钛表面含辛伐他汀药物涂层的制备及其对骨质疏松症大鼠种植体骨结合作用的研究
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摘要
缩短种植体骨结合的时间,达到种植体早期负载甚至即刻负载是种植体表面改性研究的主要目标。除了种植体的表面形貌和化学性质的影响,患者种植部位的骨量和骨质也是影响种植体成功的重要因素。我国现已进入老龄化社会,骨质疏松症患者增多,对牙种植体的需求不断上升,然而另一方面,系统性骨质疏松影响颌骨的骨量和骨密度,成为了牙种植手术的一个相对禁忌症。所以,让钛种植体和骨质疏松症患者周围骨组织之间快速牢固的结合是牙种植手术现今急需克服的一个难题。
近年来研究发现他汀类药物具有促进骨形成、抑制骨吸收的双重作用,并且能诱导骨形成蛋白-2(BMP-2),——一类已知的具有最强的骨诱导能力的生长因子的表达。本研究采用物理吸附法和仿生沉积法,在纯钛种植体表面设计、构建辛伐他汀涂层,通过体外细胞实验筛选合适浓度,然后建立骨质疏松症大鼠种植体动物模型,研究含药涂层对骨质疏松症大鼠种植体骨结合的影响。研究分两部分：
第一部分吸附辛伐他汀纯钛表面对骨质疏松症大鼠种植体骨结合作用的研究
实验方法和结果
将纯钛钛片表面打磨抛光,再喷砂酸蚀处理,得到我们所需要的基底面。然后将辛伐他汀溶解在75%乙醇溶液中,制备成终浓度为0(对照组),10-7M,10-6M,10-5M和10-4 M的辛伐他汀乙醇溶液,将钛片浸泡在上述溶液中48小时,通过物理吸附作用在钛片表面吸附上辛伐他汀。通过场发射扫描电镜FSEM对钛片表面进行表征。然后在上述各组钛片上培养小鼠前成骨细胞系MC3T3-E1,观察涂层对细胞成骨分化能力的影响。FSEM显示该涂层基本保留了原来的粗糙表面形貌。细胞实验的结果表明低浓度的辛伐他汀处理组(10-7，10-6M,尤其是10-7M)能显著促进MC3T3-E1的分化。
基于体外细胞实验的结果,我们设计螺纹型种植钉,分为对照组,实验组1(10-7M)和实验组2(10-6M)三个组,植入骨质疏松症大鼠。在术后1周、2周、4周和12周分别处死一批动物,制作硬组织切片。采用Olympus光镜显微镜和Image-Pro PlusR半自动图像处理软件对种植体-骨接触率和种植体螺纹内骨密度进行测量,并进行统计学分析。
结果显示,在各个观察点,2个实验组表面形成新骨的时间早于对照组,形成新骨的数量也高于对照组,并有统计学差异,两个实验组之间没有统计学差异。
第二部分纯钛表面磷酸钙盐仿生沉积法制备的辛伐他汀涂层对骨质疏松症大鼠种植体骨结合作用的研究
实验方法和结果
纯钛钛片基底面处理同实验第一部分。然后通过仿生沉积法在纯钛表面构建仿生涂层,这其中,我们在仿生液B中加入不同浓度的辛伐他汀(终浓度分别为0,10-7M,10-6M和10-5M),这样得到了含有不同浓度含药涂层。采用FSEM、XRD和FTIR观察分析涂层的表面形貌、晶体结构和化学组成,在上述各组钛片上培养小鼠前成骨细胞系MC3T3-E1,观察涂层对细胞增殖和成骨分化能力的影响.FSEM显示,低浓度组(10-7M组和10-6M组)的晶体的排列结构与对照组并没有显著区别；高浓度组(10-5M,10-4M和10-3M)表面的晶体形态和尺寸均有较大的改变：片状的晶体随着浓度升高而变得越薄,边缘也不规则；花状结构减少,绒毛样球状的结构出现；浓度越大,这种变化就越明显,10-3M组表面片状结构完全消失,表面呈现大小不一的球状结构。XRD和FTIR的结果显示所有涂层表面都含有磷酸八钙OCP,而且涂层内含有辛伐他汀,并且随着仿生液中辛伐他汀浓度的增高,涂层内辛伐他汀浓度也随之升高。
细胞实验的结果表明低浓度的辛伐他汀处理组10-6M能增强MC3T3-E1细胞ALP活性,OC分泌,同时对细胞增殖也没明显影响。
基于体外细胞实验的结果,我们设计螺纹型种植钉,分为对照组,实验组(10-6M)两个组,植入骨质疏松症大鼠。在术后4周和12周分别处死一批动物,制作硬组织切片。采用Olympus光镜显微镜和Image-Pro PlusR半自动图像处理软件对种植体骨接触率和种植体螺纹内骨密度进行测量,并进行统计学分析。
结果显示,在各个观察点,实验组表面形成新骨的时间和数量均早于或高于对照组,并有统计学差异。
结论
在纯钛种植体表面构建含辛伐他汀药物涂层能促进骨质疏松症大鼠的种植体表面新骨的形成,从而增强种植体-骨的结合强度,为临床开发适合骨质疏松症患者的新型种植体提供了科学实验依据。
BACKGROUND
Shortening the period of implant-bone osseointegration to achieve early loading even instant loading, is the main goal of research of surface modification of dental implants. Besides the effects of surface morphology and chemistry of implant surface, the recipient bone quantity and quality is also crucial factors. Since China is an aging society now, the population of osteoporotic people is increasing, as well as the need for dental implants. However, systematic osteoporosis affects the quantity and quality of jaw bones, which is considered as a contraindication of dental implant surgery. Thus, the urgent task of modern implantology is to achieve fast and solid osseointegration of titanium implants and surrounding osteoporotic bone tissue.
