Effects of local application of simvastatin on bone regeneration in femoral bone defects in rabbit.

Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e6

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Effects of local application of simvastatin on bone regeneration in femoral bone defects in rabbit Konstantinos Papadimitriou a, George Karkavelas b, Ioannis Vouros a, Eftichia Kessopoulou c, *, Antonis Konstantinidis a a b c

Department of Preventive Dentistry, Periodontology and Implant Biology, Dental School Aristotle University, Thessaloniki, Greece Medical School, Aristotle University, Thessaloniki, Greece Dental School, Aristotle University, Thessaloniki, Greece

a r t i c l e i n f o

a b s t r a c t

Article history: Paper received 26 February 2014 Accepted 12 November 2014 Available online xxx

Simvastatin (SIM), which is widely used in hyperlipidemia treatment, has also attracted attention due to its anabolic effects on bones. This study is designed to investigate the effectiveness of 2 mg SIM combined with 3 different carriers as delivery systems. Bone defects were surgically created in the femoral bones of 14 New Zealand white rabbits. The carriers used were the inorganic bovine bone graft (BOS), the hydroxyapatite combined with calcium sulfate (HACS), and the collagen sponge (COS). The bone defects were divided for each time period into 7 groups, as follows: passive control group (CONT), active control groups (BOS), (HACS) and (COS) (no simvastatin), and groups (BOS þ SIM), (HACS þ SIM) (carrier and simvastatin combination). Animal were sacrificed at 4 and 8 weeks postoperatively, and bone defects areas were prepared for histological examination and histomorphometric evaluation. Analysis of variance demonstrated statistically significant differences between groups depending on the carrier used. At 4 weeks, the BOS þ SIM group presented higher rates of new bone formation, whereas at 8 weeks more new bone formation was noted for the HACS þ SIM group. This study suggests that local application of simvastatin, combined with an appropriate carrier, can promote new bone formation. © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Keywords: Simvastatin Bone defects Carriers Bone regeneration

1. Introduction The development of statins (HMG-coA) as therapeutic agents in hypercholesterolemia has been of great importance due to their safe and targeted action in the reduction of high levels of cholesterol in the blood (Marou et al., 2000). Recent research data has demonstrated that the effect of statins is not limited to their lipidlowering properties, but they also present pleiotropic action. The effects of statins in the nervous, cardiovascular, and skeletal systems as well as their immunological response are well documented. Statins are also involved in several cellular functions associated with the improvement of endothelial dysfunction, namely, the antioxidant, anti-inflammatory, and anticoagulant effect, the stabilization of atherosclerotic plaques, the inhibitory effect on transplant rejection, the antitumor activity in laboratory animals,

* Corresponding author. Tel.: þ30 6945443244. E-mail address: [email protected] (E. Kessopoulou).

and the anabolic effect on bone tissue (Davignon and Laaksonen, 1999; Mundy et al., 1999; Bellosta et al., 2000). Clinical in vivo and in vitro studies revealed that statins reduce osteoclast activity and activate osteoblast differentiation and bone formation (Mundy et al., 1999; Maeda et al., 2001; Grasser et al., 2003; Song et al., 2003; Staal et al., 2003; Maeda et al., 2004; Baek et al., 2005; Hughes et al., 2007; Ayukawa et al., 2008). In particular, they increase the expression of bone morphogenetic proteine2 (BMP-2) and vascular endothelial growth factor (VEGF) (Mundy et al., 1999; Sugiyama et al., 2000; Maeda et al., 2003). Because of these properties, it is considered that statins may be an important therapeutic option in the treatment of osteoporosis, fractures, and bone defects. The systemic (oral) administration of statins presents a limited positive effect on bone healing because of their high hepatic action; statins do not accumulate on the bone as do bisphosphonates. Furthermore, high doses of oral administration may increase the risk for liver damage and kidney disease (Brown, 2008). The local application of statins directly to the bone defect area using

http://dx.doi.org/10.1016/j.jcms.2014.11.011 1010-5182/© 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Papadimitriou K, et al., Effects of local application of simvastatin on bone regeneration in femoral bone defects in rabbit, Journal of Cranio-Maxillo-Facial Surgery (2014), http://dx.doi.org/10.1016/j.jcms.2014.11.011

