辛伐他汀缓释微球影响骨组织工程成骨性能的体外实验
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摘要
背景:近年来国内外研究将辛伐他汀缓释微球组装在支架材料上,发现其可完成骨缺损的修复,取得不错的效果。目的:探讨辛伐他汀缓释微球对组织工程成骨性能的影响。方法:采用薄膜分散法制作纳米脂质体辛伐他汀缓释微球,检测其粒径和多分散系数及包封率。采用透析装置检测游离辛伐他汀溶液与辛伐他汀缓释微球溶液的缓释性能。将第3代人胎盘间充质干细胞接种于多孔羟基磷灰石陶瓷支架上,待细胞贴壁生长后分2组培养,实验组加入辛伐他汀缓释微球溶液,空白组加入普通培养基,7,14,21 d后进行转录组测序,实时荧光定量PCR验证成骨基因表达水平。结果与结论:(1)辛伐他汀缓释微球平均粒径为(77.27±6.4) nm,多分散系数为0.131±0.040,包封率为85.6%;(2)游离辛伐他汀溶液在前3 d迅速释放超过了总药量的80%,辛伐他汀缓释微球溶液在前3 d的药物释放量仅约40%,此后也一直较缓慢持续释放至14d,14d累计释放量接近80%;(3)转录组测序显示,实验组骨形态发生蛋白2、骨形态发生蛋白4和血管内皮生长因子等成骨相关基因富集表达;(4)实时荧光定量PCR检测显示,实验组7,14,21 d的骨形态发生蛋白2、骨形态发生蛋白4、血管内皮生长因子基因表达均高于空白组(P <0.05);(5)结果表明,辛伐他汀缓释微球可促进组织工程成骨性能表达。
BACKGROUND: In recent years, domestic and foreign studies have assembled simvastatin sustained-releasemicrospheres onto scaffold materials. It has been found that simvastatin can be used to repair bone defects and good results have been obtained. OBJECTIVE: To investigate the effect of simvastatin sustained-release microspheres on tissue engineering osteogenesis. METHODS: Nanoliposome simvastatin sustained-release microspheres were prepared by thin-film dispersion method. The particle size, polydispersity coefficient and encapsulation efficiency were measured. The sustained release properties of free simvastatin solution and simvastatin sustained-release microsphere solution were measured by dialysis device. The third generation of human placental mesenchymal stem cells was inoculated on porous hydroxyapatite ceramic scaffolds. The cells were cultured in two groups after adherent growth. The experimental group was treated with simvastatin sustained-release microspheres, and the normal medium was added to the blank group. Transcriptome sequencing was carried out after 7, 14, 21 days. The expression levels of osteogenic genes were verified by real-time fluorescence quantitative PCR. RESULTS AND CONCLUSION:(1) The average particle size of simvastatin sustained-release microspheres was(77.27±6.4) nm, the polydispersity coefficient was 0.131±0.040, and the encapsulation efficiency was 85.6%.(2) The rapid release of free simvastatin solution exceeded 80% total dose in the first 3 days. While the simvastatin sustained-release microsphere solution released only about 40% of the drug in the first 3 days, and had been slowly released until the 14 th day, and the cumulative release at the 14 th day was close to 80%.(3) Transcriptome sequencing showed that the osteogenesis-related genes such as bone morphogenetic protein 2, bone morphogenetic protein 4 and vascular endothelial growth factor were enriched and expressed in the experimental group.(4) Real-time quantitative PCR showed that the mRNA levels of bone morphogenetic protein 2, bone morphogenetic protein 4 and vascular endothelial growth factor were significantly higher in the experimental group than the blank group at 7, 14 and 21 days(P < 0.05). These results indicate that simvastatin sustained-release microspheres can promote osteogenic performance for tissue engineering.
