载辛伐他汀PLGA/CPC复合材料的制备及体外性能测定
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摘要
剩余牙槽嵴的骨吸收给临床的进一步治疗带来诸多问题,利用骨组织工程技术来抑制剩余牙槽嵴的骨吸收是目前的研究热点,本课题组先前合成的载辛伐他汀/PLGA复合材料能够促使新骨形成,使拔牙窝内骨量增加,但该材料的酸性降解产物易引起无菌性炎症发生,使其应用受到限制。为了对该载药支架材料的性能进行改善,在国家自然科学基金资助下(项目编号:30872912),我们将不同分子量的PLGA同时与CPC混合,通过筛选来获得一个理想的配比方案,然后将辛伐他汀载入复合支架材料。
本实验采用冷冻干燥溶剂挥发技术制备支架材料,并对该复合材料的体外性能进行了测定,结果显示该复合材料能够满足骨组织工程对支架材料的要求,并且较单一的PLGA支架材料的性能有明显的改善。本实验的创新点在于率先从仿生学角度制备具有一定缓释能力的载辛伐他汀PLGA/CPC复合材料,对于剩余牙槽嵴骨吸收防治的研究具有重要意义。
The teeth and the alveolar bone have an interdependent relationship each other, the alveolar ridge will have a continuous, progressive, and irreversible bone resorption after the tooth extraction, leading to a low level or blade form of the alveolar ridge, causing some difficulties for denture restoration and implant planting. The residual ridge resorption (RRR) leads to many aesthetic and functional problems in edentulous patients, insufficient masticatory function impacted the blood supplying of such critical organs as the heart and brain. These problems will be more obvious following with an aging population. It has clinical significance to develop a new method and material that can promote the healing of tooth socket at early stage and prevent alveolar bone resorption.
In order to prevent the atrophy of the residual ridge, the research was mainly concentrated on such methods as allografts, autografts, immediate dental implants and the guided bone regeneration technique, but secondary lesions, immunologic rejection and poor long-term results of these treatments limited their clinical applications. There was few reports about the treatment of alveolar ridge by using medicine. Recent studies have shown that statins had the function of stimulating in-vivo bone formation and increasing expression of BMP-2 in bone cells. Simvastatin was one of the widely used statins, the research showed that it could increase the bone density of menostasis woman and aged people obviously and decrease the incidence rate of bone fracture. So we assumed that whether we could promote bone formation in the early stage after tooth extraction by simv- astatin. Because simvastatin underwent extensive first-pass extraction in the liver after oral administration, the availability of the drug to the general circulation limited in bone tissue; its local concentration is too low to induce bone .It needs a great quantity of simvastain to get the effect of bone restoration for bone fracture and defective by local injection, in this way, it can cause cytotoxicity, on the other hand, it required injecting repeatedly to have the ideal pharmacodynamic action, for the low molecular weight of statins diffuse quickly and having a short half life period, this method can not be accepted by patients easily. So, it is very important to optimize the administration route of simvastain, which increase the effect of bone formation and decrease the side effects.
According to some studies, it is a practicable method to use simvastain carried by carrier to promote the restoration of the bone defective. Our team had prepared the simvastain carried by poly (DL-lactic-co-glycolic acid, PLGA) scaffold by dissolvent volatilization technique, explored the effect of simvastatin on the bone restoration in the mandible incisor extraction socket. The results showed that the drug released slowly from the scaffold could induce the expression of BMP-2 in periodontal ligameng cell (PDLC) and mesenchymical cell in the tooth extraction socket, it could also differentiate themselves to osteo- blast to induce new bone formation. The bone mass increased obviously in the tooth socket comparing with that in control group. But there were some limitations to use the homogenous material, the PLGA has the disadvantage in the poor hydrophilism, weak cell adsorbability and the acidity degradation which leaded to some aseptic inflammation. Considering the human tissue consisting of inorganic and organic compound ingredients, therefore from the angle of biomimetic, inorganic and organic compound biological material as the simvas- tatin drug carrier will be better. The calcium phosphate cements (CPC) has a satisfactory biocompatibility and higher mechanical strength, it can be molded randomly according to the shape of the bone defective and binded with the human bone tightly in its application. CPC can be solidified and molded freely in the environment and temperature of human body, its final degradation material is hydroxyapatite (HA) that is similar to the inorganic principle of human bone tissue. The CPC used as the inorganic biological activity material get more attention day by day, it has been used as the bone replacement material in clinical renovation. The base material after the degradation of CPC can neutralize the acid that was generated by the degradation of PLGA, the CPC also can enhance the mechanical strength of PLGA scaffold. There are no reports on the composite of PLGA and CPC used as the carrier of simvastain to repair bone defective in domestic and abroad up to now.