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K. Papadimitriou et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e6

appropriate grafted materials that are used as carriers and can achieve a controlled and gradual release of the substance increases their effectiveness on skeletal tissues. Mundy et al. were the first who reported the in vivo activation of bone formation by the use of statins (Mundy et al., 1999). According to in vitro and in vivo studies, simvastatin is considered a statin suitable for bone growth activation (Garrett et al., 2001). Several studies have demonstrated that high concentrations of simvastatin increase bone formation and decrease bone resorption (Maritz et al., 2001). Simvastatin presents local and systemic antiinflammatory activity, but may cause clinical symptoms (inflammation) when applied locally in high concentrations (Thylin et al., 2002). Several researchers have studied the effect of locally administered simvastatin in various concentrations (Thylin et al., 2002; Wong and Rabie, 2003, 2005; Stein et al., 2005; Nyan et al., 2007; Ma et al., 2008; Moriyama et al., 2008). Some of them are the ACS (absorbable collagen sponges) (Boyne et al., 1997; Howell et al., 1997), the DBM (decalcified bone matrix) (Toriumi et al., 1991; Sigurdsson et al., 1996), hyaluronate (hyalorunan) (Hunt et al., 2000), the inorganic bovine bone graft (deproteinized bovine bone matrix) (Jang et al., 2003; Wong and Rabie, 2003), hydroxyapatite (HA) (Koempel et al., 1998), calcium phosphate (CP) (Wikesjo et al., 2002), calcium sulfate (CS) (Nyan et al., 2007), PGA/ PLGA (polylactic acid/polyglycolic acid copolymer) (Boyne and Shabahang, 2001), and others. The purpose of this study was to investigate the local action of simvastatin in bone regeneration. More specifically, our study examined and evaluated histologically the effect of simvastatin local application on bone defects of laboratory animals (rabbits) at 4 and 8 weeks postoperatively, and evaluated the effectiveness of three grafted materials that were used as carriers. These materials were the bovine inorganic bone graft (BOS), the hydroxyapatite with calcium sulfate (HACS) and the collagen sponge (COS). 2. Materials and methods This study was conducted in a total number of 14 adult male white rabbits (New Zealand white rabbits, Oryctolacus cuniculus), weighing 3.0e3.5 kg. Fifty-six bone defects were surgically created (4 bone defects per animal, 2 bone defects per femur). Half of the animals (7) were sacrificed in the 4th week and the rest in the 8th week. The groups were as follows: BOS þ SIM, BOS, HACS þ SIM, HACS, COS þ SIM, COS, and CONT. The BOS, HACS, and COS groups were the active control groups, and CONT was the passive control group. The animals were sustained during the study period in an appropriately designed area of Experimental Surgery of the Second Surgical Clinic of the Medical School of the Aristotle University of Thessaloniki (Installation Experimentation EL 54 BIO 09) in accordance to the regulations and conditions set by the Veterinary Service of Thessaloniki Prefecture, which approved the conduct of this study (Protocol No. 13/4865, Thessaloniki 13/04/2009). The study was approved by the Ethics Committee of the Dental School of the Aristotle University of Thessaloniki, which ensures that all procedures described in this study were conducted in accordance with the principles of the Regulation Ethics Research Committee of the Aristotle University of Thessaloniki (Protocol No./Date: 185/17.4.10).

(Betadine) and proper preparation of hair removal (shaving), an incision of 4e5 cm was made on the skin and the muscle layer, until the femur was adequately exposed. Two bone defects 5 mm in diameter and 2 mm in depth were surgically prepared in each femur of each animal by a low-speed surgical motor (Kavo Intrasurg 500) with a carbide drill (2 mm), under constant saline irrigation. A titanium cylinder specifically prefabricated for this purpose with dimensions of 4 mm in diameter and 4 mm in height was placed in each bone defect, to be filled with the three different simvastatin carriers. In the passive control group, the titanium cylinders remained empty (CONT group). The purpose of their use was to achieve the same volume of carrier used in all cases (Figs. 1 and 2). The carriers that were used are the following: 1) the collagen sponge (Bioteck, Biocollagen (TO); 2) the inorganic bovine bone graft (Bio-Oss, Geistlich Pharma AG, Wolhusen, Switzerland), and 3) the hydroxyapatite with calcium sulfate (Ostim PerOssal, Biomaterials GmbH et Co. KG, Dieburg, Germany). The simvastatin concentration used was 2 mg (Artemis Biotech, Themis Medicare Limited, Industrial Development Area, Jeedimetla, Hyderabad). All bone defects were covered by Gore-Tex nonabsorbable membrane. We used 4/0 absorbable and nonabsorbable sutures for the muscle layer and the skin, respectively. Half of the animals were sacrificed in the 4th week and the rest in the 8th week. The femurs were removed, and the bone defect areas were histologically examined and analyzed by the Laboratory of Pathology of Medical School of Aristotle University of Thessaloniki. 2.2. Histological preparation and histomorphometry The bone specimens were fixed in neutral formaldehyde (volume ratio material/formaldehyde 1/10) for 48 h at room temperature. For the decalcification procedure, the bone specimens were immersed in solution (Calci-Clear Rapid of National Diagnostics) and remained at constant temperature for 2e4 days. The process was strictly controlled and 2- to 3-mm incisions were performed after achieving the appropriate composition of the specimens. Subsequently, the specimens were kept in an automatic system for 24 h and, after their inclusion in paraffin blocks, histological sections of 8e10 mm were obtained with a conventional microtome. These histological sections were stained with hematoxylineeosin. Where necessary, for technical reasons, the preparation was enriched with specific contractual stains such as Masson and Van Gieson trichromatic staining of connective tissue. The histological