Recently, it was reported that a kind of liposoluble statin, simvastatin, could induce the expression of bone morphogenetic protein (BMP)-2 mRNA and that, as a result, it promoted bone formation on the calvaria of mice. And some researchers demonstrated the topical application of statins improved bone formation around implants. In this context, we designed simvastatin-loaded porous titanium implant surface by immersion method and biomimetic method. The aim of this study was to investigate the feasibility of local delivery of simvastatin using these two methods and the effects of the delivery on implant osseointegration by in vitro and in vivo experiments. The study is divided into two parts:
Part I Preparation of simvastatin-loaded porous titanium surfaces by immersion method on Ti-based dental implant surface and its effects on implant osseointegration in osteoporotic rats
METHODS AND RESULTS
We got our substrate Ti surfaces by polishing, sandblasting and etching. The control group consisted of cells cultured on titanium disks without any intervention for different time intervals (4 d,7 d and 14 d), whereas the experimental groups (simvastatin-loaded groups) consisted of cells cultured on titanium disks that were pre-incubated in varying concentration (10-7M,10-6M, 11-5M and 10-4M) of simvastatin for the same time intervals of the control. Alkaline phosphatase activity (ALP), Type I collagen synthesis and osteocalcin release were used to measure the cellular osteoblastic activities.
All simvastatin-loaded groups showed increased ALP activity compared to control group at every time point, especially the 10-7 M group significantly increased the activity by almost 4-fold at 4 days (P＜0.05). In the Type I collagen synthesis assay, all simvastatin-loaded groups showed an increase and the effect was inverse dose dependent (maximal at 10-1M). Furthermore, this stimulatory effect of simvastatin was also observed in osteocalcin release assay (P＜0.05, at 10-7M,10-6M, maximal at 10-7 M).
Based on the in vitro results, roughened implants were divided into control groups (n=32), test 1 group (n=32), and test 2 group (n=32). Test implants were immersed into 10"7 M (Test 1 implants) or 10-6M (Test 2 implants) simvastatin solutions for drug adsorption onto implant surfaces.48 ovariectomized rats randomly received two implants in both tibiae. After 1,2,4, and 12 weeks of implantation, tibias were retrieved and prepared for histomorphometric evaluation. Bone-implant contact (BIC) and bone area around implant, as well as histological findings, were obtained. Results:New bone formation on test implant surfaces was seen after 1 week while it was seen on the control implant surface after 2 weeks. There were more bone tissue and bone-implant contact along the test implant surfaces than which along the control implant surface. The test 1 and 2 implants showed a significantly greater bone area and BIC compared to the control implant during the observation periods (p<0.05). No differences were found between the test 1 and test 2 implants after 1,2,4, and 12 weeks (p>0.05).
Part II Preparation of simvastatin-incorporated coating by biomimetic method on Ti-based dental implant surface and its effects on implant osseointegration in osteoporotic rats
METHODS AND RESULTS
We got our substrate Ti surfaces by polishing, sandblasting and etching as previously described. Simvastatin was prepared onto titanium porous surfaces by biomimetic calcium phosphate coating by adding a series of concentrations simvastation (the final concentration is 10-7M,10-6M and 10-5M) in SBF-2. FSEM, XRD and FTIR were utilized to observe the surface morphology and the crystal structure of the coatings. MC3T3-E1 cells were cultured on these disks with varying concentration of simvatatin. The total protein content, ALP activity and osteocalcin production were measured to examine the effects of coatings to the proliferation and differentiation ability.
FSEM results showed that sharp crystal flakes were deposited on the control plate surface. The crystalline coating entirely covered the substrate surface. All the crystal plates were oriented more or less perpendicularly to the surface of the plates. The flake morphology and orientation did not obviously change at the low concentration of simvastatin (10-7M and 10-6M). However, the morphology and size of crystal flakes changed at the high concentration. At the 10-5M and10-4M concentration, flake-like crystals combined into sphere-like structures. The size of flakes dramaticly decreased. At the 10-3M concentration, the sphere-like structures were seen on the sample surfaces while the flake crystals disappeared. XRD and FTIR results indicated the presence of OCP in all the coatings. Simvastatin has been incorporated into the synthetic OCP coatings. What's more, the content of simvastatin in the coating also increased with the increasing concentration of simvastatin in SBF-2 accordingly.