We divided the experiment into three groups by adjusting the quality ratio of the HMW and LMW PLGA and CPC. The method to prepare the composite is called the freeze drying and dissolvent volatilization technique. We choose the best matching propotion as the carrier for simvastain according to the result of scanning electron microscope (SEM) and the change of pH in the imitation humor, at last, the optical density (A) of simvastatin was obtained by UV spectroscopy at 238 nm and the drug release curve was drew. The results showed that this scaffold had suitable strength and integrality pore, the average aperture size was 120μm, these pores communicated to each other by micro pores, so this satisfied the requirement of scaffold about pore diameter. Adding CPC into the composite could inhibit the descent of pH that was caused by the degradation of PLGA, the pH would stabilize at 6.5 at 8 weeks, these improved the performance obviously compared with the simple PLGA scaffold. The composite had a dramatic delayed releasing effectiveness, it would last more than 48 days for the drug to release. So we drew the conclusion that the composite consisting of simvastatin carried by PLGA/CPC satisfied the requirement of bone tissue engineering (BTE), this study could bring some meaning for the bone repair of the residual ridge.
The new creativity of this study was to prepare the simvastatin carried by PLGA/CPC scaffold with slow releasing ability, and applied two different molecular weight PLGA to prepare the composite for the first time.
本实验采用冷冻干燥溶剂挥发技术制备支架材料,并对该复合材料的体外性能进行了测定,结果显示该复合材料能够满足骨组织工程对支架材料的要求,并且较单一的PLGA支架材料的性能有明显的改善。本实验的创新点在于率先从仿生学角度制备具有一定缓释能力的载辛伐他汀PLGA/CPC复合材料,对于剩余牙槽嵴骨吸收防治的研究具有重要意义。
The teeth and the alveolar bone have an interdependent relationship each other, the alveolar ridge will have a continuous, progressive, and irreversible bone resorption after the tooth extraction, leading to a low level or blade form of the alveolar ridge, causing some difficulties for denture restoration and implant planting. The residual ridge resorption (RRR) leads to many aesthetic and functional problems in edentulous patients, insufficient masticatory function impacted the blood supplying of such critical organs as the heart and brain. These problems will be more obvious following with an aging population. It has clinical significance to develop a new method and material that can promote the healing of tooth socket at early stage and prevent alveolar bone resorption.
In order to prevent the atrophy of the residual ridge, the research was mainly concentrated on such methods as allografts, autografts, immediate dental implants and the guided bone regeneration technique, but secondary lesions, immunologic rejection and poor long-term results of these treatments limited their clinical applications. There was few reports about the treatment of alveolar ridge by using medicine. Recent studies have shown that statins had the function of stimulating in-vivo bone formation and increasing expression of BMP-2 in bone cells. Simvastatin was one of the widely used statins, the research showed that it could increase the bone density of menostasis woman and aged people obviously and decrease the incidence rate of bone fracture. So we assumed that whether we could promote bone formation in the early stage after tooth extraction by simv- astatin. Because simvastatin underwent extensive first-pass extraction in the liver after oral administration, the availability of the drug to the general circulation limited in bone tissue; its local concentration is too low to induce bone .It needs a great quantity of simvastain to get the effect of bone restoration for bone fracture and defective by local injection, in this way, it can cause cytotoxicity, on the other hand, it required injecting repeatedly to have the ideal pharmacodynamic action, for the low molecular weight of statins diffuse quickly and having a short half life period, this method can not be accepted by patients easily. So, it is very important to optimize the administration route of simvastain, which increase the effect of bone formation and decrease the side effects.