2.1. Surgical procedure All animals were anaesthetized with xylazine (Rompun) and ketamine (Ketaset) injections (concentration 5 ml and 2 ml, respectively) (Rompun-Bayer AG, Leverkusen, Germany, Bayer Corporation, Agriculture Division, Animal Health, Kansas, USA, Ketaset-Fort Dodge Animal Health Division of Wyeth, Fort Dodge, IA). After disinfection of the surgical field with iodine solution

Fig. 1. Bone defects were surgically created.



Fig. 2. Titanium cylinders were applied and filled with the carriers used.

examination of bone preparations was carried out by conventional microscopy. The quantitative assessment of newly formed bone was made by the histomorphometry system PRODIT 5.2 (Quantitative image analysis and image database system, Interactive Morphometry Programme, BMA, De Meern, The Netherlands). The measurements were directly taken using a light microscope at an appropriate magnification. The newly formed bone of each bone defect area was measured in and the results were statistically compared. The histological parameters evaluated were the newly formed bone and the degree of maturation (lamellar or nonlamellar form), the quantity of newly formed bone, the presence or absence of fibrosis, and the inflammation and localization of the carriers used in the bone defect areas. 2.3. Statistical analysis To evaluate the effect of the different types of treatment at each time point (4 or 8 weeks), the data were submitted to analysis of variance (ANOVA) based on the linear mathematical model comprising the factors of time, carrier, and presence or absence of simvastatin. The criterion of least significant difference (LSD) was applied for the comparisons of averages. The significance level of statistical tests was predetermined at a ¼ 0.05 and the statistical analyses were performed with the statistical package SPSS V.15.0 (SPSS Inc., Chicago, IL).

inflammatory reaction was more intense for the simvastatin groups, especially for the HACS þ SIM group. But even in that case, a new bone formation was seen despite the inflammation. The histological features of all group samples were similar. The inorganic bovine bone graft (BOS) and the hydroxyapatite with calcium sulfate graft (HACS) were histologically found at 4 weeks, and in this time period the collagen sponge (COS) was not identified. A higher rate of new bone formation was observed for the BOS þ SIM group. In the 8th postoperative week, the newly formed bone was more mature, containing osteocytes more regularly arranged than those observed in the 4th postoperative week. A new bone formation was observed in the centre of the bone defect area. In some cases, areas of mild inflammation were still present. At 8 weeks, the inorganic bovine bone graft (BOS) and the hydroxyapatite with calcium sulfate graft (HACS) were still histologically found. More new bone formation was noted for the HACS þ SIM group (Figs. 3e6). The ANOVA results showed statistically significant interaction among the three factors examined (time, carrier, and presence or absence of simvastatin) (F (5.36) ¼ 25.10, p < 0.01). Differences in averages greater than 6.67 () are statistically significant with a significance level of p < 0.05 (least significant difference 6.67, p < 0.05). Differences in averages greater than 9.67 and 14.57 are statistically significant with a significance level of p < 0.01 and p < 0.001, respectively (9.67, p < 0.01 and 14.57, p < 0.001, respectively). The administration of simvastatin resulted in a statistically significant increase in bone surface at 4 and 8 weeks. At 4 weeks, the bone surface for the BOS4 group was measured at a mean (standard deviation [SD]) of 5.54 ± 0.37 and for the BOS þ SIM4 group was measured at 43.22 ± 8.82. Between the 2 groups there are statistically significant differences with a significance level of p < 0.001. At 8 weeks, the values for the BOS8 and BOS þ SIM8 groups were 4.88 ± 1.33 and 28 ± 2.72, respectively. These differences remained statistically significant (p < 0.001). At 4 weeks, the bone surface for HACS4 and HACS þ SIM4 groups was measured at a mean (SD) of 7.58 ± 0.44 and 16.00 ± 0.49, respectively, showing a statistically significant difference with a significance level of p < 0.05. At 8 weeks, the corresponding values for the HACS8 and HACS þ SIM8 groups were measured at 16.68 ± 0.91 and 44.31 ± 9.89, respectively. This difference is statistically significant (p < 0.001). The results for the COS4 and COS þ SIM4 groups were 8.22 ± 1.59 and 17.97 ± 3.77, respectively (a statistically significant difference, p < 0.01). At 8 weeks, no statistically significant difference was found between the COS8 and COS þ SIM8 groups (4.05 ± 0.63 and 6.66 ± 1.32, respectively).