Based on the in vitro results,10-6 M (Test implants) was chosen for the treatment of implant screws in osteoporotic animal experiments. After 4 and 12 weeks of implantation, tibias were retrieved and prepared for histomorphometric evaluation. Bone-implant contact (BIC) and bone area around implant, as well as histological findings, were obtained. New bone formation on test implant surfaces was seen earlier than control group. Also there were more bone tissue and bone-implant contact along the test implant surfaces than which along the control implant surface at 4 and 12 weeks. (p<0.05).
Conclusion:These results indicate that simvastatin-loaded porous implant surface has the potential to improve implant osseointegration in the ovariectomized model, which provide scientific proof for develop novel dental implants for osteoprotic patients clinically.
近年来研究发现他汀类药物具有促进骨形成、抑制骨吸收的双重作用,并且能诱导骨形成蛋白-2(BMP-2),——一类已知的具有最强的骨诱导能力的生长因子的表达。本研究采用物理吸附法和仿生沉积法,在纯钛种植体表面设计、构建辛伐他汀涂层,通过体外细胞实验筛选合适浓度,然后建立骨质疏松症大鼠种植体动物模型,研究含药涂层对骨质疏松症大鼠种植体骨结合的影响。研究分两部分：
第一部分吸附辛伐他汀纯钛表面对骨质疏松症大鼠种植体骨结合作用的研究
实验方法和结果
将纯钛钛片表面打磨抛光,再喷砂酸蚀处理,得到我们所需要的基底面。然后将辛伐他汀溶解在75%乙醇溶液中,制备成终浓度为0(对照组),10-7M,10-6M,10-5M和10-4 M的辛伐他汀乙醇溶液,将钛片浸泡在上述溶液中48小时,通过物理吸附作用在钛片表面吸附上辛伐他汀。通过场发射扫描电镜FSEM对钛片表面进行表征。然后在上述各组钛片上培养小鼠前成骨细胞系MC3T3-E1,观察涂层对细胞成骨分化能力的影响。FSEM显示该涂层基本保留了原来的粗糙表面形貌。细胞实验的结果表明低浓度的辛伐他汀处理组(10-7，10-6M,尤其是10-7M)能显著促进MC3T3-E1的分化。
基于体外细胞实验的结果,我们设计螺纹型种植钉,分为对照组,实验组1(10-7M)和实验组2(10-6M)三个组,植入骨质疏松症大鼠。在术后1周、2周、4周和12周分别处死一批动物,制作硬组织切片。采用Olympus光镜显微镜和Image-Pro PlusR半自动图像处理软件对种植体-骨接触率和种植体螺纹内骨密度进行测量,并进行统计学分析。
结果显示,在各个观察点,2个实验组表面形成新骨的时间早于对照组,形成新骨的数量也高于对照组,并有统计学差异,两个实验组之间没有统计学差异。
第二部分纯钛表面磷酸钙盐仿生沉积法制备的辛伐他汀涂层对骨质疏松症大鼠种植体骨结合作用的研究
实验方法和结果
纯钛钛片基底面处理同实验第一部分。然后通过仿生沉积法在纯钛表面构建仿生涂层,这其中,我们在仿生液B中加入不同浓度的辛伐他汀(终浓度分别为0,10-7M,10-6M和10-5M),这样得到了含有不同浓度含药涂层。采用FSEM、XRD和FTIR观察分析涂层的表面形貌、晶体结构和化学组成,在上述各组钛片上培养小鼠前成骨细胞系MC3T3-E1,观察涂层对细胞增殖和成骨分化能力的影响.FSEM显示,低浓度组(10-7M组和10-6M组)的晶体的排列结构与对照组并没有显著区别；高浓度组(10-5M,10-4M和10-3M)表面的晶体形态和尺寸均有较大的改变：片状的晶体随着浓度升高而变得越薄,边缘也不规则；花状结构减少,绒毛样球状的结构出现；浓度越大,这种变化就越明显,10-3M组表面片状结构完全消失,表面呈现大小不一的球状结构。XRD和FTIR的结果显示所有涂层表面都含有磷酸八钙OCP,而且涂层内含有辛伐他汀,并且随着仿生液中辛伐他汀浓度的增高,涂层内辛伐他汀浓度也随之升高。
细胞实验的结果表明低浓度的辛伐他汀处理组10-6M能增强MC3T3-E1细胞ALP活性,OC分泌,同时对细胞增殖也没明显影响。
基于体外细胞实验的结果,我们设计螺纹型种植钉,分为对照组,实验组(10-6M)两个组,植入骨质疏松症大鼠。在术后4周和12周分别处死一批动物,制作硬组织切片。采用Olympus光镜显微镜和Image-Pro PlusR半自动图像处理软件对种植体骨接触率和种植体螺纹内骨密度进行测量,并进行统计学分析。
结果显示,在各个观察点,实验组表面形成新骨的时间和数量均早于或高于对照组,并有统计学差异。
结论
在纯钛种植体表面构建含辛伐他汀药物涂层能促进骨质疏松症大鼠的种植体表面新骨的形成,从而增强种植体-骨的结合强度,为临床开发适合骨质疏松症患者的新型种植体提供了科学实验依据。
BACKGROUND
Shortening the period of implant-bone osseointegration to achieve early loading even instant loading, is the main goal of research of surface modification of dental implants. Besides the effects of surface morphology and chemistry of implant surface, the recipient bone quantity and quality is also crucial factors. Since China is an aging society now, the population of osteoporotic people is increasing, as well as the need for dental implants. However, systematic osteoporosis affects the quantity and quality of jaw bones, which is considered as a contraindication of dental implant surgery. Thus, the urgent task of modern implantology is to achieve fast and solid osseointegration of titanium implants and surrounding osteoporotic bone tissue.