According to some studies, it is a practicable method to use simvastain carried by carrier to promote the restoration of the bone defective. Our team had prepared the simvastain carried by poly (DL-lactic-co-glycolic acid, PLGA) scaffold by dissolvent volatilization technique, explored the effect of simvastatin on the bone restoration in the mandible incisor extraction socket. The results showed that the drug released slowly from the scaffold could induce the expression of BMP-2 in periodontal ligameng cell (PDLC) and mesenchymical cell in the tooth extraction socket, it could also differentiate themselves to osteo- blast to induce new bone formation. The bone mass increased obviously in the tooth socket comparing with that in control group. But there were some limitations to use the homogenous material, the PLGA has the disadvantage in the poor hydrophilism, weak cell adsorbability and the acidity degradation which leaded to some aseptic inflammation. Considering the human tissue consisting of inorganic and organic compound ingredients, therefore from the angle of biomimetic, inorganic and organic compound biological material as the simvas- tatin drug carrier will be better. The calcium phosphate cements (CPC) has a satisfactory biocompatibility and higher mechanical strength, it can be molded randomly according to the shape of the bone defective and binded with the human bone tightly in its application. CPC can be solidified and molded freely in the environment and temperature of human body, its final degradation material is hydroxyapatite (HA) that is similar to the inorganic principle of human bone tissue. The CPC used as the inorganic biological activity material get more attention day by day, it has been used as the bone replacement material in clinical renovation. The base material after the degradation of CPC can neutralize the acid that was generated by the degradation of PLGA, the CPC also can enhance the mechanical strength of PLGA scaffold. There are no reports on the composite of PLGA and CPC used as the carrier of simvastain to repair bone defective in domestic and abroad up to now.
We divided the experiment into three groups by adjusting the quality ratio of the HMW and LMW PLGA and CPC. The method to prepare the composite is called the freeze drying and dissolvent volatilization technique. We choose the best matching propotion as the carrier for simvastain according to the result of scanning electron microscope (SEM) and the change of pH in the imitation humor, at last, the optical density (A) of simvastatin was obtained by UV spectroscopy at 238 nm and the drug release curve was drew. The results showed that this scaffold had suitable strength and integrality pore, the average aperture size was 120μm, these pores communicated to each other by micro pores, so this satisfied the requirement of scaffold about pore diameter. Adding CPC into the composite could inhibit the descent of pH that was caused by the degradation of PLGA, the pH would stabilize at 6.5 at 8 weeks, these improved the performance obviously compared with the simple PLGA scaffold. The composite had a dramatic delayed releasing effectiveness, it would last more than 48 days for the drug to release. So we drew the conclusion that the composite consisting of simvastatin carried by PLGA/CPC satisfied the requirement of bone tissue engineering (BTE), this study could bring some meaning for the bone repair of the residual ridge.
The new creativity of this study was to prepare the simvastatin carried by PLGA/CPC scaffold with slow releasing ability, and applied two different molecular weight PLGA to prepare the composite for the first time.
引文
[1]Langer R, Vacanti JP. Tissue engineering. Science.1993; 260(5110): 920- 926.
[2]Stevens B, Yang YZ, Mohandas A, et al. A review of materials fabrication methods, and strategies used to enhance bone regeneration in engineered bone tissues. J Biomed Mater Res B Appl Biomater. 2008;85(2):573-582.
[3]Garrett IR, Gutierrez G, Mundy GR. Statins and bone formation. Curr Pharm Design 2001;7:715–736.
[4]Thylin MR, McConnell JC, Schmid MJ, et al. Effects of simvastatin gels on murine calvarial bone. J Periodontol 2002;73:1141–1148.
[5]Agrawal CM,Athanasiou KA. Technique to control pH in vicinity of biodegrading,PLA-PGA implants. J Biomed Mater Res.1997;38:105一114.