3. Results The BOS (inorganic bone graft), HACS (hydroxyapatite with calcium sulfate), and COS (collagen sponge) groups were used as active controls, and the CONT group (control) was used as passive control. All animals recovered well after surgery. No macroscopic infection of the wounds was noted, and at 2 weeks the wounds showed complete soft tissue healing with no appearance of swelling and/or scabbing. 3.1. Descriptive histology In the 4th postoperative week, the histological findings showed new bone formation at the edges of the defects of all groups. The newly formed bone presented osteocytes arranged in a disorganized manner and osteoblasts. Small bone islands were seen in the centre of the bone defects, which were filled with connective tissue and mild or moderate chronic inflammatory infiltrate. The

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Fig. 3. BOS þ SIM group. New bone formation (4).


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Fig. 6. Inflammatory reaction at 4 weeks (2.5). Fig. 4. HACS þ SIM group (2.5).

Between the different carriers used, there are also statistically significant differences (p < 0.001) for the BOS þ SIM (43.22 ± 8.82) and HACS þ SIM (16.00 ± 0.49) groups at 4 and 8 weeks (BOS þ SIM and HACS þ SIM 28.20 ± 2.72 and 44.31 ± 9.89, respectively). Statistically significant differences with a significance level of p < 0.001 resulted for the BOS þ SIM (43.22 ± 8.82) and COS þ SIM (17.97 ± 3.77, respectively) groups at 4 and 8 weeks (BOS þ SIM and COS þ SIM 28.20 ± 2.72 and 6.66 ± 1.32, respectively). At 4 weeks, there is no statistically significant difference between the HACS þ SIM and COS þ SIM groups (16.00 ± 0.49 and 17.97 ± 3.77, respectively), whereas at 8 weeks, the differences are statistically significant (p < 0.001) (HACS þ SIM and COS þ SIM 44.31 ± 9.89 and 6.66 ± 1.32, respectively). All results are illustrated in Tables 1 and 2. 4. Discussion The effect of local administration of statins in bone tissue in combination with various transport systems has been reported by many experimental researchers. Simvastatin increases osteoblast and inhibits osteoclast activity. The mechanisms by which this activity is achieved concerns the induction of the osteoblast differentiation by the BMPs (through competition of TNF-a-to-Ras/Rho/ mitogen-activated protein kinase), the increase of BMP-Smad activation (BMP-Smad signaling) (Yamashita et al., 2008), the increased alkaline phosphatase activity, the increased expression of bone sialoprotein, osteocalcin, type I collagen, and the anti-

Fig. 5. COS þ SIM group (2).