Recently, it was reported that a kind of liposoluble statin, simvastatin, could induce the expression of bone morphogenetic protein (BMP)-2 mRNA and that, as a result, it promoted bone formation on the calvaria of mice. And some researchers demonstrated the topical application of statins improved bone formation around implants. In this context, we designed simvastatin-loaded porous titanium implant surface by immersion method and biomimetic method. The aim of this study was to investigate the feasibility of local delivery of simvastatin using these two methods and the effects of the delivery on implant osseointegration by in vitro and in vivo experiments. The study is divided into two parts:
Part I Preparation of simvastatin-loaded porous titanium surfaces by immersion method on Ti-based dental implant surface and its effects on implant osseointegration in osteoporotic rats
METHODS AND RESULTS
We got our substrate Ti surfaces by polishing, sandblasting and etching. The control group consisted of cells cultured on titanium disks without any intervention for different time intervals (4 d,7 d and 14 d), whereas the experimental groups (simvastatin-loaded groups) consisted of cells cultured on titanium disks that were pre-incubated in varying concentration (10-7M,10-6M, 11-5M and 10-4M) of simvastatin for the same time intervals of the control. Alkaline phosphatase activity (ALP), Type I collagen synthesis and osteocalcin release were used to measure the cellular osteoblastic activities.
All simvastatin-loaded groups showed increased ALP activity compared to control group at every time point, especially the 10-7 M group significantly increased the activity by almost 4-fold at 4 days (P＜0.05). In the Type I collagen synthesis assay, all simvastatin-loaded groups showed an increase and the effect was inverse dose dependent (maximal at 10-1M). Furthermore, this stimulatory effect of simvastatin was also observed in osteocalcin release assay (P＜0.05, at 10-7M,10-6M, maximal at 10-7 M).
Based on the in vitro results, roughened implants were divided into control groups (n=32), test 1 group (n=32), and test 2 group (n=32). Test implants were immersed into 10"7 M (Test 1 implants) or 10-6M (Test 2 implants) simvastatin solutions for drug adsorption onto implant surfaces.48 ovariectomized rats randomly received two implants in both tibiae. After 1,2,4, and 12 weeks of implantation, tibias were retrieved and prepared for histomorphometric evaluation. Bone-implant contact (BIC) and bone area around implant, as well as histological findings, were obtained. Results:New bone formation on test implant surfaces was seen after 1 week while it was seen on the control implant surface after 2 weeks. There were more bone tissue and bone-implant contact along the test implant surfaces than which along the control implant surface. The test 1 and 2 implants showed a significantly greater bone area and BIC compared to the control implant during the observation periods (p<0.05). No differences were found between the test 1 and test 2 implants after 1,2,4, and 12 weeks (p>0.05).
Part II Preparation of simvastatin-incorporated coating by biomimetic method on Ti-based dental implant surface and its effects on implant osseointegration in osteoporotic rats
METHODS AND RESULTS
We got our substrate Ti surfaces by polishing, sandblasting and etching as previously described. Simvastatin was prepared onto titanium porous surfaces by biomimetic calcium phosphate coating by adding a series of concentrations simvastation (the final concentration is 10-7M,10-6M and 10-5M) in SBF-2. FSEM, XRD and FTIR were utilized to observe the surface morphology and the crystal structure of the coatings. MC3T3-E1 cells were cultured on these disks with varying concentration of simvatatin. The total protein content, ALP activity and osteocalcin production were measured to examine the effects of coatings to the proliferation and differentiation ability.
FSEM results showed that sharp crystal flakes were deposited on the control plate surface. The crystalline coating entirely covered the substrate surface. All the crystal plates were oriented more or less perpendicularly to the surface of the plates. The flake morphology and orientation did not obviously change at the low concentration of simvastatin (10-7M and 10-6M). However, the morphology and size of crystal flakes changed at the high concentration. At the 10-5M and10-4M concentration, flake-like crystals combined into sphere-like structures. The size of flakes dramaticly decreased. At the 10-3M concentration, the sphere-like structures were seen on the sample surfaces while the flake crystals disappeared. XRD and FTIR results indicated the presence of OCP in all the coatings. Simvastatin has been incorporated into the synthetic OCP coatings. What's more, the content of simvastatin in the coating also increased with the increasing concentration of simvastatin in SBF-2 accordingly.
