BMP-2明胶/壳聚糖水凝胶缓释系统复合羟基磷灰石/二氧化锆泡沫陶瓷与诱导多能干细胞来源MSCs的体外研究
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摘要
目的构建BMP-2明胶/壳聚糖水凝胶缓释系统,与诱导多能干细胞(induced pluripotent stem cells,iPS)来源MSCs复合种植至羟基磷灰石(hydroxyapatite,HA)/二氧化锆(zirconium dioxide,ZrO2)生物多孔泡沫陶瓷材料,体外共培养,探索缓释系统对iPS-MSCs成骨分化的作用。方法运用油包水相溶液制备BMP-2明胶/壳聚糖水凝胶微球,检测微球的药物包封率、载药率和体外缓释速率。建立HA/ZrO2多孔生物泡沫陶瓷材料复合iPS-MSCs及BMP-2明胶/壳聚糖水凝胶缓释系统共培养体系,作为实验组;以未复合BMP-2明胶/壳聚糖水凝胶缓释系统的细胞-支架复合物作为对照组。两组培养3、7、10、14 d,检测细胞的ALP分泌量,RT-PCR检测核心结合因子α1(core binding factorα1,Cbfa1)、Ⅰ型胶原和锌指结构转录因子(Osterix,OSX)基因表达水平;培养14 d时行免疫组织化学染色观察Ⅰ型胶原表达,并通过扫描电镜观察细胞爬行及黏附状态。结果 BMP-2明胶/壳聚糖水凝胶缓释系统具有较好的药物包封率及载药率,可延长BMP-2的活性时间。共培养体系体外培养各时间点实验组ALP分泌量及Cbfa1、Ⅰ型胶原、OSX基因相对表达量均显著高于对照组,差异有统计学意义(P<0.05)。免疫组织化学染色观察示,实验组荧光数量明显多于对照组,即Ⅰ型胶原表达水平高于对照组;细胞能较均匀地分布于材料上,细胞形态良好。扫描电镜观察示缓释系统能较好地黏附于细胞之间。结论 iPS-MSCs具有促成骨分化能力,在BMP-2明胶/壳聚糖水凝胶缓释系统作用下其促成骨能力显著增强。iPS-MSCs与缓释系统结合后能良好黏附于材料上,且细胞活性较好。
Objective To construct bone morphogenetic protein 2(BMP-2) gelatin/chitosan hydrogel sustainedrelease system, co-implant with induced pluripotent stem cells(iPS) derived mesenchymal stem cells(MSCs) to hydroxyapatite(HA)/zirconium dioxide(ZrO2) bio porous ceramic foam, co-culture in vitro, and to explore the effect of sustained-release system on osteogenic differentiation of iPS-MSCs. Methods BMP-2 gelatin/chitosan hydrogel microspheres were prepared by water-in-oil solution. Drug encapsulation efficiency, drug loading, and in vitro sustained release rate of the microspheres were tested. HA/ZrO2 bio porous ceramic foam composite iPS-MSCs and BMP-2 gelatin/chitosan hydrogel sustained release system co-culture system was established as experimental group, and cell s caffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After3, 7, 10, and 14 days of co-culture in the two groups, ALP secretion of cells was detected; gene expression levels of core binding factor alpha 1(Cbfa1), collagen type Ⅰ, and Osterix(OSX) were detected by RT-PCR; the expression of collagen type Ⅰ was observed by immunohistochemical staining at 14 days of culture; and cell creep and adhesion were observed by scanning electron microscopy. Results BMP-2 gelatin/chitosan hydrogel sustained-release system had better drug encapsulation efficiency and drug loading, and could prolong the activity time of BMP-2. The secretion of ALP and the relative expression of Cbfa1, collagen type Ⅰ, and OSX genes in the experimental group were significantly higher than those in the control group at different time points in the in vitro co-culture system(P<0.05). Immunohistochemical staining showed that the amount of fluorescence in the experimental group was significantly more than that in the control group, i.e. the expression level of collagen type Ⅰ was higher than that in the control group. The cells could be more evenly distributed on the materials, and the cell morphology was good. Scanning electron microscopy showed that the sustained-release system could adhere to cells well. Conclusion iPS-MSCs have the ability of osteogenic differentiation,which is significantly enhanced by BMP-2 gelatin/chitosan hydrogel sustained-release system. The combination of iPSMSCs and sustained-release system can adhere to the materials well, and the cell activity is better.