[6]Ambard AJ, Mueninghoff L. Calcium phosphate cement: review of mechanical and biological properties. J Prosthodont. 2006;15(5):321-328.
[7]Link DP, van den Dolder J, Jurgens WJ, et al. Mechanical evaluation of implanted calcium phosphate cement incorporated with PLGA microparticles. Biomaterials. 2006;27(28):4941-4947.
[8]Fei Z, Hu Y, Wu D,et al. Preparation and property of a novel bone graft composite consisting of rhBMP-2 loaded PLGA microspheres and calcium phosphate cement J Mater Sci: Mater Med. 2008;19:1109–1116
[9]Habraken WJ, Wolke JG, Mikos AG, et al. PLGA microsphere/calcium phosphate cement composites for tissue engineering: in vitro release and degradation characteristics.J. Biomater. Sci. Polymer Edn,2008;19(9):1171-1188.
[10]Becker W, Becker BE. Guide tissue regeneration of implants placed into extraction sockets and for implant dehisences: Surgical techniques and case reports. Int J Peridontol, 1990,10:376-391.
[11]Buser D, Dula D, Hirt HP, et al. Lateral ridge augmentation using autografts and barrier membranes. J Oral Maxillofacial Surg,1996,54:420-433.
[12]Scheller EL, Krebsbach PH, Kohn DH.Tissue engineering: state of the art in oral rehabilitation. Joural of Oral Rehabitation, 2009,36;368-389.
[13]Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005;26:5474–5491
[14]Freed LE. Vunjak-Novakovic G. Langer R. Cultivation of cell-polymer cartilage implants in bioreactors. J Cell Biochem. 1993,51:257-264.
[15]Jain RA. The manufacturing techniques of various drug loaded biodegr- adable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials.2000;21 2475-2490
[16]Tice TR, Cowsar DR. Biodegradable controlled-release parenteral systems. Pharm Technol,1984;11:26-35.
[17]刘竞龙,余斌,高成杰.骨组织工程修复骨缺损:大鼠成骨细胞与聚乳酸和聚乙醇共聚合物支架联合培养观察.中国临床康复.2002.6:2371-2372.
[18]Ignatius AA, Betz O, Augat P, Claes LE. In vivo investigations on composites made of resorbable ceramics and poly(lactide) used as bone graft substitutes. J Biomed Mater Res. 2001;58(6):701-709.
[19]Habraken WJ, Wolke JG, Mikos AG, Jansen JA. Injectable PLGAmicrosphere/calcium phosphate cements: physical properties and degradation characteristics. J Biomater Sci Polym Ed. 2006;17(9):1057-1074.
[20]RuhéPQ, Boerman OC, Russel FGM,et al. Controlled release of rhBMP-2 loaded poly(DL-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo. Journal of Controlled Release.2005;106:162– 171.
[21]Cohen S, AlonsoMJ, Langer R. Novel approaches to controlled release antigen delivery. Int J Technol Assessment Health Care.1994;10(1):121-130.
[22]倪建光,刘丹平.骨组织工程支架材料的研究进展.中国组织工程研究与临床康复.2009;(13 )8:1525-1528.
[23]Brown WE , Chow LC. A new calcium phosphate water setting cement. [J ]. Cement Research Progress.1986;352-379.
[24]Larsson S. Bauer TW. Use of injectable calcium phosphate cement for fracture fixation: a review. Clin Orthop. 2002;(395):23-32.
[25]Blom EJ. Klein-Nulend J. Wolke JG. Et al. Transforming growth factor-betal incorporation in a calcium phosphate bone cement: material prop- erties and release characteristics. J Biomed Mater Res .2002:59(2):265-272.
[26]Bohner M,Lemaitre J,Mekle HP. et al. Control of gentamicin release from a calcium phosphate cement by admixed poly(acrylic acid). J PHann Sci 2000;89(10)1262-1270.
[27]Pandya NM,Dhalla NS, Santani DD. Angiogenesis a new target for future therapy. Vascul Pharmacol. 2006,44(5):265-274.