BACKGROUND: In recent years, domestic and foreign studies have assembled simvastatin sustained-releasemicrospheres onto scaffold materials. It has been found that simvastatin can be used to repair bone defects and good results have been obtained. OBJECTIVE: To investigate the effect of simvastatin sustained-release microspheres on tissue engineering osteogenesis. METHODS: Nanoliposome simvastatin sustained-release microspheres were prepared by thin-film dispersion method. The particle size, polydispersity coefficient and encapsulation efficiency were measured. The sustained release properties of free simvastatin solution and simvastatin sustained-release microsphere solution were measured by dialysis device. The third generation of human placental mesenchymal stem cells was inoculated on porous hydroxyapatite ceramic scaffolds. The cells were cultured in two groups after adherent growth. The experimental group was treated with simvastatin sustained-release microspheres, and the normal medium was added to the blank group. Transcriptome sequencing was carried out after 7, 14, 21 days. The expression levels of osteogenic genes were verified by real-time fluorescence quantitative PCR. RESULTS AND CONCLUSION:(1) The average particle size of simvastatin sustained-release microspheres was(77.27±6.4) nm, the polydispersity coefficient was 0.131±0.040, and the encapsulation efficiency was 85.6%.(2) The rapid release of free simvastatin solution exceeded 80% total dose in the first 3 days. While the simvastatin sustained-release microsphere solution released only about 40% of the drug in the first 3 days, and had been slowly released until the 14 th day, and the cumulative release at the 14 th day was close to 80%.(3) Transcriptome sequencing showed that the osteogenesis-related genes such as bone morphogenetic protein 2, bone morphogenetic protein 4 and vascular endothelial growth factor were enriched and expressed in the experimental group.(4) Real-time quantitative PCR showed that the mRNA levels of bone morphogenetic protein 2, bone morphogenetic protein 4 and vascular endothelial growth factor were significantly higher in the experimental group than the blank group at 7, 14 and 21 days(P < 0.05). These results indicate that simvastatin sustained-release microspheres can promote osteogenic performance for tissue engineering.
引文
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[7]邱晓明,甄平,李松凯.聚乳酸-羟基乙酸共聚物载药微球制备工艺研究进展[J].中国组织工程研究,2018,22(10):1599-1604.
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[9]宋效庆,刘红,陈天杰,等.淫羊藿苷/载明胶纳米复合物-PLGA缓释系统的制备及工艺优化[J].吉林大学学报(医学版),2018,60(2):438-443.
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[12]Li J,Zhi W,Xu T,et al.Ectopic osteogenesis and angiogenesis regulated by porous architecture of hydroxyapatite scaffolds with similar interconnecting structure in vivo.Regen Biomater.2016;3(5):285-297.
[13]Zhang B,He L,Han Z,et al.Enhanced osteogenesis of multilayered pore-closed microsphere-immobilizated hydroxyapatite scaffold via sequential delivery of osteogenic growth peptide and BMP-2.J Mater Chem B.2017;5(41).DOI:10.1039/C7TB01970J
[14]邵苗苗,刘钟西,许诺,等.牙髓干细胞在组织工程研究中的进展和应用前景[J].中国组织工程研究,2018,22(13):2126-2132.
[15]杨超,申才良.诱导多能干细胞治疗颈节段脊髓损伤的研究进展[J].安徽医科大学学报,2018,53(2):323-326.
[16]Bernardo AS,Hay CW,Docherty K.Pancreatic transcription factors and their role in the birth,life and survival of the pancreatic beta cell.Mol Cell Endocrinol.2008;294(1):1-9.
[17]Xie J,Peng C,Zhao Q,et al.Osteogenic Differentiation and Bone Regeneration of the iPSC-MSCs Supported by a Biomimetic Nanofibrous Scaffold.Acta Biomater.2016;29:365-379.
[18]Jaiswal N,Haynesworth SE,Caplan AI,et al.Osteogenic differentiation of purified,culture-expanded human mesenchymal stem cells in vitro.J Cell Biochem.2015;64(2):295-312.
[19]伍佳,温永梅,吕欣荣,等.人胎盘来源间充质干细胞的生物学性能及体外生物相容性[J].中国组织工程研究,2017,21(5):755-759.
