Osteogenic differentiation of bone marrow mesenchymal stem cells induced by gene-loaded lipopolysaccharide-amine nanopolymersomes_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Jing ^1,2 , CHEN Ying ¹ , TENG Wei ³ ,  WANG Qinmei ¹

1. Key Laboratory on Assisted Circulation of Health Ministry, First Affiliated Hospital, Sun Yat-sen University, Guangzhou Guangdong, 510089, P.R.China;
2. School of Biomedical Engineering, Sun Yat-sen University, Guangzhou Guangdong, 510006, P.R.China;
3. Department of Prosthodontics, Hospital of Stomatology, Sun Yat-sen University, Guangzhou Guangdong, 510060, P.R.China;

Corresponding?author：

WANG Qinmei, Email: wangqinm@mail.sysu.edu.cn

Keywords：

Gene carrier; lipopolysaccharide-amine nanopolymersomes; gene transfection; directional differentiation; bone marrow mesenchymal stem cells

DOI：

10.7507/1002-1892.201804125

Video：

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Objective To investigate the ability of gene-loaded lipopolysaccharide-amine nanopolymersomes (LNPs) in inducing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by in vitro gene transfection, where LNPs were used as a non-viral cationic carrier, and their properties were optimized during synthesis. Methods LNPs were synthesized by a graft-copolymerization method, and the effects of different pH environments during synthesis on physicochemical properties of LNPs and LNPs/plasmid of bone morphogenetic protein 2-green fluorescent protein (pBMP-2-GFP) complexes were explored. Then, optimized LNPs with maximum transfection efficiency and safe cytotoxicity in rat BMSCs were identified by cytotoxicity and transfection experiments in vitro. Thereafter, the optimized LNPs were used to mediate pBMP-2-GFP to transfect rat BMSCs, and the influences of LNPs/pBMP-2-GFP on osteogenic differentiation of BMSCs were evaluated by monitoring the cell morphology, concentration of BMP-2 protein, activity of alkaline phosphatase (ALP), and the formation of calcium nodules. Results The nitrogen content, particle size, and zeta potential of LNPs synthesized at pH 8.5 were lower than those of the other pH groups, with the lowest cytotoxicity (96.5%±1.4%) and the highest transfection efficiency (98.8%±0.1%). After transfection treatment, within the first 4 days, BMSCs treated by LNPs/pBMP-2-GFP expressed BMP-2 protein significantly higher than that treated by Lipofectamine2000 (Lipo)/pBMP-2-GFP, polyethylenimine 25K/pBMP-2-GFP, and the blank (non-treated). At 14 days after transfection, ALP activity in BMSCs treated by LNPs/pBMP-2-GFP was higher than that treated by Lipo/pBMP-2-GFP and the blank, comparable to that induced by osteogenic medium; with alizarin red staining, visible calcium nodules were found in BMSCs treated by LNPs/pBMP-2-GFP or osteogenic medium, but absent in BMSCs treated by Lipo/pBMP-2-GFP or the blank with apoptosis. At 21 days after transfection, transparent massive nodules were discovered in BMSCs treated by LNPs/pBMP-2-GFP, and BMSCs exhibited the morphologic features of osteoblasts. Conclusion LNPs synthesized at pH 8.5 has optimal transfection efficiency and cytotoxicity, they can efficiently mediate pBMP-2-GFP to transfect BMSCs, and successfully induce their directional osteogenic differentiation, whose inducing effect is comparable to the osteogenic medium. The results suggest that gene transfection mediated by LNPs may be a convenient and effective strategy in inducing directional differentiation of stem cells.

Citation： LI Jing, CHEN Ying, TENG Wei, WANG Qinmei. Osteogenic differentiation of bone marrow mesenchymal stem cells induced by gene-loaded lipopolysaccharide-amine nanopolymersomes. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(11): 1469-1476. doi: 10.7507/1002-1892.201804125 Copy