[28]RuhéPQ, Kroese-Deutman HC, Wolke JG. et al. Bone inductive prop-erties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials. 2004;25(11):2123-2132.
[29]Li RH, Bouxsein ML, Blake CA. er al. rhBMP-2 injected in a calcium phosphate paste (alpha-BSM) accelerates healing in the rabbit ulnar osteotomy model. J Orthop Res. 2003;21(6):997-1004.
[30]Friedman CD, Costantino PD, Jones K. et al. Hydroxyapatite cement. II. Obliteration and reconstruction of the cat frontal sinus. Arch Otolaryngol Head Neck Surg. 1991;117(4):385-389.
[31]Shindo ML, Costantino PD, Friedman CD, Chow LC. Facial skeletal augmentation using hydroxyapatite cement. Arch Otolaryngol Head Neck Surg. 1993;119(2):185-190.
[32]Ruhe PQ, Hedberg EL, Padron NT. et al. Biocompatibility and degr- adation of poly(DL-lactic-co-glycolic acid)/calcium phosphate cement comp- osites. Wiley Periodicals, Inc. 2005;533-544.
[33]Kitchell JP, Wise DL. Poly(lactic/glycolic acid) biodegradable drug polymer matrix systems. Methods Enzymol. 1985; 112: 436-48.
[34]Hing KA,Best SM,Tanner KE,et al. Quantification of bone ingrowth within bone-derived Porous hydroxyapatite implants of varying density. J Mater Sci Mater Med,1999;10(10/11):663-670.
[35]Dahl SG,Allain P,Marie PJ,et al. Incorporation and distribution of str- ontium in bone. Bone 2001,28(4):446-453.
[36]Lane JM,Bostrom MP. Bone grafting and new composite biosyntheticgraft materials. Instr Course Lect,1998,47:525-534.
[37]Lu JX,Flautre B,Anselme K,et al. Role of interconnections in Porous bioceramics on bone recolonization in vitro and in vivo. J Mater Sci Mater Med,1999,10(2),111-120.
[38]Nam YS,Park TG. Biodegradable polymeric microcellular foams by modified thermally induced phase separation method. Biomaterials,1999,20(19):1783-1790.
[39]Hempel U, Reinstorf A, Poppe M, et al. Proliferation and differentiation of osteoblasts on Biocement D modified with collagen type I and citric acid. J Biomed Mater Res B Appl Biomater. 2004;71(1):130-143.
[40]Wong RWK, Rabie ABM. Statin collagen grafts used to repair defects in the parietal bone of rabbits. Brit J Oral Max Surg 2003;41:244–248.
[41]Nyan M, Daisuke Sato, Hidemichi Kihara,et al. Effects of the combi- nation withα-tricalcium phosphate and simvastatin on bone regeneration .Clin. Oral Impl. Res. 2009;20:280–287.
[42]Nishimura K. Local application of simvastatin to rat incisor sockets augments bone Kokubyo Gakkai Zasshi. 2008;75(1):49-54.
[43]Sugiyama M, Kodama T, Konishi K, et al. Compactin and simvastatin, but not pravastatin, induce bone morphogenetic protein-2 in human osteosa- rcoma cells. Biochem Biophys Res Comm,2000,271:688-692.
[44]Maeda T, Matsunuma A, Kawane T. Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Biochem Biophys ResComm,2001;280:874-877.
[45]Garrett IR, Mundy GR. The role of statins as potential targets for bone formation. Arthritis Res,200;24:237-240.
[46]Yang YM, Huang WD, Xie QM, et al. Simvastatin attenuates TNF- alpHa-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells. Inflamm Res. In?amm. Res. 2010,59:151–157.
[47]Maeda T, Kawane T, Horiuchi N. Statins augment vascular endothelial growth factor expression in osteoblastic cells via inhibition of protein prenylation. Endocrinology, 2003,144:681-692.
[2]Stevens B, Yang YZ, Mohandas A, et al. A review of materials fabrication methods, and strategies used to enhance bone regeneration in engineered bone tissues. J Biomed Mater Res B Appl Biomater. 2008;85(2):573-582.
