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β-Tricalcium phosphate 3D scaffold promote alone osteogenic differentiation of human adipose stem cells: in vitro study

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Abstract

Human adipose tissues surgically resected from the subcutaneous abdominal region were enzymatically processed to obtain Human Adipose Stem cells (fibroblast-like adipose tissue-derived stromal cells—ADSC-FL) that were immunophenotypically characterized using a panel of mesenchymal markers by flow cytometry. The formation of new hydroxyapatite crystals in culture dishes, by differentiating cells, further demonstrate the osteogenic potential of purified cells. The aim of this study was to evaluate the osteogenic differentiation potential of ADSC-FL seeded onto a porous β-tricalcium phosphate (β-TCP) matrix. ADSC-FL was cultured on the β-TCP matrix in medium with or without osteogenic differentiation additives. Time-dependent cell differentiation was monitored using osteogenic markers such as alkaline phosphatase (activity assay), osteocalcin and ostopontin (ELISA method) expression. Our results reveal that β-TCP triggers the differentiation of ADSC-FL toward an osteoblastic phenotype irrespective of whether the cells are grown in a proliferative or a differentiative medium. Hence, a β-TCP matrix is sufficient to promote osteoblastic differentiation of ADSC-FL. However, in proliferative medium, alkaline phosphatase activity was detected at lower level respect to differentiative medium and osteocalcin and osteopontin showed an expression delay in cells cultured in proliferative medium respect to differentiative one. Moreover, we observed an increase in FAK phosphorylation at level of tyrosine residue in position 397 (Western-blot) that indicates a good cell adhesion to β-TCP scaffold. In conclusion, our paper demonstrates that a three-dimensional β-TCP scaffold in vitro triggers on its own the differentiation of ADSC-FL toward an osteoblastic phenotype without the need to use differentiative media.

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References

  1. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211–8.

    Article  CAS  PubMed  Google Scholar 

  2. Kern S, Eichler H, Stoeve J. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006;24:1294–301.

    Article  CAS  PubMed  Google Scholar 

  3. Gronthos S, Franklin DM, Leddy HA, Robey PG, Storms RW, Gimble JM. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol. 2001;189(1):54–63.

    Article  CAS  PubMed  Google Scholar 

  4. Tocci A, Forte L. Mesenchymal stem cell: use and perspectives. Hematol J. 2003;4:92–6.

    Article  PubMed  Google Scholar 

  5. Schäffler A, Büchler C. Concise review: adipose tissue-derived stromal cells-basic and clinical implications for novel cell-based therapies. Stem Cells. 2007;25:818–27.

    Article  PubMed  Google Scholar 

  6. El-Ghannam A. Bone reconstruction: from bioceramics to tissue engineering. Expert Rev Med Devices. 2005;2:87–101.

    Article  PubMed  Google Scholar 

  7. Müller P, Bulnheim U, Diener A, Lüthen F, Teller M, Klinkenberg ED, et al. Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells. J Cell Mol Med. 2008;12(1):281–91.

    Article  PubMed  Google Scholar 

  8. Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992;70(3):389–99.

    Article  CAS  PubMed  Google Scholar 

  9. Parsons JT. Integrin-mediated signaling: regulation by protein tyrosine kinases and small GTP-binding proteins. Curr Opin Cell Biol. 1996;11:146–52.

    Article  Google Scholar 

  10. Schwartz M, Schaller M, Ginsberg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol. 1995;11:549–99.

    Article  CAS  PubMed  Google Scholar 

  11. Hynes RO. Cell adhesion: old and new questions. Trends Cell Biol. 1999;9(12):M33–7.

    Article  CAS  PubMed  Google Scholar 

  12. Schlaepfer DD, Jones KC, Hunter T. Multiple Grb2-mediated integrinstimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol Cell Biol. 1998;18:2571–85.

    CAS  PubMed  Google Scholar 

  13. Short SM, Talbott GA, Juliano RL. Integrin-mediated signalling events in human endothelial cells. Mol Biol Cell. 1998;9:1969–80.

    CAS  PubMed  Google Scholar 

  14. Hanks SK, Polte TR. Signalling through focal adhesion kinase. Bioessays. 1997;19:137–45.

    Article  CAS  PubMed  Google Scholar 

  15. Zhao J-H, Reiske H, Guan J-L. Regulation of the cell cycle by focal adhesion kinase. J Cell Biol. 1998;143:1997–2008.

    Article  CAS  PubMed  Google Scholar 

  16. Thomas Parsons J. Focal adhesion kinase: the first ten years. J Cell Sci. 2003;116:1409–16.

    Article  PubMed  Google Scholar 

  17. Rosso F, Marino G, Muscariello L, Cafiero G, Favia P, D’Aloia E, et al. Adhesion, proliferation of fibroblasts on RF plasma-deposited nanostructured fluorocarbon coatings: evidence of FAK activation. J Cell Physiol. 2006;207(3):636–43.

    Article  CAS  PubMed  Google Scholar 

  18. Birk RZ, Abramovitch-Gottlib L, Margalit I, Aviv M, Forti E, Geresh S, et al. Conversion of adipogenic to osteogenic phenotype using crystalline porous biomatrices of marine origin. Tissue Eng. 2006;12:21–31.

    Article  CAS  PubMed  Google Scholar 

  19. Hicok KC, Du Laney TV, Zhou YS, Halvorsen YD, Hitt DC, Cooper LF, et al. Human adipose-derived adult stem cells produce osteoid in vivo. Tissue Eng. 2004;10(3–4):371–80.