inflammatory action by reducing the production of interleukin 6 (IL-6) and 8 (IL-8) (Sakoda et al., 2006). Many authors have reported that statins have a local anabolic effect on bones (Wu et al., 2008; Nyan et al., 2007, 2009). The ideal doses and appropriate carriers are still under research. Low doses of statins have no impact on bone regeneration, whereas high doses may cause a local inflammatory response. Statins in elevated local concentrations can be cytotoxic due to a drastic reduction in the production of cholesterol, which is important for the integrity of cell membranes (Benoit et al., 2006). The results of this study showed that a 2-mg concentration of simvastatin may cause a local inflammatory reaction in the bone defect. This reaction is histologically more intense during the first weeks, whereas, according to the histological findings, the inflammation is milder at 8 weeks. Inflammatory response was also reported by other researchers who used 2.2 mg of statin in methylcellulose gel in rat calvaria (Thylin et al., 2002), 1 mg of statin in calcium sulfate in rat calvaria (Nyan et al., 2007), 0.5 mg, 1.0 mg, 1.5 mg, and 2.2 mg of statin in rat mandible (Stein et al., 2005), 2 mg of simvastatin in calcium sulfate in rat alveolar bone after incisor extraction (Sato et al., 2005). The effectiveness of simvastatin depends on the rhythm in which the carrier releases the drug, as well as on its local concentration. Between the simvastatin carriers that were evaluated, the HACS þ SIM group showed the best results in 8 weeks, whereas the BOS þ SIM group showed the best results in 4 weeks, compared to the other groups. This is explained by the fact that apart from the local inflammatory response caused by simvastatin, hydroxyapatite with calcium sulfate may also cause an inflammatory response, lasting until about the fifth week, as reported in relevant studies (Stein et al., 2005; Nyan et al., 2007). The bone production rate is significantly increased for the HACS þ SIM group from the fourth to the eighth week. However, even during the first weeks with the inflammatory response being present, new bone formation is observed. Calcium sulfate and phosfate materials have been shown to be osteoconductive and biocompatible (LeGeros, 2002) and these materials have been successfully used as carriers for growth factors such as BMPs (Yan et al., 2004; Ginebra et al., 2006; Saito et al., 2006). Studies on the release of substances from calcium sulfate and phosfate materials reported a biphasic manner of release, an initial rapid phase followed by a gradual long-lasting one (Ziegler et al., 2002). Such a release of simvastatin may be advantageous for providing an initial stimulation of the local cells to express BMP-2. The second phase of simvastatin release from the gradually degrading calcium sulfate/phosfate may provide continued exposure of local osteoblasts to simvastatin, resulting in the osteoblastic differentiation.



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Table 1 Mean and standard deviation of new bone formation in mm2 between the study groups at both times related to the level of significance p. (LSD0.05 ¼ 6.67, p < 0.05, LSD0,01 ¼ 9.67, p < 0.01 and LSD0.001 ¼ 14.57, p < 0.001, *means no statistically significant differences). 4 weeks

8 weeks

BOS þ SIM 43.22 ± 8.82 BOS þ SIM 43.22 ± 8.82 HACS þ SIM 16.00 ± 0.49

HACS þ SIM 16.00 ± 0.49 COS þ SIM 17.97 ± 3.77 COS þ SIM 17.97 ± 3.77

BOS þ SIM 43.22 ± 8.82 HACS þ SIM 16.00 ± 0.49 COS þ SIM 17.97 ± 3.77

BOS 5.54 ± 0.37 HACS 7.58 ± 0.44 COS 8.22 ± 1.59

p < 0.001 p < 0.001 * p < 0.001 p < 0.05 p < 0.01

A significant reduction of bone formation is observed in the BOS þ SIM group from the fourth to the eighth week, which is attributed to the gradual absorption of the material. As reported in similar studies, the inorganic bone graft cannot be completely integrated (Lima et al., 2011). With regard to the inflammatory response, this is less intense than in the HACS þ SIM group at both times (4 and 8 weeks). However, other studies have mentioned a more intense inflammatory response during the first 30 days, which might be related to factors such as incomplete decalcification of the graft (Lima et al., 2011). Although inorganic bovine bone graft presents osteoconductive and osteoinductive properties, osteoinductivity varies depending on the commercial preparation. Collagen is widely used as a carrier material for protein and drug delivery or as a scaffold in bone engineering, since it is particularly biocompatible, is biodegradable, and enhances cellular penetration in extracellular matrix formation (Ruszczak and Friess, 2003). Collagen is a vehicle for the local delivery of statins when grafted in bony defects. In this study, the COS þ SIM group presented higher rates of new bone formation compared with the COS group at weeks 4 and 8. However, a gradual decrease in new bone formation from weeks 4e8 is observed that might be explained by the high and rapid absorption of this material, which is not histologically detected at weeks 4 and 8. The results of the COS þ SIM group were limited compared to the other carrier groups.