[20]Li J,Xu T,Wang Q,et al.Integrating surface topography of stripe pattern on pore surface of 3-dimensional hydroxyapatiye scaffolds.Mater Lett.2016;169:148-152.
[21]Maciel-Oliveira N,Bradaschia-Correa V,Arana-Chavez VE.Early alveolar bone regeneration in rats after topical administration of simvastatin.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2011;112(2):170-179.
[22]Kwon YD,Yang DH,Lee DW.A Titanium Surface-Modified with Nano-Sized Hydroxyapatite and Simvastatin Enhances Bone Formation and Osseintegration.J Biomed Nanotechnol.2015;11(6):1007.
[23]Gentile P,Nandagiri VK,Daly J,et al.Localised controlled release of simvastatin from porous chitosan-gelatin scaffolds engrafted with simvastatin loaded PLGA-microparticles for bone tissue engineering application.Mater Sci Eng C Mater Biol Appl.2016;59:249-257.
[24]Zhang HX,Xiao GY,Wang X,et al.Biocompatibility and osteogenesis of calcium phosphate composite scaffolds containing simvastatin-loaded PLGA microspheres for bone tissue engineering.J Biomed Mater Res A.2015;103(10):3250-3258..
[25]Liu T,Gao Y,Sakamoto K,et al.BMP-2 promotes differentiation of osteoblasts and chondroblasts in Runx2-deficient cell lines.J Cell Physiol.2010;211(3):728-735.
[26]Wang Z,Gerstein M,Snyder M.RNA-Seq:a revolutionary tool for transcriptomics.Nat Rev Genet.2009;10(1):57-63.
[27]Ferreira CL,Abreu FAMD,Silva GAB,et al.TGF-β1 and BMP-4 carried by liposomes enhance the healing process in alveolar bone.Arch Oral Biol.2013;58(6):646-656.
[28]Pullisaar H,Reseland JE,Haugen HJ,et al.Simvastatin coating of TiO?scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.Biochem Biophys Res Commun.2014;447(1):139-144.
[2]Jiang L,Sun H,Yuan A,et al.Enhancement of osteoinduction by continual simvastatin release from poly(lactic-co-glycolic acid)-hydroxyapatite-simvastatin nano-fibrous scaffold.JBiomed Nanotechnol.2013;9(11):1921-1928.
[3]Mundy G,Garrett R,Harris S,et al.Stimulation of Bone Formation in Vitro and in Rodents by Statins.Science.1999286(5446):1946-1949.
[4]Webber MA,Mahmud W,Lightfoot JD,et al.Rhabdomyolysis and compartment syndrome with coadministration of risperidone and simvastatin.J Psychopharmacol.2004;18(3)432.
[5]Law M,Rudnicka AR.Statin safety:a systematic review.Am JCardiol.2006;97(8):S52-52S60.
[6]Ochoa L,Igartua M,Hernández RM,et al.Novel extended-release formulation of lovastatin by one-step melt granulation:in vitro and in vivo evaluation.Eur J Pharm Biopharm.2011;77(2):306-312.
[7]邱晓明,甄平,李松凯.聚乳酸-羟基乙酸共聚物载药微球制备工艺研究进展[J].中国组织工程研究,2018,22(10):1599-1604.
[8]朱林,王聿栋,董艳梅,等.缓释米诺环素的介孔纳米生物玻璃载药系统[J].北京大学学报(医学版),2018,50(2):249-255.
[9]宋效庆,刘红,陈天杰,等.淫羊藿苷/载明胶纳米复合物-PLGA缓释系统的制备及工艺优化[J].吉林大学学报(医学版),2018,60(2):438-443.
[10]Davison NL,Luo X,Schoenmaker T,et al.Submicron-scale surface architecture of tricalcium phosphate directs osteogenesis in vitro and in vivo.Eur Cell Mater.2014;27(20):281.
