg (C), OVX-5 mg (D), HS-20 mg (E), and OVX 20 mg (F). c, cartilaginous tissue; ct, connective tissue, to – bone. Color: Masson Trichrome. Scale bars: 100 mm.

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DOI: 10.3109/08977194.2015.1011270

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Figure 4. Histological characteristics of the fracture callus of the femurs using masson trichrome staining at 14 postoperative days, in different experimental groups. Groups: SH (A), OVX (B), SH-5 mg (C), OVX-5 mg (D), HS-20 mg (E), and OVX 20 mg (F). c, cartilaginous tissue; ct, connective tissue, to – bone. Scale bars: 100 mm.

On the 28th day of post-surgery, all the experimental groups showed on the fracture area a prevalence of new bone tissue that extended to the bone surface, forming callus. A varied amount of cartilaginous tissue still remained on the majority of specimens, also small areas occupied by connective tissue. The quantity of new bone tissue was evidently higher in the OVX-20 mg group, which showed consolidation of the facture and callus formation (Figure 5). Immunohistochemistry analysis The immunohistochemistry for BSP, OCN, VEGF, MMP-2, and MMP-9 staining (Figure 6) showed high specificity on detecting these proteins, which was proved by the complete absence of immunostaining on negative control of the immunohistochemistry reaction (Tables 3 and 4). The immunostaining showed a brownish color confined predominantly on the cytoplasm of some cells and extracellular matrix.

Discussion Simvastatin is a drug used for the treatment of hypercholesterolemia, however, its effects are observed in various organs and tissues including bone tissue (Fassbender et al., 2001; Kwak et al., 2000). Recent studies have shown interesting results for this drug in this tissue, suggesting that this medication might be relevant for the treatment of osteoporosis, since it has been shown to be effective in preventing bone loss (Luckman et al., 1998; Van Beek et al., 1999). This study analyzed the effects of this medication on bone healing (femur) in groups of ovariectomized and nonovariectomized rats. We observed on non-ovariectomized rats that the intake of simvastatin for the variation of trabecular bone, according to the simvastatin dose for the same period, was not able to change this parameter for the periods of 7, 14, and 28 d. Thus, we found that ingesting or not ingesting low and high doses of this medication will

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Figure 5. Histological characteristics of the fracture callus of the femurs using masson trichrome staining at 28 postoperative days, in different experimental groups. Groups: SH (A), OVX (B), SH-5 mg (C), OVX-5 mg (D), SH-20 mg (E), and OVX 20 mg (F). c, cartilaginous tissue; ct, connective tissue, to – bone. Scale bars: 100 mm.

not result in local changes in bone repair. This suggests that this drug in rats with normal estrogen levels does not alter bone formation. Unlike the OVX group, performing the same comparison, we noted that the trabecular bone changes when different doses of simvastatin are taken for the same period as in 14 and 28 d. At 14 d, the trabecular bone increased with the dose of the drug, thus the concentration of 20 mg of the drug showed the greatest results found for BV/TV. At 28 d, the concentration of 5 mg observed a significant BV/TV decrease and at the concentration of 20 mg a significant increase in this value. These results corroborate with the literature showing that statins may have beneficial effects in protecting against osteoporosis due to moderate increase in the formation of cortical bone on the surface of periosteal bone, reduction of oxidative stress, and nitric oxide restoration in aged and ovariectomized rats (Oxlund & Andreassen, 2004; Yin et al., 2012). This indicates that simvastatin may prove

to have an attenuating effect on bone loss in estrogendeficient situations, without preventive effects on normal hormone levels. These results were consistent with the histological findings in which it was observed that statins have accelerated the repair process of the wound area. This suggests that these findings may be due to direct stimulation of endothelial cell proliferation, angiogenesis, and the migration and activation of osteoblasts originating from monocytes generated by statins (Namkung-Matthai et al., 2001; Xu et al., 2003). This also correlates the forming effects mentioned here, with the capacity of simvastatin in reducing apoptosis of osteoblasts and inhibiting osteoclast activity with consequent increase in bone formation (Hernandes et al., 2012; Pohlemann & Menger, 2012; Salazar et al., 2011; Xu et al., 2003). The immunohistochemical reaction showed that the bone growth factors BSP, OCN, and the angiogenic VEGF were expressed in OVX and SHAM.