Objective To construct bone morphogenetic protein 2(BMP-2) gelatin/chitosan hydrogel sustainedrelease system, co-implant with induced pluripotent stem cells(iPS) derived mesenchymal stem cells(MSCs) to hydroxyapatite(HA)/zirconium dioxide(ZrO2) bio porous ceramic foam, co-culture in vitro, and to explore the effect of sustained-release system on osteogenic differentiation of iPS-MSCs. Methods BMP-2 gelatin/chitosan hydrogel microspheres were prepared by water-in-oil solution. Drug encapsulation efficiency, drug loading, and in vitro sustained release rate of the microspheres were tested. HA/ZrO2 bio porous ceramic foam composite iPS-MSCs and BMP-2 gelatin/chitosan hydrogel sustained release system co-culture system was established as experimental group, and cell s caffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After3, 7, 10, and 14 days of co-culture in the two groups, ALP secretion of cells was detected; gene expression levels of core binding factor alpha 1(Cbfa1), collagen type Ⅰ, and Osterix(OSX) were detected by RT-PCR; the expression of collagen type Ⅰ was observed by immunohistochemical staining at 14 days of culture; and cell creep and adhesion were observed by scanning electron microscopy. Results BMP-2 gelatin/chitosan hydrogel sustained-release system had better drug encapsulation efficiency and drug loading, and could prolong the activity time of BMP-2. The secretion of ALP and the relative expression of Cbfa1, collagen type Ⅰ, and OSX genes in the experimental group were significantly higher than those in the control group at different time points in the in vitro co-culture system(P<0.05). Immunohistochemical staining showed that the amount of fluorescence in the experimental group was significantly more than that in the control group, i.e. the expression level of collagen type Ⅰ was higher than that in the control group. The cells could be more evenly distributed on the materials, and the cell morphology was good. Scanning electron microscopy showed that the sustained-release system could adhere to cells well. Conclusion iPS-MSCs have the ability of osteogenic differentiation,which is significantly enhanced by BMP-2 gelatin/chitosan hydrogel sustained-release system. The combination of iPSMSCs and sustained-release system can adhere to the materials well, and the cell activity is better.
引文
1 Chijimatsu R, Ikeya M, Yasui Y, et al. Characterization of mesenchymal stem cell-like cells derived from human iPSCs via neural crest development and their application for osteochondral repair. Stem Cells Int,2017,2017:1960965.
2 Okita K, Yamakawa T, Matsumura Y, et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells,2013, 31(3):458-466.
3 Simonson OE, Domogatskaya A, Volchkov P, et al. The safety of human pluripotent stem cells in clinical treatment. Ann Med, 2015,47(5):370-380.
4康明,黄杰华,张理选,等.壳聚糖/胡须/磷酸钙骨水泥复合生物材料的力学性能及对诱导多能干细胞成骨潜能的影响.中国修复重建外科杂志,2018, 32(7):959-967.
5 Jung Y, Bauer G, Nolta JA. Concise review:Induced pluripotent stem cell-derived mesenchymal stem cells:progress toward safe clinical products. Stem Cells, 2012, 30(1):42-47.
6 de Peppo GM, Marolt D. Modulating the biochemical and biophysical culture environment to enhance osteogenic differentiation and maturation of human pluripotent stem cellderived mesenchymal progenitors. Stem Cell Res Ther, 2013, 4(5):106.
7 Barberi T, Willis LM, Socci ND, et al.Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med, 2005, 2(6):e161.
8 Hwang NS, Varghese S, Lee HJ, et al. In vivo commitment and functional tissue regeneration using human embryonic stem cellderived mesenchymal cells.Proc Natl Acad Sci U S A, 2008,105(52):20641-20646.
9 Villa-Diaz LG, Brown SE, Liu Y, et al. Derivation of mesenchymal stem cells from human induced pluripotent stem cells cultured on synthetic substrates. Stem Cells, 2012, 30(6):1174-1181.
10 Martinez A, Arana P, Fernandez A, et al. Synthesis and characterisation of alginate/chitosan nanoparticles as tamoxifen controlled delivery systems. J Microencapsul, 2013, 30(4):398-408.
11 Cicciu M, Herford AS, Cicciu D, et al. Recombinant human bone morphogenetic protein-2 promote and stabilize hard and soft tissue healing for large mandibular new bone reconstruction defects. J Craniofacial Surg, 2014, 25(3):860-862.
12 Mohajel N, Najafabadi AR, Azadmanesh K, et al. Drying of a plasmid containing formulation:chitosan as a protecting agent.Darn, 2012,20(1):29.
13 Jun SH, Lee EJ, Jang TS, et al. Bone morphogenic protein-2(BMP-2)loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering. J Mat Sci Mat Med,2013, 24(3):773-782.
14 Nitta SK, Numata K. Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering. Int J Mol Sci, 2013,14(1):1629-1654.
15 Bastami F, Paknejad Z, Jafari M, et al. Fabrication of a threedimensionalβ-tricalcium-phosphate/gelatin containing chitosanbased nanoparticles for sustained release of bone morphogenetic protein-2:Implication for bone tissue engineering. Mater Sci Eng C Mater Biol Appl, 2017, 72:481-491.