[11]Li J,Wang Q,Zhi W,et al.Immobilization of salvianolic acid B-loaded chitosan microspheres distributed three-dimensionally and homogeneously on the porous surface of hydroxyapatite scaffolds.Biomed Mater.2016;11(5):055014.
[12]Li J,Zhi W,Xu T,et al.Ectopic osteogenesis and angiogenesis regulated by porous architecture of hydroxyapatite scaffolds with similar interconnecting structure in vivo.Regen Biomater.2016;3(5):285-297.
[13]Zhang B,He L,Han Z,et al.Enhanced osteogenesis of multilayered pore-closed microsphere-immobilizated hydroxyapatite scaffold via sequential delivery of osteogenic growth peptide and BMP-2.J Mater Chem B.2017;5(41).DOI:10.1039/C7TB01970J
[14]邵苗苗,刘钟西,许诺,等.牙髓干细胞在组织工程研究中的进展和应用前景[J].中国组织工程研究,2018,22(13):2126-2132.
[15]杨超,申才良.诱导多能干细胞治疗颈节段脊髓损伤的研究进展[J].安徽医科大学学报,2018,53(2):323-326.
[16]Bernardo AS,Hay CW,Docherty K.Pancreatic transcription factors and their role in the birth,life and survival of the pancreatic beta cell.Mol Cell Endocrinol.2008;294(1):1-9.
[17]Xie J,Peng C,Zhao Q,et al.Osteogenic Differentiation and Bone Regeneration of the iPSC-MSCs Supported by a Biomimetic Nanofibrous Scaffold.Acta Biomater.2016;29:365-379.
[18]Jaiswal N,Haynesworth SE,Caplan AI,et al.Osteogenic differentiation of purified,culture-expanded human mesenchymal stem cells in vitro.J Cell Biochem.2015;64(2):295-312.
[19]伍佳,温永梅,吕欣荣,等.人胎盘来源间充质干细胞的生物学性能及体外生物相容性[J].中国组织工程研究,2017,21(5):755-759.
[20]Li J,Xu T,Wang Q,et al.Integrating surface topography of stripe pattern on pore surface of 3-dimensional hydroxyapatiye scaffolds.Mater Lett.2016;169:148-152.
[21]Maciel-Oliveira N,Bradaschia-Correa V,Arana-Chavez VE.Early alveolar bone regeneration in rats after topical administration of simvastatin.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2011;112(2):170-179.
[22]Kwon YD,Yang DH,Lee DW.A Titanium Surface-Modified with Nano-Sized Hydroxyapatite and Simvastatin Enhances Bone Formation and Osseintegration.J Biomed Nanotechnol.2015;11(6):1007.
[23]Gentile P,Nandagiri VK,Daly J,et al.Localised controlled release of simvastatin from porous chitosan-gelatin scaffolds engrafted with simvastatin loaded PLGA-microparticles for bone tissue engineering application.Mater Sci Eng C Mater Biol Appl.2016;59:249-257.
[24]Zhang HX,Xiao GY,Wang X,et al.Biocompatibility and osteogenesis of calcium phosphate composite scaffolds containing simvastatin-loaded PLGA microspheres for bone tissue engineering.J Biomed Mater Res A.2015;103(10):3250-3258..
[25]Liu T,Gao Y,Sakamoto K,et al.BMP-2 promotes differentiation of osteoblasts and chondroblasts in Runx2-deficient cell lines.J Cell Physiol.2010;211(3):728-735.
[26]Wang Z,Gerstein M,Snyder M.RNA-Seq:a revolutionary tool for transcriptomics.Nat Rev Genet.2009;10(1):57-63.
[27]Ferreira CL,Abreu FAMD,Silva GAB,et al.TGF-β1 and BMP-4 carried by liposomes enhance the healing process in alveolar bone.Arch Oral Biol.2013;58(6):646-656.
[28]Pullisaar H,Reseland JE,Haugen HJ,et al.Simvastatin coating of TiO?scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.Biochem Biophys Res Commun.2014;447(1):139-144.