Skip to main content

Advertisement

SpringerLink
Log in

BMP-2 Gene Modified Canine bMSCs Promote Ectopic Bone Formation Mediated by a Nonviral PEI Derivative

  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

The study was to explore the effects of BMP-2 gene modified canine bone marrow stromal cells (bMSCs) mediated by a nonviral PEI derivative (GenEscort™ II) in promoting bone formation in vitro and in vivo. Canine bMSCs were cultured and transfected with plasmids containing bone morphogenetic protein-2 gene (pBMP-2) or enhanced green fluorescent protein gene (pEGFP). Gene transfection conditions were initially optimized by varying GenEscort™ II/plasmid ratios. Osteogenic differentiation of gene modified bMSCs was investigated via alkaline phosphatase (ALP) activity analysis and real-time quantitative PCR (RT-qPCR) analysis in vitro. The bone formation ability of pBMP-2 transfected bMSCs combined with apatite-coated silk scaffolds (mSS) was explored and compared with pEGFP transfected bMSCs/mSS or untreated bMSCs/mSS at 8, 12 weeks after operation. Results showed that gene transfection efficiency reached up to 36.67 ± 4.12% as demonstrated by EGFP expression. ALP staining and activity assay were stronger with pBMP-2 gene transfection, and the mRNA expression of BMP-2, bone sialoprotein (BSP), Runt-related transcription factor 2 (Runx-2), and osteopontin (OPN) up-regulated in bMSCs 3, 6, 9 days in pBMP-2 group. Besides, the tissue-engineered bone complex with pBMP-2 modified bMSCs achieved significantly increased de novo bone formation compared with control groups (p < 0.01). We conclude that pBMP-2 transfection mediated by GenEscort™ II could enhance the osteogenic differentiation of canine bMSCs and promote the ectopic new bone formation in nude mice. GenEscort™ II mediated pBMP-2 gene transfer appears to be a safe and effective nonviral method for gene enhanced bone tissue engineering.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  1. Abdallah, B., A. Hassan, C. Benoist, D. Goula, J. P. Behr, and B. A. Demeneix. A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum. Gene Ther. 7:1947–1954, 1996.

    Article  PubMed  CAS  Google Scholar 

  2. Ahn, H. H., M. S. Lee, M. H. Cho, Y. N. Shin, J. H. Lee, and K. S. Kim. DNA/PEI nano-particles for gene delivery of rat bone marrow stem cells. Colloids Surf. A Physicochem. Eng. Asp. 313:116–120, 2008.

    Article  Google Scholar 

  3. Baltzer, A. W., and J. R. Lieberman. Regional gene therapy to enhance bone repair. Gene Ther. 11:344–350, 2004.

    Article  PubMed  CAS  Google Scholar 

  4. Betz, V. M., O. B. Betz, M. B. Harris, M. S. Vrahas, and C. H. Evans. Bone tissue engineering and repair by gene therapy. Front. Biosci. 13:833–841, 2008.

    Article  PubMed  CAS  Google Scholar 

  5. Boussif, O., F. Lezoualc’h, M. A. Zanta, M. Mergny, D. Scherman, B. Demeneix, and J. P. Behr. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA 92:7297–7301, 1995.

    Article  PubMed  CAS  Google Scholar 

  6. Boussif, O., M. A. Zanta, and J. P. Behr. Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther. 3:1074–1080, 1996.

    PubMed  CAS  Google Scholar 

  7. Cancedda, R., P. Giannoni, and M. Mastrogiacomo. A tissue engineering approach to bone repair in large animal models and in clinical practice. Biomaterials 28:4240–4250, 2007.

    Article  PubMed  CAS  Google Scholar 

  8. Farrell, L. L., J. Pepin, C. Kucharski, X. Lin, Z. Xu, and H. Uludag. A comparison of the effectiveness of cationic polymers poly-l-lysine (PLL) and polyethylenimine (PEI) for non-viral delivery of plasmid DNA to bone marrow stromal cells (BMSC). Eur. J. Pharm. Biopharm. 65:388–397, 2007.

    Article  PubMed  CAS  Google Scholar 

  9. Fischer, W., M. Calderón, and R. Haag. Hyperbranched polyamines for transfection. Top. Curr. Chem. 296:95–129, 2010.

    Article  PubMed  CAS  Google Scholar 

  10. Glover, D. J., H. J. Lipps, and D. A. Jans. Towards safe, non-viral therapeutic gene expression in humans. Nat. Rev. Genet. 6:299–310, 2005.