[3]Garrett IR, Gutierrez G, Mundy GR. Statins and bone formation. Curr Pharm Design 2001;7:715–736.
[4]Thylin MR, McConnell JC, Schmid MJ, et al. Effects of simvastatin gels on murine calvarial bone. J Periodontol 2002;73:1141–1148.
[5]Agrawal CM,Athanasiou KA. Technique to control pH in vicinity of biodegrading,PLA-PGA implants. J Biomed Mater Res.1997;38:105一114.
[6]Ambard AJ, Mueninghoff L. Calcium phosphate cement: review of mechanical and biological properties. J Prosthodont. 2006;15(5):321-328.
[7]Link DP, van den Dolder J, Jurgens WJ, et al. Mechanical evaluation of implanted calcium phosphate cement incorporated with PLGA microparticles. Biomaterials. 2006;27(28):4941-4947.
[8]Fei Z, Hu Y, Wu D,et al. Preparation and property of a novel bone graft composite consisting of rhBMP-2 loaded PLGA microspheres and calcium phosphate cement J Mater Sci: Mater Med. 2008;19:1109–1116
[9]Habraken WJ, Wolke JG, Mikos AG, et al. PLGA microsphere/calcium phosphate cement composites for tissue engineering: in vitro release and degradation characteristics.J. Biomater. Sci. Polymer Edn,2008;19(9):1171-1188.
[10]Becker W, Becker BE. Guide tissue regeneration of implants placed into extraction sockets and for implant dehisences: Surgical techniques and case reports. Int J Peridontol, 1990,10:376-391.
[11]Buser D, Dula D, Hirt HP, et al. Lateral ridge augmentation using autografts and barrier membranes. J Oral Maxillofacial Surg,1996,54:420-433.
[12]Scheller EL, Krebsbach PH, Kohn DH.Tissue engineering: state of the art in oral rehabilitation. Joural of Oral Rehabitation, 2009,36;368-389.
[13]Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005;26:5474–5491
[14]Freed LE. Vunjak-Novakovic G. Langer R. Cultivation of cell-polymer cartilage implants in bioreactors. J Cell Biochem. 1993,51:257-264.
[15]Jain RA. The manufacturing techniques of various drug loaded biodegr- adable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials.2000;21 2475-2490
[16]Tice TR, Cowsar DR. Biodegradable controlled-release parenteral systems. Pharm Technol,1984;11:26-35.
[17]刘竞龙,余斌,高成杰.骨组织工程修复骨缺损:大鼠成骨细胞与聚乳酸和聚乙醇共聚合物支架联合培养观察.中国临床康复.2002.6:2371-2372.
[18]Ignatius AA, Betz O, Augat P, Claes LE. In vivo investigations on composites made of resorbable ceramics and poly(lactide) used as bone graft substitutes. J Biomed Mater Res. 2001;58(6):701-709.
[19]Habraken WJ, Wolke JG, Mikos AG, Jansen JA. Injectable PLGAmicrosphere/calcium phosphate cements: physical properties and degradation characteristics. J Biomater Sci Polym Ed. 2006;17(9):1057-1074.
[20]RuhéPQ, Boerman OC, Russel FGM,et al. Controlled release of rhBMP-2 loaded poly(DL-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo. Journal of Controlled Release.2005;106:162– 171.
[21]Cohen S, AlonsoMJ, Langer R. Novel approaches to controlled release antigen delivery. Int J Technol Assessment Health Care.1994;10(1):121-130.
[22]倪建光,刘丹平.骨组织工程支架材料的研究进展.中国组织工程研究与临床康复.2009;(13 )8:1525-1528.
[23]Brown WE , Chow LC. A new calcium phosphate water setting cement. [J ]. Cement Research Progress.1986;352-379.
[24]Larsson S. Bauer TW. Use of injectable calcium phosphate cement for fracture fixation: a review. Clin Orthop. 2002;(395):23-32.
