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Gene delivery of osteoinductive signals to a human fetal osteoblast cell line induces cell death in a dose-dependent manner

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Abstract

Gene delivery provides a powerful tool for regulating tissue regeneration by activating or inhibiting specific genes associated with targeted signaling pathways. Up-regulating bone morphogenetic protein-2 (BMP-2) or silencing GNAS and Noggin gene expression in stem cells has been shown to enhance osteogenic differentiation and bone tissue formation. However, few studies have examined how such gene delivery would influence other differentiated cell types residing in the bone. In this study, we examined the effects of DNA delivery of BMP-2 and siRNA delivery of GNAS or Noggin on a widely used human fetal osteoblast cell line (hFOB1.19) using biomaterials-mediated gene delivery. Our results showed that both GNAS and Noggin siRNA delivery increased cell death in hFOB1.19 in a dose-dependent manner. In particular, groups treated with the highest doses of BMP-2, siGNAS or siNoggin showed a more than 50 % decline in cell proliferation and a 90 % decline in cell viability compared to untransfected and sham DNA/siRNA-transfected controls. TUNEL staining showed that BMP-2, siGNAS or siNoggin induced cell apoptosis in hFOBs. In contrast, cells transfected using sham DNA or siRNA showed no noticeable cell death or apoptosis. These results elucidate the nuanced responses of progenitor and immortalized cell populations to the delivery of exogenous osteoinductive genes. In particular, they highlight the differences between immortalized and primary cell lines and underscore the importance of targeted gene delivery mechanisms in the regeneration of injured bone tissue.

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Abbreviations

hFOBs:

Human fetal osteoblast cell line 1.19

BMP-2:

Bone morphogenetic protein-2

GNAS:

Guanine nucleotide binding protein alpha stimulating activity polypeptide

ELISA:

Enzyme-linked immunosorbent assay

qPCR:

Quantitative Real-Time Polymerase Chain Reaction

References

  1. Stein GS, Lian JB, Stein JL, Van Wijnen AJ, Montecino M. Transcriptional control of osteoblast growth and differentiation. Physiol Rev. 1996;76:593–629.

    CAS  PubMed  Google Scholar 

  2. Clines GA. Prospects for osteoprogenitor stem cells in fracture repair and osteoporosis. Curr Opin Organ Transplant. 2010;15(1):73–8.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Subramaniam M, Jalal SM, Rickard DJ, Harris SA, Bolander ME, Spelsberg TC. Further characterization of human fetal osteoblastic hFOB 1.19 and hFOB/ER alpha cells: bone formation in vivo and karyotype analysis using multicolor fluorescent in situ hybridization. J Cell Biochem. 2002;87:9–15.

    Article  CAS  PubMed  Google Scholar 

  4. Gautschi OP, Cadosch D, Zellweger R, Joesbury KA, Filgueira L. Apoptosis induction and reduced proliferation in human osteoblasts by rhBMP-2, -4 and -7. J Musculoskelet Neuronal Interact. 2009;9:53–60.

    CAS  PubMed  Google Scholar 

  5. Strulovici Y, Leopold PL, O'Connor TP, Pergolizzi RG, Crystal RG. Human embryonic stem cells and gene therapy. Mol Ther : J Am Soc Gene Ther. 2007;15:850–66.

    CAS  Google Scholar 

  6. Jabbarzadeh E, Starnes T, Khan YM, et al. Induction of angiogenesis in tissue-engineered scaffolds designed for bone repair: a combined gene therapy–cell transplantation approach. Proc Natl Acad Sci U S A. 2008;105:11099–104.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gardlik R, Palffy R, Hodosy J, Lukacs J, Turna J, Celec P. Vectors and delivery systems in gene therapy. Med Sci Monit. 2005;11:RA110–21.

    CAS  PubMed  Google Scholar 

  8. Urist MR. Bone: formation by autoinduction. Science. 1965;150:893–9.

    Article  CAS  PubMed  Google Scholar 

  9. Wan DC, Pomerantz JH, Brunet LJ, et al. Noggin suppression enhances in vitro osteogenesis and accelerates in vivo bone formation. J Biol Chem. 2007;282:26450–9.