Based on the in vitro results,10-6 M (Test implants) was chosen for the treatment of implant screws in osteoporotic animal experiments. After 4 and 12 weeks of implantation, tibias were retrieved and prepared for histomorphometric evaluation. Bone-implant contact (BIC) and bone area around implant, as well as histological findings, were obtained. New bone formation on test implant surfaces was seen earlier than control group. Also there were more bone tissue and bone-implant contact along the test implant surfaces than which along the control implant surface at 4 and 12 weeks. (p<0.05).
Conclusion:These results indicate that simvastatin-loaded porous implant surface has the potential to improve implant osseointegration in the ovariectomized model, which provide scientific proof for develop novel dental implants for osteoprotic patients clinically.
引文
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16. Maeda T, Matsunuma A, Kawane T, Horiuchi N:Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Biochem Biophys Res Commun 2001;280:874
17. Maeda T, Matsunuma A, Kurahashi I et al.:Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells. J Cell Biochem 2004;92:458
18. Song C, Guo Z, Ma Q et al.:Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells. Biochem Biophys Res Commun 2003;308:458
19. Baek KH, Lee WY, Oh KW et al.:The effect of simvastatin on the proliferation and differentiation of human bone marrow stromal cells. J Korean Med Sci 2005;20:438
20. Thunyakitpisal PD, Chaisuparat R:Simvastatin, an HMG-CoA reductase inhibitor, reduced the expression of matrix metalloproteinase-9 (Gelatinase B) in osteoblastic cells and HT1080 fibrosarcoma cells. J Pharmacol Sci 2004;94:403
21. Hwang R, Lee EJ, Kim MH et al.:Calcyclin, a Ca2+ ion-binding protein, contributes to the anabolic effects of simvastatin on bone. J Biol Chem 2004;279:21239
22. Wang X, Tokuda H, Hatakeyama D et al.:Mechanism of simvastatin on induction of heat shock protein in osteoblasts. Arch Biochem Biophys 2003;415:6
23. Ruiz-Gaspa S, Nogues X, Enjuanes A et al.:Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG-63 cultures. J Cell Biochem 2007;101:1430
24. Edwards CJ, Hart DJ, Spector TD:Oral statins and increased bone-mineral density in postmenopausal women. Lancet 2000;355:2218
25. Ohno T, Shigetomi M, Ihara K et al.:Effects of cerivastatin on vascularized allogenic bone transplantation in rats treated with cyclosporine A. Calcif Tissue Int 2003;72:50
26. Oxlund H, Dalstra M, Andreassen TT:Statin given perorally to adult rats increases cancellous bone mass and compressive strength. Calcif Tissue Int 2001;69:299
27. Staal A, Frith JC, French MH 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 2003;18:88
28. Wang JW, Xu SW, Yang DS, Lv RK:Locally applied simvastatin promotes fracture healing in ovariectomized rat. Osteoporos Int 2007;18:1641
29. Wada Y, Nakamura Y, Koshiyama H:Lack of positive correlation between statin use and bone mineral density in Japanese subjects with type 2 diabetes. Arch Intern Med 2000;160:2865
30. Chung YS, Lee MD, Lee SK et al.:HMG-CoA reductase inhibitors increase BMD in type 2 diabetes mellitus patients. J Clin Endocrinol Metab 2000;85:1137
31. Bellosta S, Ferri N, Bernini F et al.:Non-lipid-related effects of statins. Ann Med 2000;32:164
32. Shibli JA, Aguiar KC, Melo L et al.:Histological comparison between implants retrieved from patients with and without osteoporosis. Int J Oral Maxillofac Surg 2008;37:321
33. Qi MC, Zhou XQ, Hu J et al.:Oestrogen replacement therapy promotes bone healing around dental implants in osteoporotic rats. Int J Oral Maxillofac Surg 2004;33:279
34. Jeffcoat MK, Chesnut CH,3rd:Systemic osteoporosis and oral bone loss:evidence shows increased risk factors. J Am Dent Assoc 1993;124:49
35. Duarte PM, de Vasconcelos Gurgel BC, Sallum AW et al.:Alendronate therapy may be effective in the prevention of bone loss around titanium implants inserted in estrogen-deficient rats. JPeriodontol 2005;76:107
36. Duarte PM, Goncalves PF, Casati MZ et al.:Age-related and surgically induced estrogen deficiencies may differently affect bone around titanium implants in rats. J Periodontol 2005;76:1496
37. Ayukawa Y, Okamura A, Koyano K:Simvastatin promotes osteogenesis around titanium implants. Clin Oral Implants Res 2004;15:346
38. Du Z, Chen J, Yan F, Xiao Y:Effects of Simvastatin on bone healing around titanium implants in osteoporotic rats. Clin Oral Implants Res 2009;20:145
39. Basarir K, Erdemli B, Can A et al.:Osseointegration in arthroplasty:can simvastatin promote bone response to implants? Int Orthop 2009;33:855
