<i>In vitro</i> study of bone morphogenetic protein 2 gelatin/chitosan hydrogel sustained-release system composite hydroxyapatite/zirconium dioxide foam ceramics and induced pluripotent stem cells derived mesenchymal stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHAI Le ¹ ,  QUAN Renfu ² , HU Jintao ¹ , HUANG Xiaolong ² , LÜ Jianlan ¹ , ZHANG Can ¹ , QIU Rui ² , CAI Bingbing ²

1. Zhejiang University of Traditional Chinese Medicine, Hangzhou Zhejiang, 310053, P.R.China;
2. Department of Spine Surgery, Jiangnan Hospital of Zhejiang Chinese Medicine College, Hangzhou Zhejiang, 311200, P.R.China;

Corresponding author：

QUAN Renfu, Email: quanrenfu@163.com

Keywords：

Hydroxyapatite; zirconium dioxide; bone morphogenetic protein 2; induced pluripotent stem cells; mesenchymal stem cells; sustained-release system

DOI：

10.7507/1002-1892.201809060

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Objective To construct bone morphogenetic protein 2 (BMP-2) gelatin/chitosan hydrogel sustained-release 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 scaffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After 3, 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 iPS-MSCs and sustained-release system can adhere to the materials well, and the cell activity is better.

Citation： CHAI Le, QUAN Renfu, HU Jintao, HUANG Xiaolong, LÜ Jianlan, ZHANG Can, QIU Rui, CAI Bingbing. In vitro study of bone morphogenetic protein 2 gelatin/chitosan hydrogel sustained-release system composite hydroxyapatite/zirconium dioxide foam ceramics and induced pluripotent stem cells derived mesenchymal stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(2): 252-258. doi: 10.7507/1002-1892.201809060 Copy

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 cell-derived 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 cell-derived 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.	Martínez A, Arana P, Fernández A, et al. Synthesis and characterisation of alginate/chitosan nanoparticles as tamoxifen controlled delivery systems. J Microencapsul, 2013, 30(4): 398-408.
11.	Cicciù M, Herford AS, Cicciù 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. Daru, 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 three-dimensional β-tricalcium-phosphate/gelatin containing chitosan-based 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 mineral-based 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.

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 cell-derived 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 cell-derived 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. Martínez A, Arana P, Fernández A, et al. Synthesis and characterisation of alginate/chitosan nanoparticles as tamoxifen controlled delivery systems. J Microencapsul, 2013, 30(4): 398-408.
11. Cicciù M, Herford AS, Cicciù 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. Daru, 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 three-dimensional β-tricalcium-phosphate/gelatin containing chitosan-based 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 mineral-based 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.

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Chinese Journal of Reparative and Reconstructive Surgery

In vitro study of bone morphogenetic protein 2 gelatin/chitosan hydrogel sustained-release system composite hydroxyapatite/zirconium dioxide foam ceramics and induced pluripotent stem cells derived mesenchymal stem cells

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