16江涛.可加工复相陶瓷材料的研究现状与发展.材料导报,2012,26(17):49-53.
17宁聪琴,戴尅戎.硬组织替换用羟基磷灰石复合材料的研究进展.生物医学工程学杂志,2003, 20(3):550-554.
18 Gravel M, Gross T, Vago R, et al. Responses of mesenchymal stem cell to chitosan-coralline composites microstructured using coralline as gas forming agent. Biomaterials, 2006, 9(27):1899-1906.
19 Hutmacher DW, Schantz T, Zein I, et al. Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling. J Biomed Mat Res,2001, 55(2):203-216.
20全仁夫,张亮,许世超.BMP-2/VEGF165双基因修饰的骨髓间充质干细胞及其制备方法:中国,104250655 A. 2014.
21周传利.骨形态发生蛋白2基因表达异常与脊柱融合的相关性研究.山东青岛:青岛大学,2008.
22谢尚举,全仁夫,李长明,等.复合rhBMP-2壳聚糖水凝胶的制备及其缓释性能研究.中国海洋药物,2015, 34(4):31-36.
23 Xu Y, Du Y. Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles. Int J Pharm, 2003,250(1):215-226.
24黄鑫,孟国林,刘建,等.rh-BMP-2壳聚糖微球的制备及体外检测,中国矫形外科杂志,2009,17(15):1172-1174.
25 Park J, Ries J, Gelse K, et al. Bone regeneration in critical size defects by cell-mediated BMP-2 gene transfer:a comparison of adenoviral vectors and liposomes. Gene Ther, 2003, 10(13):1089-1098.
26 Tsuda H, Wada T, Yamashita T, et al. Enhanced osteoinduction by mesenchymal stem cells transfected with a fiber-mutant adenoviral BMP2 gene. J Gene Med,2005, 7(10):1322-1334.
27 Baldwin T. Morality and human embryo research. Introduction to the talking point on morality and human embryo research. EMBO Rep, 2009, 10(4):299-300.
28 Gaspar-Maia A, Alajem A, Meshorer E, et al. Open chromatin in pluripotency and reprogramming. Nat Rev Mol Cell Biol, 2011,12(1):36-47.
29 Strong M, Farrugia A, Rebulla P. Stem cell and cellular therapy developments. Biologicals, 2009, 37(2):103-107.
30 Nakamura A,Akahane M,Shigematsu H, et al. Cell sheet transplantation of cultured mesenchynmal stem cells enhances bone formation in a rat nonunion model. Bone, 2010, 46(2):418-424.
31 Lee DW,Yun YP,Park K,et al. Gentamicin and bone morphogenic protein-2(BMP-2)-delivering heparinized-titanium implant with enhanced antibacterial activity and osteointegration.Bone,2012, 50(4):974-982.
32 Luong LN, Ramaswamy J, Kohn DH. Effects of osteogenic growth factors on bone marrow stromal cell differentiation in a mineralbased delivery system. Biomaterials, 2012, 33(1):283-294.
33 Liu Y, Goldberg AJ, Dennis JE, et al. One-step derivation of mesenchymal stem cell(MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating. PLoS One, 2012, 7(3):e33225.
34 Li B, Hu RY, Sun L, et al. Potential role of andrographolide in the proliferation of osteoblasts mediated by the ERK signaling pathway. Biomed Pharmacother, 2016, 83:1335-1344.
2 Okita K, Yamakawa T, Matsumura Y, et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells,2013, 31(3):458-466.
3 Simonson OE, Domogatskaya A, Volchkov P, et al. The safety of human pluripotent stem cells in clinical treatment. Ann Med, 2015,47(5):370-380.
4康明,黄杰华,张理选,等.壳聚糖/胡须/磷酸钙骨水泥复合生物材料的力学性能及对诱导多能干细胞成骨潜能的影响.中国修复重建外科杂志,2018, 32(7):959-967.
5 Jung Y, Bauer G, Nolta JA. Concise review:Induced pluripotent stem cell-derived mesenchymal stem cells:progress toward safe clinical products. Stem Cells, 2012, 30(1):42-47.
6 de Peppo GM, Marolt D. Modulating the biochemical and biophysical culture environment to enhance osteogenic differentiation and maturation of human pluripotent stem cellderived mesenchymal progenitors. Stem Cell Res Ther, 2013, 4(5):106.
7 Barberi T, Willis LM, Socci ND, et al.Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med, 2005, 2(6):e161.
8 Hwang NS, Varghese S, Lee HJ, et al. In vivo commitment and functional tissue regeneration using human embryonic stem cellderived mesenchymal cells.Proc Natl Acad Sci U S A, 2008,105(52):20641-20646.