40. Moriyama Y, Ayukawa Y, Ogino Y et al.:Topical application of statin affects bone healing around implants. Clin Oral Implants Res 2008;19:600
41. Monjo M, Rubert M, Wohlfahrt JC et al.:In vivo performance of absorbable collagen sponges with rosuvastatin in critical-size cortical bone defects. Acta Biomater 2010;6:1405
42. Masuzaki T, Ayukawa Y, Moriyama Y et al.:The effect of a single remote injection of statin-impregnated poly (lactic-co-glycolic acid) microspheres on osteogenesis around titanium implants in rat tibia. Biomaterials 2010;31:3327
43. Yang GL, He FM, Yang XF et al.:Bone responses to titanium implants surface-roughened by sandblasted and double etched treatments in a rabbit model. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:516
44. He FM, Yang GL, Zhao SF, Cheng ZP:Mechanical and histomorphometric evaluations of rough titanium implants treated with hydrofluoric acid/nitric acid solution in rabbit tibia. Int J Oral Maxillofac Implants 2011;26:115
45. Zhang F, Yang GL, He FM et al.:Cell response of titanium implant with a roughened surface containing titanium hydride:an in vitro study. J Oral Maxillofac surg 2010;68:1131
46. Li X, Cui Q, Kao C et al.:Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPARgamma2 and increasing Cbfal/Runx2 expression in bone marrow mesenchymal cell cultures. Bone 2003;33:652
47. Horiuchi N, Maeda T:Statins and bone metabolism. Oral Dis 2006;12:85
48. Woo JT, Kasai S, Stern PH, Nagai K:Compactin suppresses bone resorption by inhibiting the fusion of prefusion osteoclasts and disrupting the actin ring in osteoclasts. J Bone Miner Res 2000;15:650
49. Maeda T, Kawane T, Horiuchi N:Statins augment vascular endothelial growth factor expression in osteoblastic cells via inhibition of protein prenylation. Endocrinology 2003;144:681
50. Takenaka M, Hirade K, Tanabe K et al.:Simvastatin stimulates VEGF release via p44/p42 MAP kinase in vascular smooth muscle cells. Biochem Biophys Res Commun 2003;301:198
51. Sakou T:Bone morphogenetic proteins:from basic studies to clinical approaches. Bone 1998;22:591
52. Seto H. Ohba H, Tokunaga K et al.:Topical administration of simvastatin recovers alveolar bone loss in rats. JPeriodontal Res 2008;43:261
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15. Maeda T, Kawane T, Horiuchi N:Statins augment vascular endothelial growth factor expression in osteoblastic cells via inhibition of protein prenylation. Endocrinology 2003;144:681
16. Takenaka M, Hirade K, Tanabe K et al.:Simvastatin stimulates VEGF release via p44/p42 MAP kinase in vascular smooth muscle cells. Biochem Biophys Res Commun 2003;301:198
17. Li X, Cui Q, Kao C et al.:Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPARgamma2 and increasing Cbfal/Runx2 expression in bone marrow mesenchymal cell cultures. Bone 2003;33:652
18. Song C, Guo Z, Ma Q et al.:Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells. Biochem Biophys Res Commun 2003;308:458
19. Woo JT, Kasai S, Stern PH, Nagai K:Compactin suppresses bone resorption by inhibiting the fusion of prefusion osteoclasts and disrupting the actin ring in osteoclasts. J Bone Miner Res 2000;15:650
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26. Ayukawa Y, Okamura A, Koyano K:Simvastatin promotes osteogenesis around titanium implants. Clin Oral Implants Res 2004;15:346
27. Du Z, Chen J, Yan F, Xiao Y:Effects of Simvastatin on bone healing around titanium implants in osteoporotic rats. Clin Oral Implants Res 2009;20:145
28. Basarir K, Erdemli B, Can A et al.:Osseointegration in arthroplasty:can simvastatin promote bone response to implants? Int Orthop 2009;33:855
29. Moriyama Y, Ayukawa Y, Ogino Y et al.:Topical application of statin affects bone healing around implants. Clin Oral Implants Res 2008;19:600
30. Monjo M, Rubert M, Wohlfahrt JC et al.:In vivo performance of absorbable collagen sponges with rosuvastatin in critical-size cortical bone defects. Acta Biomater 2010;6:1405
31. Masuzaki T, Ayukawa Y, Moriyama Y et al.:The effect of a single remote injection of statin-impregnated poly (lactic-co-glycolic acid) microspheres on osteogenesis around titanium implants in rat tibia. Biomaterials 2010;31:3327
32. Ayukawa Y, Ogino Y, Moriyama Y et al.:Simvastatin enhances bone formation around titanium implants in rat tibiae. J Oral Rehabil 2010;37:123
33. Shibli JA, Aguiar KC, Melo L et al.:Histological comparison between implants retrieved from patients with and without osteoporosis. Int J Oral Maxillofac Surg 2008;37:321