Table 2 Means and standard deviation of new bone formation on postoperative weeks 4 and 8 in mm2 for each group (LSD0.05 ¼ 6.67, p < 0.05). 50 45 40 35 30 -SIM

25

+SIM

20 15 10 5 0 BOS4

HACS4

COS4

CONT4

BOS8

HACS8

COS8

CONT8

BOS þ SIM 28.20 ± 2.72 BOS þ SIM 28.20 ± 2.72 HACS þ SIM 44.31 ± 9.89

HACS þ SIM 44.31 ± 9.89 COS þ SIM 6.66 ± 1.32 COS þ SIM 6.66 ± 1.32

BOS þ SIM 28.20 ± 2.72 HACS þ SIM 44.31 ± 9.89 COS þ SIM 6.66 ± 1.32

BOS 4.88 ± 1.33 HACS 16.68 ± 0.91 COS 4.05 ± 0.63

p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 *

5. Conclusion The results of this study revealed that the local application of simvastatin in the bone defect areas leads to an increase in bone formation. In addition, the 2-mg concentration of simvastatin increases the total amount of new bone without causing a strong immune response, which may endanger bone formation. The inflammatory response is histologically present at 4 weeks but not at 8 weeks. The inorganic bovine bone graft (BOS) and the hydroxyapatite with calcium sulfate (HACS) are histologically detected at weeks 4 and 8, in contrast with the collagen sponge (COS). The best results for the BOS are shown after 4 weeks, whereas more new bone formation for the HACS is observed after 8 weeks. References Ayukawa Y, Yasukawa E, Moriyama Y, Ogino Y, Wada H, Atsuta I, et al: Local application of statin promotes bone repair through the suppression of osteoclasts and the enhancement of osteoblasts at bone healing sites in rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 107(3): 336e342, 2008 Baek KH, Lee WY, Oh KW, Tae HJ, Lee JM, Lee EJ, et al: The effect of simvastatin on the proliferation and differentiation of human bone marrow stromal cells. J Korean Med Sci 20(3): 438e444, 2005 Bellosta S, Ferri N, Bernini F, Paoletti R, Corsini A: Non-lipid-related effects of statins. Ann Med 32: 164e176, 2000 Benoit DS, Nuttelman CR, Collins SD, Anseth KS: Synthesis and characterization of a fluvastatin-releasing hydrogel delivery system to modulate hMSC differentiation and function for bone regeneration. Biomaterials 27(36): 6102e6110, 2006 Boyne PJ, Shabahang S: An evaluation of bone induction delivery materials in conjunction with root-form implant placement. Int J Periodontics Restorative Dent 21: 333e343, 2001 Boyne PJ, Marx RE, Nevins M, Triplett G, Lazaro E, Lilly LC, et al: A feasibility study evaluating rhBMP-2/absorbable collagen sponge for maxillary sinus floor augmentation. Int J Periodontics Restorative Dent 17: 10e25, 1997 Brown WV: Safety of statins. Curr Opin Lipidol 19(6): 558e562, 2008 Davignon J, Laaksonen R: Low-density lipoprotein-independent effects of statins. Curr Opin Lipidol 10: 543e559, 1999 Garrett IR, Gutierrez G, Mundy GR: Statins and bone formation. Curr Pharm Des 7: 715e736, 2001 Ginebra MP, Traykova T, Planell JA: Calcium phosphate cements as bone drug delivery systems: a review. J Control Release 113: 102e110, 2006 Grasser WA, Baumann AP, Petras SF, Harwood Jr HJ, Devalaraja R, Renkiewicz R, et al: Regulation of osteoclast differentiation by statins. J Musculoskelet Neuronal Interact 3(1): 53e62, 2003 Howell TH, Fiorellini J, Jones A, Alder M, Nummikoski P, Lazaro M, et al: A feasibility study evaluating rhBMP-2/absorbable collagen sponge device for local alveolar ridge preservation or augmentation. Int J Periodontics Restorative Dent 17: 124e139, 1997 Hughes A, Rogers MJ, Idris Al, Crockett JC: A comparison between the effects of hydrophobic and hydrophilic statins on osteoclast function in vitro and ovariectomy-induced bone loss in-vivo. Calcif Tissue Int 81(5): 403e413, 2007 Hunt DR, Jovanovich SA, Wikesjo UME, Wozney JM, Bernard GW: Hyaluronan supports recombinant human bone morphogenetic protein-2 induced bone reconstruction of advanced alveolar ridge defects in dogs: a pilot study. J Periodontol 72: 651e658, 2000