9 Villa-Diaz LG, Brown SE, Liu Y, et al. Derivation of mesenchymal stem cells from human induced pluripotent stem cells cultured on synthetic substrates. Stem Cells, 2012, 30(6):1174-1181.
10 Martinez A, Arana P, Fernandez A, et al. Synthesis and characterisation of alginate/chitosan nanoparticles as tamoxifen controlled delivery systems. J Microencapsul, 2013, 30(4):398-408.
11 Cicciu M, Herford AS, Cicciu D, et al. Recombinant human bone morphogenetic protein-2 promote and stabilize hard and soft tissue healing for large mandibular new bone reconstruction defects. J Craniofacial Surg, 2014, 25(3):860-862.
12 Mohajel N, Najafabadi AR, Azadmanesh K, et al. Drying of a plasmid containing formulation:chitosan as a protecting agent.Darn, 2012,20(1):29.
13 Jun SH, Lee EJ, Jang TS, et al. Bone morphogenic protein-2(BMP-2)loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering. J Mat Sci Mat Med,2013, 24(3):773-782.
14 Nitta SK, Numata K. Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering. Int J Mol Sci, 2013,14(1):1629-1654.
15 Bastami F, Paknejad Z, Jafari M, et al. Fabrication of a threedimensionalβ-tricalcium-phosphate/gelatin containing chitosanbased nanoparticles for sustained release of bone morphogenetic protein-2:Implication for bone tissue engineering. Mater Sci Eng C Mater Biol Appl, 2017, 72:481-491.
16江涛.可加工复相陶瓷材料的研究现状与发展.材料导报,2012,26(17):49-53.
17宁聪琴,戴尅戎.硬组织替换用羟基磷灰石复合材料的研究进展.生物医学工程学杂志,2003, 20(3):550-554.
18 Gravel M, Gross T, Vago R, et al. Responses of mesenchymal stem cell to chitosan-coralline composites microstructured using coralline as gas forming agent. Biomaterials, 2006, 9(27):1899-1906.
19 Hutmacher DW, Schantz T, Zein I, et al. Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling. J Biomed Mat Res,2001, 55(2):203-216.
20全仁夫,张亮,许世超.BMP-2/VEGF165双基因修饰的骨髓间充质干细胞及其制备方法:中国,104250655 A. 2014.
21周传利.骨形态发生蛋白2基因表达异常与脊柱融合的相关性研究.山东青岛:青岛大学,2008.
22谢尚举,全仁夫,李长明,等.复合rhBMP-2壳聚糖水凝胶的制备及其缓释性能研究.中国海洋药物,2015, 34(4):31-36.
23 Xu Y, Du Y. Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles. Int J Pharm, 2003,250(1):215-226.
24黄鑫,孟国林,刘建,等.rh-BMP-2壳聚糖微球的制备及体外检测,中国矫形外科杂志,2009,17(15):1172-1174.
25 Park J, Ries J, Gelse K, et al. Bone regeneration in critical size defects by cell-mediated BMP-2 gene transfer:a comparison of adenoviral vectors and liposomes. Gene Ther, 2003, 10(13):1089-1098.
26 Tsuda H, Wada T, Yamashita T, et al. Enhanced osteoinduction by mesenchymal stem cells transfected with a fiber-mutant adenoviral BMP2 gene. J Gene Med,2005, 7(10):1322-1334.
27 Baldwin T. Morality and human embryo research. Introduction to the talking point on morality and human embryo research. EMBO Rep, 2009, 10(4):299-300.
28 Gaspar-Maia A, Alajem A, Meshorer E, et al. Open chromatin in pluripotency and reprogramming. Nat Rev Mol Cell Biol, 2011,12(1):36-47.
29 Strong M, Farrugia A, Rebulla P. Stem cell and cellular therapy developments. Biologicals, 2009, 37(2):103-107.
30 Nakamura A,Akahane M,Shigematsu H, et al. Cell sheet transplantation of cultured mesenchynmal stem cells enhances bone formation in a rat nonunion model. Bone, 2010, 46(2):418-424.
31 Lee DW,Yun YP,Park K,et al. Gentamicin and bone morphogenic protein-2(BMP-2)-delivering heparinized-titanium implant with enhanced antibacterial activity and osteointegration.Bone,2012, 50(4):974-982.
32 Luong LN, Ramaswamy J, Kohn DH. Effects of osteogenic growth factors on bone marrow stromal cell differentiation in a mineralbased delivery system. Biomaterials, 2012, 33(1):283-294.
33 Liu Y, Goldberg AJ, Dennis JE, et al. One-step derivation of mesenchymal stem cell(MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating. PLoS One, 2012, 7(3):e33225.
34 Li B, Hu RY, Sun L, et al. Potential role of andrographolide in the proliferation of osteoblasts mediated by the ERK signaling pathway. Biomed Pharmacother, 2016, 83:1335-1344.