    Article  PubMed  CAS  Google Scholar 

  11. Hsu, W. K., O. Sugiyama, S. H. Park, A. Conduah, B. T. Feeley, N. Q. Liu, and L. Krenek. Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats. Bone 40:931–938, 2007.

    Article  PubMed  CAS  Google Scholar 

  12. Huh, S. H., H. J. Do, H. Y. Lim, D. K. Kim, S. J. Choi, H. Song, N. H. Kim. Optimization of 25 kDa linear polyethylenimine for efficient gene delivery. Biologicals 35:165–171, 2007.

    Article  PubMed  CAS  Google Scholar 

  13. Jere, D., H. L. Jiang, R. Arote, Y. K. Kim, Y. J. Choi, M. H. Cho, and T. Akaike. Degradable polyethylenimines as DNA and small interfering RNA carriers. Expert Opin. Drug Deliv. 6:827–834, 2009.

    Article  PubMed  CAS  Google Scholar 

  14. Jiang, X. Q., J. G. Chen, S. Gittens, C. J. Chen, X. L. Zhang, and Z. Y. Zhang. The ectopic study of tissue-engineered bone with hBMP-4 gene modified bone marrow stromal cells in rabbits. Chin. Med. J. 118:281–288, 2005.

    PubMed  CAS  Google Scholar 

  15. Jiang, X. Q., X. J. Sun, H. C. Lai, J. Zhao, S. Y. Wang, and Z. Y. Zhang. Maxillary sinus floor elevation using a tissue-engineered bone complex with beta-TCP and BMP-2 gene-modified bMSCs in rabbits. Clin. Oral Implants Res. 20:1333–1340, 2009.

    Article  PubMed  Google Scholar 

  16. Jiang, X., J. Zhao, S. Y. Wang, X. J. Sun, X. L. Zhang, J. Chen, and D. L. Kaplan. Mandibular repair in rats with premineralized silk scaffolds and BMP-2-modified bMSCs. Biomaterials 30:4522–4532, 2009.

    Article  PubMed  CAS  Google Scholar 

  17. John, G. H., and R. S. Thomas. Composite materials based on silk proteins. Prog. Polym. Sci. 35:1093–1115, 2010.

    Article  Google Scholar 

  18. Kawai, M., K. Bessho, S. Kaihara, J. Sonobe, K. Oda, T. Iizuka, and H. Maruyama. Ectopic bone formation by human bone morphogenetic protein-2 gene transfer to skeletal muscle using transcutaneous electroporation. Hum. Gene Ther. 14:1547–1556, 2003.

    Article  PubMed  CAS  Google Scholar 

  19. Kim, H. J., U. J. Kim, H. S. Kim, C. Li, M. Wada, G. G. Leisk, and D. L. Kaplan. Bone tissue engineering with premineralized silk scaffolds. Bone 42:1226–1234, 2008.

    Article  PubMed  CAS  Google Scholar 

  20. Kim, S. W., T. Ogawa, Y. Tabata, and I. Nishimura. Efficacy and cytotoxicity of cationic-agent-mediated nonviral gene transfer into osteoblasts. J. Biomed. Mater. Res. A 71A:308–315, 2004.

    Article  CAS  Google Scholar 

  21. Lu, J. X., A. Gallur, B. Flautre, K. Anselme, M. Descamps, B. Thierry, and P. Hardouin. Comparative study of tissue reactions to calcium phosphate ceramics among cancellous, cortical, and medullar bone sites in rabbits. J. Biomed. Mater. Res. 42:357–367, 1998.

    Article  PubMed  CAS  Google Scholar 

  22. Merdan, T., K. Kunath, D. Fischer, J. Kopecek, and T. Kissel. Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments. Pharm. Res. 19:140–146, 2002.

    Article  PubMed  CAS  Google Scholar 

  23. Muschler, G. F., V. P. Raut, T. E. Patterson, J. C. Wenke, and J. O. Hollinger. The design and use of animal models for translational research in bone tissue engineering and regenerative Medicine. Tissue Eng. Part B Rev. 16:123–145, 2010.

    Article  PubMed  Google Scholar 

  24. O’Loughlin, P. F., S. Morr, L. Bogunovic, A. D. Kim, B. Park, and J. M. Lane. Selection and development of preclinical models in fracture-healing research. J. Bone Joint Surg. Am. 90(Suppl 1):79–84, 2008.