[25]Blom EJ. Klein-Nulend J. Wolke JG. Et al. Transforming growth factor-betal incorporation in a calcium phosphate bone cement: material prop- erties and release characteristics. J Biomed Mater Res .2002:59(2):265-272.
[26]Bohner M,Lemaitre J,Mekle HP. et al. Control of gentamicin release from a calcium phosphate cement by admixed poly(acrylic acid). J PHann Sci 2000;89(10)1262-1270.
[27]Pandya NM,Dhalla NS, Santani DD. Angiogenesis a new target for future therapy. Vascul Pharmacol. 2006,44(5):265-274.
[28]RuhéPQ, Kroese-Deutman HC, Wolke JG. et al. Bone inductive prop-erties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials. 2004;25(11):2123-2132.
[29]Li RH, Bouxsein ML, Blake CA. er al. rhBMP-2 injected in a calcium phosphate paste (alpha-BSM) accelerates healing in the rabbit ulnar osteotomy model. J Orthop Res. 2003;21(6):997-1004.
[30]Friedman CD, Costantino PD, Jones K. et al. Hydroxyapatite cement. II. Obliteration and reconstruction of the cat frontal sinus. Arch Otolaryngol Head Neck Surg. 1991;117(4):385-389.
[31]Shindo ML, Costantino PD, Friedman CD, Chow LC. Facial skeletal augmentation using hydroxyapatite cement. Arch Otolaryngol Head Neck Surg. 1993;119(2):185-190.
[32]Ruhe PQ, Hedberg EL, Padron NT. et al. Biocompatibility and degr- adation of poly(DL-lactic-co-glycolic acid)/calcium phosphate cement comp- osites. Wiley Periodicals, Inc. 2005;533-544.
[33]Kitchell JP, Wise DL. Poly(lactic/glycolic acid) biodegradable drug polymer matrix systems. Methods Enzymol. 1985; 112: 436-48.
[34]Hing KA,Best SM,Tanner KE,et al. Quantification of bone ingrowth within bone-derived Porous hydroxyapatite implants of varying density. J Mater Sci Mater Med,1999;10(10/11):663-670.
[35]Dahl SG,Allain P,Marie PJ,et al. Incorporation and distribution of str- ontium in bone. Bone 2001,28(4):446-453.
[36]Lane JM,Bostrom MP. Bone grafting and new composite biosyntheticgraft materials. Instr Course Lect,1998,47:525-534.
[37]Lu JX,Flautre B,Anselme K,et al. Role of interconnections in Porous bioceramics on bone recolonization in vitro and in vivo. J Mater Sci Mater Med,1999,10(2),111-120.
[38]Nam YS,Park TG. Biodegradable polymeric microcellular foams by modified thermally induced phase separation method. Biomaterials,1999,20(19):1783-1790.
[39]Hempel U, Reinstorf A, Poppe M, et al. Proliferation and differentiation of osteoblasts on Biocement D modified with collagen type I and citric acid. J Biomed Mater Res B Appl Biomater. 2004;71(1):130-143.
[40]Wong RWK, Rabie ABM. Statin collagen grafts used to repair defects in the parietal bone of rabbits. Brit J Oral Max Surg 2003;41:244–248.
[41]Nyan M, Daisuke Sato, Hidemichi Kihara,et al. Effects of the combi- nation withα-tricalcium phosphate and simvastatin on bone regeneration .Clin. Oral Impl. Res. 2009;20:280–287.
[42]Nishimura K. Local application of simvastatin to rat incisor sockets augments bone Kokubyo Gakkai Zasshi. 2008;75(1):49-54.
[43]Sugiyama M, Kodama T, Konishi K, et al. Compactin and simvastatin, but not pravastatin, induce bone morphogenetic protein-2 in human osteosa- rcoma cells. Biochem Biophys Res Comm,2000,271:688-692.
[44]Maeda T, Matsunuma A, Kawane T. Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Biochem Biophys ResComm,2001;280:874-877.
[45]Garrett IR, Mundy GR. The role of statins as potential targets for bone formation. Arthritis Res,200;24:237-240.
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