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4.	Paschos NK, Sennett ML. Update on mesenchymal stem cell therapies for cartilage disorders. World J Orthop, 2017, 8(12): 853-860.
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7.	朱勇, 陳良萬, 林若柏, 等. SD大鼠骨髓間充質干細胞體外培養、表型鑒定及標記. 吉林大學學報: 醫學版, 2009, 35(2): 326-329.
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9.	Elsabahy M, Nazarali A, Foldvari M. Non-viral nucleic acid delivery: key challenges and future directions. Curr Drug Deliv, 2011, 8(3): 235-244.
10.	Munsell EV, Kurpad DS, Freeman TA, et al. Histone-targeted gene transfer of bone morphogenetic protein-2 enhances mesenchymal stem cell chondrogenic differentiation. Acta Biomater, 2018, 71: 156-167.
11.	Cui ZK, Sun JA, Baljon JJ, et al. Simultaneous delivery of hydrophobic small molecules and siRNA using Sterosomes to direct mesenchymal stem cell differentiation for bone repair. Acta Biomater, 2017, 58: 214-224.
12.	Huang ZH, Teng W, Liu LS, et al. Efficient cytosolic delivery mediated by polymersomes facilely prepared from a degradable, amphiphilic, and amphoteric copolymer. Nanotechnology, 2013, 24(26): 265104.
13.	Teng W, Huang ZH, Chen Y, et al. pVEGF-loaded lipopolysaccharide-amine nanopolymersomes for therapeutic angiogenesis. Nanotechnology, 2014, 25(6): 065702.
14.	王琴梅, 張亦霞, 李卓萍, 等. 多醛基海藻酸鈉交聯劑的制備及性能. 應用化學, 2010, 27(2): 155-158.
15.	譚琦, 黃政德, 王立鳳. 大鼠骨髓間充質干細胞分離和培養方法的研究. 湖南中醫藥大學學報, 2011, 31(7): 74-76.
16.	Carroll J, Douarre P, Coffey A, et al. Optimization of a rapid viability assay for Mycobacterium avium subsp. paratuberculosis by using alamarBlue. Appl Environ Microbiol, 2009, 75(24): 7870-7872.
17.	Song HM, Wang G, He B, et al. Cationic lipid-coated PEI/DNA polyplexes with improved efficiency and reduced cytotoxicity for gene delivery into mesenchymal stem cells. Int J Nanomedicine, 2012, 7: 4637-4648.
18.	劉儻, 張湘生, 張慶, 等. 骨髓間充質干細胞成骨分化中血管內皮生長因子和骨形態發生蛋白2的表達. 中國組織工程研究, 2012, 16(36): 6651-6657.
19.	張靜, 詹琪, 黃艷. 磁場對骨髓間充質干細胞分化的影響. 中國組織工程研究, 2015, 19(23): 3744-3749.
20.	Jsiswal RK, Jaiswal N, Bruder SP, et al. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem, 2000, 275(13): 9645-9652.
21.	Dunbar CE, High KA, Joung JK, et al. Gene therapy comes of age. Science, 2018, 359(6372): 175.
22.	Wolski H, Bogacz A, Bartkowiak-Wieczorek J, et al. Polymorphism of bone morphogenetic protein (BMP2) and osteoporosis etiology. Ginekol Pol, 2015, 86(3): 203-209.
23.	Grgurevic L, Macek B, Mercep M, et al. Bone morphogenetic protein (BMP) 1-3 enhances bone repair. Biochem Biophys Res Commun, 2011, 408(1): 25-31.
24.	Yazici C, Takahata M, Reynolds DG, et al. Self-complementary AAV2.5-BMP2-coated femoral allografts mediated superior bone healing versus live autografts in mice with equivalent biomechan-ics to unfractured femur. Mol Ther, 2011, 19(8): 1416-1425.
25.	Song K, Huang M, Shi Q, et al. Cultivation and identification of rat bone marrow-derived mesenchymal stem cells. Mol Med Rep, 2014, 10(2): 755-760.
26.	李雪峰, 劉亮, 王東. 大鼠骨髓間充質干細胞體外向成骨細胞分化培養的生物學特性. 中國組織工程研究與臨床康復, 2011, 15(23): 4185-4188.
27.	Kotobuki N, Ioku K, Kawagoe D, et al. Observation of osteogenic differentiation cascade of living mesenchymal stem cells on transparent hydroxyapatite ceramics. Biomaterials, 2005, 26(7): 779-785.