34. Qi MC, Zhou XQ, Hu J et al.:Oestrogen replacement therapy promotes bone healing around dental implants in osteoporotic rats. Int J Oral Maxillofac Surg 2004;33:279
35. Jeffcoat MK, Chesnut CH,3rd:Systemic osteoporosis and oral bone loss:evidence shows increased risk factors. J Am Dent Assoc 1993;124:49
36. Duarte PM, de Vasconcelos Gurgel BC, Sallum AW et al.:Alendronate therapy may be effective in the prevention of bone loss around titanium implants inserted in estrogen-deficient rats. J Periodontol 2005;76:107
37. Duarte PM, Goncalves PF, Casati MZ et al.:Age-related and surgically induced estrogen deficiencies may differently affect bone around titanium implants in rats. J Periodontol 2005;76:1496
38. Yamazaki M, Shirota T, Tokugawa Y et al.:Bone reactions to titanium screw implants in ovariectomized animals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:411
39. Giro G, Goncalves D, Sakakura CE et al.:Influence of estrogen deficiency and its treatment with alendronate and estrogen on bone density around osseointegrated implants:radiographic study in female rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:162
2. Lips P:Epidemiology and predictors of fractures associated with osteoporosis. Am J Med 1997;103:3S
3. Yamazaki M, Shirota T, Tokugawa Y et al.:Bone reactions to titanium screw implants in ovariectomized animals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:411
4. Giro G, Goncalves D, Sakakura CE et al.:Influence of estrogen deficiency and its treatment with alendronate and estrogen on bone density around osseointegrated implants:radiographic study in female rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:162
5. Paganini-Hill A:The benefits of estrogen replacement therapy on oral health. The Leisure World cohort. Arch Intern Med 1995;155:2325
6. Riggs BL, Hartmann LC:Selective estrogen-receptor modulators--mechanisms of action and application to clinical practice. N Engl J Med 2003;348:618
7. Christiansen C, Lindsay R:Estrogens, bone loss and preservation. Osteoporos Int 1990;1:7
8. Margolis RN, Canalis E, Partridge NC:Invited review of a workshop:anabolic hormones in bone:basic research and therapeutic potential. J Clin Endocrinol Metab 1996;81:872
9. Mundy G, Garrett R, Harris S et al.:Stimulation of bone formation in vitro and in rodents by statins. Science 1999;286:1946
10. Goldstein JL, Brown MS:Regulation of the mevalonate pathway. Nature 1990;343:425
11. Istvan ES, Deisenhofer J:Structural mechanism for statin inhibition of HMG-CoA reductase. Science 2001;292:1160
12. Casey PJ, Seabra MC:Protein prenyltransferases. J Biol Chem 1996;271:5289
13. Eto M, Kozai T, Cosentino F et al.:Statin prevents tissue factor expression in human endothelial cells:role of Rho/Rho-kinase and Akt pathways. Circulation 2002;105:1756
14. Laufs U, La Fata V, Plutzky J, Liao JK:Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 1998;97:1129
15. Laufs U, Fata VL, Liao JK:Inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase blocks hypoxia-mediated down-regulation of endothelial nitric oxide synthase. J Biol Chem 1997;272:31725
16. Maeda T, Matsunuma A, Kawane T, Horiuchi N:Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Biochem Biophys Res Commun 2001;280:874
17. Maeda T, Matsunuma A, Kurahashi I et al.:Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells. J Cell Biochem 2004;92:458
18. Song C, Guo Z, Ma Q et al.:Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells. Biochem Biophys Res Commun 2003;308:458
19. Baek KH, Lee WY, Oh KW et al.:The effect of simvastatin on the proliferation and differentiation of human bone marrow stromal cells. J Korean Med Sci 2005;20:438
20. Thunyakitpisal PD, Chaisuparat R:Simvastatin, an HMG-CoA reductase inhibitor, reduced the expression of matrix metalloproteinase-9 (Gelatinase B) in osteoblastic cells and HT1080 fibrosarcoma cells. J Pharmacol Sci 2004;94:403
21. Hwang R, Lee EJ, Kim MH et al.:Calcyclin, a Ca2+ ion-binding protein, contributes to the anabolic effects of simvastatin on bone. J Biol Chem 2004;279:21239
22. Wang X, Tokuda H, Hatakeyama D et al.:Mechanism of simvastatin on induction of heat shock protein in osteoblasts. Arch Biochem Biophys 2003;415:6
23. Ruiz-Gaspa S, Nogues X, Enjuanes A et al.:Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG-63 cultures. J Cell Biochem 2007;101:1430
24. Edwards CJ, Hart DJ, Spector TD:Oral statins and increased bone-mineral density in postmenopausal women. Lancet 2000;355:2218