    Article  PubMed  Google Scholar 

  25. Park, T. G., J. H. Jeong, and S. W. Kim. Current status of polymeric gene delivery systems. Adv. Drug Deliv. Rev. 58:467–486, 2006.

    Article  PubMed  CAS  Google Scholar 

  26. Park, J., J. Ries, K. Gelse, F. Kloss, K. von der Mark, J. Wiltfang, and F. W. Neukam. Bone regeneration in critical size defects by cell-mediated BMP-2 gene transfer: a comparison of adenoviral vectors and liposomes. Gene Ther. 10:1089–1098, 2003.

    Article  PubMed  CAS  Google Scholar 

  27. Peng, L., M. Liu, Y. N. Xue, S. W. Huang, R. X. Zhuo. Transfection and intracellular trafficking characteristics for poly(amidoamine)s with pendant primary amine in the delivery of plasmid DNA to bone marrow stromal cells. Biomaterials 30:5825–5833, 2009.

    Article  PubMed  CAS  Google Scholar 

  28. Sun, X. J., L. G. Xia, L. L. Chou, W. Zhong, X. L. Zhang, S. Y. Wang. Maxillary sinus floor elevation using a tissue engineered bone complex with BMP-2 gene modified bMSCs and a novel porous ceramic scaffold in rabbits. Arch. Oral Biol. 55:195–202, 2010.

    Article  PubMed  CAS  Google Scholar 

  29. Sun, X. J., Z. Y. Zhang, S. Y. Wang, S. A. Gittens, X. Q. Jiang, and L. L. Chou. Maxillary sinus floor elevation using a tissue-engineered bone complex with OsteoBone (TM) and bMSCs in rabbits. Clin. Oral Implants Res. 19:804–813, 2008.

    Article  PubMed  Google Scholar 

  30. Yamano, S., J. Dai, and A. M. Moursi. Comparison of transfection efficiency of nonviral gene transfer reagents. Mol. Biotechnol. 46:287–300, 2010.

    Article  PubMed  CAS  Google Scholar 

  31. Yang, F., C. G. Williams, D. A. Wang, H. Lee, P. N. Manson, and J. Elisseeff. The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells. Biomaterials 26:5991–5998, 2005.

    Article  PubMed  CAS  Google Scholar 

  32. Yu, W. Q., X. Q. Jiang, F. Q. Zhang, and L. Xu. The effect of anatase TiO2 nanotube layers on MC3T3-E1 preosteoblast adhesion, proliferation, and differentiation. J. Biomed. Mater. Res. A 94:1012–1022, 2010.

    PubMed  Google Scholar 

  33. Zhao, J., Z. Y. Zhang, S. Y. Wang, X. J. Sun, X. L. Zhang, J. Chen, and D. L. Kaplan. Apatite-coated silk fibroin scaffolds to healing mandibular border defects in canines. Bone 45:517–527, 2009.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Carmen Preda for fabricating the silk scaffolds. This work was supported by National Natural Science Foundation of China 30772431, 30973342; Science and Technology Commission of Shanghai Municipality 08DZ2271100, 0852nm02900, 0952nm04000, 10430710900, 10dz2211600, 1052nm04300, 10JC1408600, 1052nm04300, and 10JC1408600.

Author information

Authors and Affiliations

  1. Department of Prosthodontics, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, 639 Zhizaoju Road, Shanghai, 200011, People’s Republic of China

    Kaige Lü, Deliang Zeng, Yilin Zhang, Ling Xu, Xinquan Jiang & Fuqiang Zhang

  2. Oral Bioengineering Lab, Shanghai Research Institute of Stomatology, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhizaoju Road, Shanghai, 200011, People’s Republic of China

    Deliang Zeng, Lunguo Xia & Xinquan Jiang

  3. Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA, 02155, USA

    David L. Kaplan

Authors
  1. Kaige Lü
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deliang Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yilin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lunguo Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ling Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David L. Kaplan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xinquan Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fuqiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Xinquan Jiang or Fuqiang Zhang.

Additional information

Associate Editor Mona Kamal Marei oversaw the review of this article.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lü, K., Zeng, D., Zhang, Y. et al. BMP-2 Gene Modified Canine bMSCs Promote Ectopic Bone Formation Mediated by a Nonviral PEI Derivative. Ann Biomed Eng 39, 1829–1839 (2011). https://doi.org/10.1007/s10439-011-0276-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-011-0276-7

Keywords

Search

Navigation