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DOI: 10.3109/08977194.2015.1011270

The effect of simvastatin treatment on bone repair

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Figure 6. Photomicrographs showing the pattern of immunostaining for immunohistochemical expression for the factors BSP, OCN, VEGF, and MMP9 in bone, represented by arrows head. Bar scale: 30 mm..

In biochemically analyzing bone structure, we describe MMPs representing proteases that degrade extracellular matrix components. These enzymes are extracellular regulators of tissue contributing to homeostasis of many tissues, participating in physiological processes. Its deregulation may lead to pathological conditions and can therefore be highlighted as promising therapeutic targets (Loffek et al., 2011). Seeking better correlation of these factors, the present study examined the levels of MMP-2 and MMP-9 by the zymography technique and found that in the different groups, MMP-2 levels for the OVX groups, concentration of 5 and 20 mg, showed significantly increased MMP-2 with 14 d of treatment, subsequently decreasing after 28 d. This change was not observed for the OVX group that did not ingest the drug, which showed similar levels of the enzyme during treatment. For SHAM group, the MMP-2 levels during treatment differed only in the control group, presenting a significant increase of MMP-2 levels, at 7 d, with subsequent reduction after 28 d. It was noted that the effect of simvastatin on the MMP-2 levels in the OVX and SHAM animals is altered differently. For MMP-9 in the OVX group with 0 mg and 5 mg, enzyme levels of simvastatin showed a significant increase after 28 d, but for 20 mg, there was no statistical difference over time. Moreover, we observed a significant reduction in the levels of MMP-9 with the use of 20 mg after 28 d.

These results confirm the literature reports that MMP-9 secretion by macrophages is reduced by the use of statins (Bellosta et al., 1998). In SHAM, ingestion of simvastatin in different doses showed increased enzyme levels of MMP-9 over time, reaching higher levels at 28 d. We suggest that simvastatin in this situation is not able to modify MMP-9 levels and this may be related to normal hormone levels in these animals that follow the control group parameters. Some other studies (Kamio et al., 2010; Massaro et al., 2010; Souza-Costa et al., 2007a,b) state that simvastatin promotes direct stimulation of endothelial cell proliferation, angiogenesis, and the migration and activation of osteoblasts and cells originating from monocytes. Angiogenesis is a fundamental phenomenon for the repair of fractures, and one of the early stages of bone healing is the reconstruction of intraosseous circulation, as well as increased levels of MMP1, 3, and 9. Our general study for enzyme values demonstrated that enzyme levels of MMP-2 were initially larger and decrease over time, and MMP-9 levels had increased these values with time especially at 28-d mean values. Analyzing BMD, it was observed in the period of 7 d a concentration of 20 mg for OVX showed significantly lower bone density; however, in the period of 28 d for the 20 mg dose of simvastatin, it was observed that mineral density

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J.P.M. Issa et al.

increased compared to the 0 and 5 mg dose. This result suggests that simvastatin may have later effects on the increase in BMD, however, for this parameter, analyzing simvastatin variation according to the concentration over time demonstrated no effect on the increase in BMD. Similarly, Yao et al. (2006) found no effect of simvastatin on bone formation. The SH-0 mg and SH-20 mg groups had BMD values similar and higher than in the SH-5 mg (7 and 14 d). This leads us to believe that initially the concentration of 5 mg of simvastatin can affect bone density, and this effect is then repaired after 28 d of treatment, demonstrating that the time may be decisive in BMD. Based on the results found in the present experimental study, treatment with 20 mg of simvastatin was the concentration with the best results in bone recovery. However, more work must be performed in order to better understand the effects of this medicine in bone repair.

Declaration of interest The authors report that they have no conflicts of interest. The research for this paper was financially supported by the FAPESP Process: number 2011/13449-0, all the authors are gratefully to the FAPESP.

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The effect of simvastatin treatment on bone repair of femoral fracture in animal model.

The aim of this research was to evaluate the fracture healing area in osteoporotic femur of female rats restrained by stainless steel wire by statin a...
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