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Jang RE, Glauser R, Scharer P, Hammerle CHF, Weber FE: The effect of rh-BMP-2 on guided bone regeneration in humans. A randomized, controlled clinical and histomorphometric study. Clin Oral Implants Res 14: 556e568, 2003 Koempel JA, Patt BS, O'Grady K, Wozney J, Toriumi DM: The effect of recombinant human bone morphogenetic protein-2 on the integration of porous hydroxyapatite implants with bone. J Biomed Mater Res 41: 359e363, 1998 LeGeros RZ: Properties of osteoconductive biomaterials: calcium phosphates. Clin Orthop Relat Res 395: 81e98, 2002 Lima CE, Calixto JC, Andinder AL: Influence of the association between simvastatin and demineralized bovine bone matrix on bone repair in rats. Braz Oral Res 25(1): 42e48, 2011 Ma B, Clarke SA, Brooks RA, Rushton N: The effect of simvastatin on bone formation and ceramic resorption in a peri-implant defect model. Acta Biomater 4(1): 149e155, 2008 Maeda T, Matsunuma A, Kawane T, Horiuchi N: Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Biochem Biophys Res Commun 280: 874e877, 2001 Maeda T, Kawane T, Horiuchi N: Statins augment vascular endothelial growth factor expression in osteoblastic cells via inhibition of protein prenylation. Endocrinology 144: 681e692, 2003 Maeda T, Matsunuma A, Kurahashi I, Yanagawa T, Yoshida H, Horiuchi N: Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells. J Cell Biochem 92: 458e471, 2004 Maritz FJ, Conradie MM, Hulley PA, Gopal R, Hough S: Effect of statins on bone mineral density and bone histomorphometry in rodents. Arterioscler Thromb Vasc Biol 21: 1636e1643, 2001 Marou DJ, Fazio S, Linton MF: Current perspectives on statins. Circulation 101: 207e213, 2000 Moriyama Y, Ayukawa Y, Ogino Y, Atsuta I, Koyano K: Topical application of statin affects bone healing around implants. Clin Oral Implants Res 19(6): 600e605, 2008 Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, et al: Stimulation of bone formation in vitro and in rodents by statins. Science 286: 1946e1949, 1999 Nyan M, Sato D, Oda M, Machida T, Kobayashi H, Nakamura T, et al: Bone formation with the combination of simvastatin and calcium sulphate in critical sized rat calvarial defect. J Pharmacol Sci 104: 384e386, 2007 Nyan M, Sato D, Kihara H, Machida T, Ohya K, Kasugai S: Effects of the combination with alpha-tricalcium phosphate and simvastatin on bone regeneration. Clin Oral Implants Res 20(3): 280e287, 2009 Ruszczak Z, Friess W: Collagen as a carrier for on-site delivery of antibacterial drugs. Adv Drug Deliv Rev 55(12): 1679e1698, 2003 Saito A, Suzuki Y, Kitamura M, Ogata S, Yoshihara Y, Masuda S, et al: Repair of 20 mm long rabbit radial bone defects using BMP-derived peptide combined with an a-tricalcium phosphate scaffold. J Biomed Mater Res 77A: 700e706, 2006 Sakoda K, Yamamoto M, Negishi Y, Liao JK, Node K, Izumi Y: Simvastatin decreases IL-6 and IL-8 production in epithelial cells. J Dent Res 85: 520e523, 2006