    Article  CAS  PubMed  Google Scholar 

  10. Zhao Y, Ding S. A high-throughput siRNA library screen identifies osteogenic suppressors in human mesenchymal stem cells. Proc Natl Acad Sci U S A. 2007;104:9673–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mikami Y, Asano M, Honda MJ, Takagi M. Bone morphogenetic protein 2 and dexamethasone synergistically increase alkaline phosphatase levels through JAK/STAT signaling in C3H10T1/2 cells. J Cell Physiol. 2010;223(1):123–33.

    CAS  PubMed  Google Scholar 

  12. Lee SJ, Kang SW, Do HJ, Han I, Shin DA, Kim JH, et al. Enhancement of bone regeneration by gene delivery of BMP2/Runx2 bicistronic vector into adipose-derived stromal cells. Biomaterials. 2010;31(21):5652–9.

    Google Scholar 

  13. Ramasubramanian A, Shiigi S, Lee GK, Yang F. Non-viral delivery of inductive and suppressive genes to adipose-derived stem cells for osteogenic differentiation. Pharm Res. 2011;28(6):1328–37.

    Article  CAS  PubMed  Google Scholar 

  14. Dragoo JL, Choi JY, Lieberman JR, et al. Bone induction by BMP-2 transduced stem cells derived from human fat. J Orthop res : Publ Orthop Res Soc. 2003;21:622–9.

    Article  CAS  Google Scholar 

  15. Jeon O, Rhie JW, Kwon IK, Kim JH, Kim BS, Lee SH. In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically. Tissue Eng Part A. 2008;14:1285–94.

    Article  CAS  PubMed  Google Scholar 

  16. Lutolf MP, Gilbert PM, Blau HM. Designing materials to direct stem-cell fate. Nature. 2009;462:433–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jilka RL, Weinstein RS, Bellido T, Parfitt AM, Manolagas SC. Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Mineral res : J Am Soc Bone Mineral Res. 1998;13:793–802.

    Article  CAS  Google Scholar 

  18. Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest. 1998;102:274–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hay E, Lemonnier J, Fromigue O, Marie PJ. Bone morphogenetic protein-2 promotes osteoblast apoptosis through a Smad-independent, protein kinase C-dependent signaling pathway. J Biol Chem. 2001;276:29028–36.

    Article  CAS  PubMed  Google Scholar 

  20. Schwartz LM, Smith SW, Jones ME, Osborne BA. Do all programmed cell deaths occur via apoptosis? PNAS. 1993;90:980–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Weinstein RS, Nicholas RW, Manolagas SC. Apoptosis of osteocytes in glucocorticoid-induced osteonecrosis of the hip. J Clin Endocrinol Metab. 2000;85:2907–12.

    CAS  PubMed  Google Scholar 

  22. Almonte-Becerril M, Navarro-Garcia F, Gonzalez-Robles A, Vega-Lopez MA, Lavalle C, Kouri JB. Cell death of chondrocytes is a combination between apoptosis and autophagy during the pathogenesis of osteoarthritis within an experimental model. Apoptosis. 2010;15(5):631–8.

    Article  CAS  PubMed  Google Scholar 

  23. Youm YS, Lee SY, Lee SH. Apoptosis in the osteonecrosis of the femoral head. Clin Orthop Surg. 2010;2(4):250–5.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992;119:493–501.

    Article  CAS  PubMed  Google Scholar 

  25. Hyzy SL, Olivares-Navarrete R, Schwartz Z, Boyan BD. BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner. J Cell Biochem. 2012;113(10):3236–45.

    Article  CAS  PubMed  Google Scholar 

  26. Cheema SK, Chen E, Shea LD, Mathur AB. Regulation and guidance of cell behavior for tissue regeneration via the siRNA mechanism. Wound Repair Regen. 2007;15(3):286–95.