1. Madigan M, Atoui R. Therapeutic use of stem cells for myocardial infarction. Bioengineering (Basel), 2018, 5(2): pii. E28.
2. Budd E, Waddell S, de Andrés MC, et al. The potential of microRNAs for stem cell-based therapyfor degenerative skeletal diseases. Curr Mol Bio Rep, 2017, 3(4): 263-275.
3. Heissig B, Dhahri D, Eiamboonsert S, et al. Role of mesenchymal stem cell-derived fibrinolytic factor in tissue regeneration and cancer progression. Cell Mol Life Sci, 2015, 72(24): 4759-4770.
4. Paschos NK, Sennett ML. Update on mesenchymal stem cell therapies for cartilage disorders. World J Orthop, 2017, 8(12): 853-860.
5. White AP, Vaccaro AR, Hall JA, et al. Clinical applications of BMP-7/OP-1 in fractures, nonunions and spinal fusion. Int Orthop, 2007, 31(6): 735-741.
6. Gonzalez-Fernandez T, Sathy BN, Hobbs C, et al. Mesenchymal stem cell fate following non-viral gene transfection strongly depends on the choice of delivery vector. Acta Biomaterialia, 2017, 55: 226-238.
7. 朱勇, 陳良萬, 林若柏, 等. SD大鼠骨髓間充質干細胞體外培養、表型鑒定及標記. 吉林大學學報: 醫學版, 2009, 35(2): 326-329.
8. Pathak A, Patnaik S, Gupta KC. Recent trends in non-viral vector-mediated gene delivery. Biotechnol J, 2009, 4(11): 1559-1572.
9. Elsabahy M, Nazarali A, Foldvari M. Non-viral nucleic acid delivery: key challenges and future directions. Curr Drug Deliv, 2011, 8(3): 235-244.
10. Munsell EV, Kurpad DS, Freeman TA, et al. Histone-targeted gene transfer of bone morphogenetic protein-2 enhances mesenchymal stem cell chondrogenic differentiation. Acta Biomater, 2018, 71: 156-167.
11. Cui ZK, Sun JA, Baljon JJ, et al. Simultaneous delivery of hydrophobic small molecules and siRNA using Sterosomes to direct mesenchymal stem cell differentiation for bone repair. Acta Biomater, 2017, 58: 214-224.
12. Huang ZH, Teng W, Liu LS, et al. Efficient cytosolic delivery mediated by polymersomes facilely prepared from a degradable, amphiphilic, and amphoteric copolymer. Nanotechnology, 2013, 24(26): 265104.
13. Teng W, Huang ZH, Chen Y, et al. pVEGF-loaded lipopolysaccharide-amine nanopolymersomes for therapeutic angiogenesis. Nanotechnology, 2014, 25(6): 065702.
14. 王琴梅, 張亦霞, 李卓萍, 等. 多醛基海藻酸鈉交聯劑的制備及性能. 應用化學, 2010, 27(2): 155-158.
15. 譚琦, 黃政德, 王立鳳. 大鼠骨髓間充質干細胞分離和培養方法的研究. 湖南中醫藥大學學報, 2011, 31(7): 74-76.
16. Carroll J, Douarre P, Coffey A, et al. Optimization of a rapid viability assay for Mycobacterium avium subsp. paratuberculosis by using alamarBlue. Appl Environ Microbiol, 2009, 75(24): 7870-7872.
17. Song HM, Wang G, He B, et al. Cationic lipid-coated PEI/DNA polyplexes with improved efficiency and reduced cytotoxicity for gene delivery into mesenchymal stem cells. Int J Nanomedicine, 2012, 7: 4637-4648.
18. 劉儻, 張湘生, 張慶, 等. 骨髓間充質干細胞成骨分化中血管內皮生長因子和骨形態發生蛋白2的表達. 中國組織工程研究, 2012, 16(36): 6651-6657.
19. 張靜, 詹琪, 黃艷. 磁場對骨髓間充質干細胞分化的影響. 中國組織工程研究, 2015, 19(23): 3744-3749.
20. Jsiswal RK, Jaiswal N, Bruder SP, et al. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem, 2000, 275(13): 9645-9652.
21. Dunbar CE, High KA, Joung JK, et al. Gene therapy comes of age. Science, 2018, 359(6372): 175.
22. Wolski H, Bogacz A, Bartkowiak-Wieczorek J, et al. Polymorphism of bone morphogenetic protein (BMP2) and osteoporosis etiology. Ginekol Pol, 2015, 86(3): 203-209.
23. Grgurevic L, Macek B, Mercep M, et al. Bone morphogenetic protein (BMP) 1-3 enhances bone repair. Biochem Biophys Res Commun, 2011, 408(1): 25-31.
24. Yazici C, Takahata M, Reynolds DG, et al. Self-complementary AAV2.5-BMP2-coated femoral allografts mediated superior bone healing versus live autografts in mice with equivalent biomechan-ics to unfractured femur. Mol Ther, 2011, 19(8): 1416-1425.
25. Song K, Huang M, Shi Q, et al. Cultivation and identification of rat bone marrow-derived mesenchymal stem cells. Mol Med Rep, 2014, 10(2): 755-760.
26. 李雪峰, 劉亮, 王東. 大鼠骨髓間充質干細胞體外向成骨細胞分化培養的生物學特性. 中國組織工程研究與臨床康復, 2011, 15(23): 4185-4188.
27. Kotobuki N, Ioku K, Kawagoe D, et al. Observation of osteogenic differentiation cascade of living mesenchymal stem cells on transparent hydroxyapatite ceramics. Biomaterials, 2005, 26(7): 779-785.

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Osteogenic differentiation of bone marrow mesenchymal stem cells induced by gene-loaded lipopolysaccharide-amine nanopolymersomes

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