25. Ohno T, Shigetomi M, Ihara K et al.:Effects of cerivastatin on vascularized allogenic bone transplantation in rats treated with cyclosporine A. Calcif Tissue Int 2003;72:50
26. Oxlund H, Dalstra M, Andreassen TT:Statin given perorally to adult rats increases cancellous bone mass and compressive strength. Calcif Tissue Int 2001;69:299
27. Staal A, Frith JC, French MH 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 2003;18:88
28. Wang JW, Xu SW, Yang DS, Lv RK:Locally applied simvastatin promotes fracture healing in ovariectomized rat. Osteoporos Int 2007;18:1641
29. Wada Y, Nakamura Y, Koshiyama H:Lack of positive correlation between statin use and bone mineral density in Japanese subjects with type 2 diabetes. Arch Intern Med 2000;160:2865
30. Chung YS, Lee MD, Lee SK et al.:HMG-CoA reductase inhibitors increase BMD in type 2 diabetes mellitus patients. J Clin Endocrinol Metab 2000;85:1137
31. Bellosta S, Ferri N, Bernini F et al.:Non-lipid-related effects of statins. Ann Med 2000;32:164
32. Shibli JA, Aguiar KC, Melo L et al.:Histological comparison between implants retrieved from patients with and without osteoporosis. Int J Oral Maxillofac Surg 2008;37:321
33. Qi MC, Zhou XQ, Hu J et al.:Oestrogen replacement therapy promotes bone healing around dental implants in osteoporotic rats. Int J Oral Maxillofac Surg 2004;33:279
34. Jeffcoat MK, Chesnut CH,3rd:Systemic osteoporosis and oral bone loss:evidence shows increased risk factors. J Am Dent Assoc 1993;124:49
35. Duarte PM, de Vasconcelos Gurgel BC, Sallum AW et al.:Alendronate therapy may be effective in the prevention of bone loss around titanium implants inserted in estrogen-deficient rats. JPeriodontol 2005;76:107
36. Duarte PM, Goncalves PF, Casati MZ et al.:Age-related and surgically induced estrogen deficiencies may differently affect bone around titanium implants in rats. J Periodontol 2005;76:1496
37. Ayukawa Y, Okamura A, Koyano K:Simvastatin promotes osteogenesis around titanium implants. Clin Oral Implants Res 2004;15:346
38. Du Z, Chen J, Yan F, Xiao Y:Effects of Simvastatin on bone healing around titanium implants in osteoporotic rats. Clin Oral Implants Res 2009;20:145
39. Basarir K, Erdemli B, Can A et al.:Osseointegration in arthroplasty:can simvastatin promote bone response to implants? Int Orthop 2009;33:855
40. Moriyama Y, Ayukawa Y, Ogino Y et al.:Topical application of statin affects bone healing around implants. Clin Oral Implants Res 2008;19:600
41. Monjo M, Rubert M, Wohlfahrt JC et al.:In vivo performance of absorbable collagen sponges with rosuvastatin in critical-size cortical bone defects. Acta Biomater 2010;6:1405
42. Masuzaki T, Ayukawa Y, Moriyama Y et al.:The effect of a single remote injection of statin-impregnated poly (lactic-co-glycolic acid) microspheres on osteogenesis around titanium implants in rat tibia. Biomaterials 2010;31:3327
43. Yang GL, He FM, Yang XF et al.:Bone responses to titanium implants surface-roughened by sandblasted and double etched treatments in a rabbit model. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:516
44. He FM, Yang GL, Zhao SF, Cheng ZP:Mechanical and histomorphometric evaluations of rough titanium implants treated with hydrofluoric acid/nitric acid solution in rabbit tibia. Int J Oral Maxillofac Implants 2011;26:115
45. Zhang F, Yang GL, He FM et al.:Cell response of titanium implant with a roughened surface containing titanium hydride:an in vitro study. J Oral Maxillofac surg 2010;68:1131
46. Li X, Cui Q, Kao C et al.:Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPARgamma2 and increasing Cbfal/Runx2 expression in bone marrow mesenchymal cell cultures. Bone 2003;33:652
47. Horiuchi N, Maeda T:Statins and bone metabolism. Oral Dis 2006;12:85
48. Woo JT, Kasai S, Stern PH, Nagai K:Compactin suppresses bone resorption by inhibiting the fusion of prefusion osteoclasts and disrupting the actin ring in osteoclasts. J Bone Miner Res 2000;15:650
49. Maeda T, Kawane T, Horiuchi N:Statins augment vascular endothelial growth factor expression in osteoblastic cells via inhibition of protein prenylation. Endocrinology 2003;144:681
50. Takenaka M, Hirade K, Tanabe K et al.:Simvastatin stimulates VEGF release via p44/p42 MAP kinase in vascular smooth muscle cells. Biochem Biophys Res Commun 2003;301:198
51. Sakou T:Bone morphogenetic proteins:from basic studies to clinical approaches. Bone 1998;22:591
52. Seto H. Ohba H, Tokunaga K et al.:Topical administration of simvastatin recovers alveolar bone loss in rats. JPeriodontal Res 2008;43:261
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