    Article  PubMed  Google Scholar 

  27. Evans CH, Gouze JN, Gouze E, Robbins PD, Ghivizzani SC. Osteoarthritis gene therapy. Gene Ther. 2004;11:379–89.

    Article  CAS  PubMed  Google Scholar 

  28. Zaidi M. Skeletal remodeling in health and disease. Nat Med. 2007;13:791–801.

    Article  CAS  PubMed  Google Scholar 

  29. Wang XX, Allen Jr RJ, Tutela JP, Sailon A, Allori AC, Davidson EH, et al. Progenitor cell mobilization enhances bone healing by means of improved neovascularization and osteogenesis. Plast Reconstr Surg. 2011;128(2):395–405.

  30. Hughes SM, Moussavi-Harami F, Sauter SL, Davidson BL. Viral-mediated gene transfer to mouse primary neural progenitor cells. Mol Ther. 2002;5:16–24.

    Article  CAS  PubMed  Google Scholar 

  31. Wang X, Cui F, Madhu V, Dighe AS, Balian G, Cui Q. Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation. Growth Factors. 2011;29(1):36–48.

    Article  PubMed  Google Scholar 

  32. Zachos T, Diggs A, Weisbrode S, Bartlett J, Bertone A. Mesenchymal stem cell-mediated gene delivery of bone morphogenetic protein-2 in an articular fracture model. Mol Ther. 2007;15:1543–50.

    Article  CAS  PubMed  Google Scholar 

  33. Long F. Building strong bones: molecular regulation of the osteoblast lineage. Nat Rev Mol Cell Biol. 2011;13(1):27–38.

    Article  PubMed  Google Scholar 

  34. Harris SA, Enger RJ, Riggs BL, Spelsberg TC. Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. J Bone Mineral res : J Am Soc Bone Mineral Res. 1995;10:178–86.

    Article  CAS  Google Scholar 

  35. Yang F, Williams CG, Wang DA, Lee H, Manson PN, Elisseeff J. The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells. Biomaterials. 2005;26:5991–8.

    Article  CAS  PubMed  Google Scholar 

  36. Cho SW, Goldberg M, Son SM, et al. Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells. Adv Funct Mater. 2009;19:3112–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yang F, Green JJ, Dinio T, et al. Gene delivery to human adult and embryonic cell-derived stem cells using biodegradable nanoparticulate polymeric vectors. Gene Ther. 2009;16:533–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Yang F, Cho SW, Son SM, Bogatyrev SR, Singh D, Green JJ, et al. Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles. Proc Natl Acad Sci USA. 2010;107(8):3317–22.

    Google Scholar 

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Acknowledgments

A.R. and S.J. would like to thank the Stanford Undergraduate Advising and Research Office for funding. S.J. would also like to thank the Stanford School of Engineering for funding.

Conflict of interest

The authors have no competing financial interests.

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Authors and Affiliations

  1. Program in Biomechanical Engineering, Stanford University, Stanford, CA, 94305, USA

    Anusuya Ramasubramanian

  2. Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA

    Shaheen Jeeawoody & Fan Yang

  3. Department of Orthopaedic Surgery, Stanford University School of Medicine, 300 Pasteur Dr., Edwards R105, Stanford, CA, 94305-5301, USA

    Fan Yang

Authors
  1. Anusuya Ramasubramanian
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  2. Shaheen Jeeawoody
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  3. Fan Yang
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Corresponding author

Correspondence to Fan Yang.

Additional information

Anusuya Ramasubramanian and Shaheen Jeeawoody contributed equally to this work.

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Ramasubramanian, A., Jeeawoody, S. & Yang, F. Gene delivery of osteoinductive signals to a human fetal osteoblast cell line induces cell death in a dose-dependent manner. Drug Deliv. and Transl. Res. 5, 160–167 (2015). https://doi.org/10.1007/s13346-013-0163-x

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  • DOI: https://doi.org/10.1007/s13346-013-0163-x

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