Sato D, Nishimura K, Ishioka T, Hondo H, Kuroda S, Kasugai S: Local application of simvastatin to rat incisor socket: carrier dependent effect on bone augmentation. J Oral Tiss Engin 2(2): 81e85, 2005 Sigurdsson EF, Nygaard L, Tatakis DN, Fu E, Turek TJ, Jin L, et al: Periodontal repair in dogs; evaluation of rh-BMP-2 carriers. Int J Periodontics Restorative Dent 16: 524e537, 1996 Song C, Guo Z, Ma Q, Chen Z, Liu Z, Jia H, et al: Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells. Biochem Biophys Res Commun 308(3): 458e462, 2003 Staal A, Frith JC, French MH, Swartz J, Gungor T, Harrity TW, et al: The ability of statins to inhibit bone resorption is directly related to their inhibitory effect on HMG-CoA-reductase activity. J Bone Miner Res 18(1): 88e96, 2003 Stein D, Lee Y, Schmid MJ, Killpack B, Genrich MA, Narayana N, et al: Local simvastatin effects on mandibular bone growth and inflammation. J Periodontol 76(11): 1861e1870, 2005 Sugiyama M, Kodama T, Konishi K, Abe K, Asami S, Oikawa S: Compactin and simvastatin, but not pravastatin, induce bone morphogenetic protein-2 in human osteosarcoma cells. Biochem Biophys Res Commun 271: 688e692, 2000 Thylin MR, McConnell JC, Schmid MJ, Reckling RR, Ojha J, Bhattacharyya I, et al: Effects of simvastatin gels on murine calvarial bone. J Periodontol 76: 1861e1870, 2002 Toriumi DM, Kotler HS, Luxenberg DP, Holtrop ME, Wang EA: Mandibular reconstruction with a recombinant bone-inducing factor. Functional, histologic, and biomechanical evaluation. Arch Otolaryngol Head Neck Surg 117: 1101e1112, 1991 Wikesjo UM, Sorensen RG, Kinosita A, Wozney JM: Rh-BMP-2/alphaBSM induces significant vertical alveolar ridge augmentation and dental implant osseointegration. Clin Implant Dent Relat Res 4: 174e182, 2002 Wong RWK, Rabie ABM: Statin collagen grafts used to repair bone defects in the parietal bone of rabbits. Br J Oral Maxillofac Surg 41: 244e248, 2003 Wong RW, Rabie AB: Histologic and ultrastructural study on statin graft in rabbit skulls. J Oral Maxillofac Surg 63(10): 1515e1521, 2005 Wu Z, Liu C, Zang G, Sun H: The effect of simvastatin on remodelling of the alveolar bone following tooth extraction. Int J Oral Maxillofac Surg 37: 170e176, 2008 Yamashita M, Otsuka F, Mukai T, Otani H, Inagaki K, Miyoshi T, et al: Simvastatin antagonizes tumor necrosis factor-alpha inhibition of bone morphogenetic proteins-2 induced osteoblast differentiation by regulating Smad signaling and Ras/Rho-mitogen-activated protein kinase pathway. J Endocrinol 196: 601e603, 2008 Yan WZ, Zhou DL, Yin SY, Yin GF, Gao LD, Zhang Y: Osteogenesis capacity of a novel BMP/aTCP bioactive composite bone cement. J Wuhan Uni Tech-Mater Sci 19: 30e34, 2004 Ziegler J, Mayr-Wohlfart U, Kessler S, Breitig D, Gunther KP: Adsorption and release properties of growth factors from biodegradable implants. J Biomed Mater Res 59: 422e428, 2002


Effects of bovine lactoferrin in surgically created bone defects on bone regeneration around implants.

The influence of cortical perforation on guided bone regeneration using synthetic bone substitutes: a study of rabbit cranial defects.

Effects of Simvastatin and Combination of Simvastatin and Nylestriol on Bone Metabolism in Ovariectomized Rats.

The Effects of Desferroxamine on Bone and Bone Graft Healing in Critical-Size Bone Defects.

Alveolar bone regeneration in response to local application of calcitriol in vitamin D deficient rats.

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

Biodegradable guide for bone regeneration. Polyurethane membranes tested in rabbit radius defects.

Effects of two types of anorganic bovine bone on bone regeneration: a histological and histomorphometric study of rabbit calvaria.

Method to investigate bioelectric regeneration of bone in periodontal defects.

Increased bone formation in a rabbit long-bone defect model after single local and single systemic application of erythropoietin.

The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using xenogenic bone substitutes: a study of rabbit cranial defects.

Osteostatin-coated porous titanium can improve early bone regeneration of cortical bone defects in rats.

The effect of simvastatin on chemotactic capability of SDF-1α and the promotion of bone regeneration.

Dexamethasone-loaded hydroxyapatite enhances bone regeneration in rat calvarial defects.

Histologic evaluation of combining tetracycline and allogeneic freeze-dried bone on bone regeneration in experimental defects in baboons.

Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration.

Application of AMOR in craniofacial rabbit bone bioengineering.

The efficacy of BMP-2 preloaded on bone substitute or hydrogel for bone regeneration at peri-implant defects in dogs.

Combination of calcium sulfate and simvastatin-controlled release microspheres enhances bone repair in critical-sized rat calvarial bone defects.

acellular bone matrix composite for bone regeneration in cranial defects.

The role of topical simvastatin on bone regeneration: A systematic review.

Role of mechanical compression on bone regeneration around a particulate bone graft material: an experimental study in rabbit calvaria.

Effect of platelet-rich plasma combined with demineralised bone matrix on bone healing in rabbit ulnar defects.

The effect of L-PRF membranes on bone healing in rabbit tibiae bone defects: micro-CT and biomarker results.