    Article  CAS  PubMed  Google Scholar 

  20. Justesen J, Pedersen SB, Stenderup K, Kassem M. Subcutaneous adipocytes can differentiate into bone-forming cells in vitro, in vivo. Tissue Eng. 2004;10(3–4):381–91.

    Article  CAS  PubMed  Google Scholar 

  21. Hattori H, Masuoka K, Sato M, Ishihara M, Asazuma T, Takase B, et al. Bone formation using human adipose tissue-derived stromal cells and a biodegradable scaffold. J Biomed Mater Res B: Appl Biomater. 2006;76(1):230–9.

    Google Scholar 

  22. Liu Q, Cen L, Yin S, Chen L, Liu G, Chang J, et al. A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and beta-TCP ceramics. Biomaterials. 2008;29(36):4792–9. Epub 2008 Sep 26.

    Article  CAS  PubMed  Google Scholar 

  23. Leong DT, Nah WK, Gupta A, Hutmacher DW, Woodruff MA. The osteogenic differentiation of adipose tissue-derived precursor cells in a 3D scaffold/matrix environment. Curr Drug Discov Technol. 2008;5(4):319–27.

    Article  CAS  PubMed  Google Scholar 

  24. McCullen SD, Zhu Y, Bernacki SH, Narayan RJ, Pourdeyhimi B, Gorga RE, Loboa EG. Electrospun composite poly(L-lactic acid)/tricalcium phosphate scaffolds induce proliferation and osteogenic differentiation of human adipose-derived stem cells. Biomed Mater. 2009;4(3):35002. Epub 2009 Apr 24.

    Google Scholar 

  25. Hao W, Hu YY, Wei YY, Pang L, Lv R, Bai JP, et al. Collagen I gel can facilitate homogenous bone formation of adipose-derived stem cells in PLGA-beta-TCP scaffold. Cells Tissues Organs. 2008;187(2):89–102. Epub 2007 Oct 15.

    Article  CAS  PubMed  Google Scholar 

  26. Mitchell J, Micintosh k, Zavonic S, Garrett S, Floyd E, Loster A, et al. Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell–associated markers. Stem Cells. 2006;24:376–85.

    Article  PubMed  Google Scholar 

  27. Salazar EP, Hunger-Glaser I, Rozengurt E. Dissociation of focal adhesion kinase and paxillin tyrosine phosphorylation induced by Bombesin and Lysophosphatidic acid from epidermal growth factor receptor transactivation in Swiss 3T3 Cells. J Cell Physiol. 2003;194:314–24.

    Article  CAS  PubMed  Google Scholar 

  28. Walsh MF, Thamilselvana V, Grotelueschena R, Farhanab L, Bassona MD. Absence of adhesion triggers differential FAK and SAPKp38 signals in SW620 human colon cancer cells that may inhibit adhesiveness and lead to cell death. Cell Physiol Biochem. 2003;13:135–46.

    Article  CAS  PubMed  Google Scholar 

  29. Kim H-M, Kishimoto K, Miyaji F, Kokubo T, et al. Composition and structure of apatite formed on organic polymer in simulated body fluid with a high content of carbonate ion. J Mater Sci: Mater Med. 2000;11:421–6.

    Article  CAS  Google Scholar 

  30. Legeros RZ, Legeros JP. In: Hench LL, Wilson J, editors. An introduction to bioceramics. Singapore: World Scientific; 1993. p. 139.

    Google Scholar 

  31. Betre H, Ong SR, Guilak F, Chilkoti A, Fermor B, Setton LA. Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide. Biomaterials. 2006;27:91–9.

    Article  CAS  PubMed  Google Scholar 

  32. Halvorsen YD, Franklin D, Bond AL, Hitt DC, Auchter C, Boskey AL, et al. Extracellular matrix mineralization, osteoblast gene expression by human adipose tissue-derived stromal cells. Tissue Eng. 2001;7(6):729–41.

    Article  CAS  PubMed  Google Scholar 

  33. Kawaguchi J, Mee PJ, Smith AG. Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone. 2005;36(5):758–69. Epub 2005 Mar 24.

    Article  CAS  PubMed  Google Scholar 

  34. Curran JM, Chen R, Hunt JA. The guidance of human mesenchymal stem cell differentiation in vitro by controlled modifications to the cell substrate. Biomaterials. 2006;27:4783–93.

    Article  CAS  PubMed  Google Scholar 

  35. Keselowsky BG, Collard DM, Garcia AJ. Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion. J Biomed Mater Res A. 2003;66:247–59.

    Article  PubMed  Google Scholar 

  36. Wollenweber M, Domaschke H, Hanke T, Boxberger S, Schmack G, Gliesche K. Scharnweber et al. Mimicked bioartificial matrix containing chondroitin sulphate on a textile scaffold of poly(3-hydroxybutyrate) alters the differentiation of adult human mesenchymal stem cells. Tissue Eng. 2006;12:345–59.

    Article  CAS  PubMed  Google Scholar 

  37. Datta N, Pham QP, Sharma U, Sikavitsas VI, Jansen JA, Mikos AG. In vitro generated extracellular matrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation. Proc Natl Acad Sci USA. 2006;103:2488–93.

    Article  CAS  PubMed  ADS  Google Scholar 

  38. Muscariello L, Rosso F, Marino G, Cafiero G, Barbarisi A. A critical overview of ESEM applications in the biological field. J Cell Physiol. 2005;205:328–34.

    Article  CAS  PubMed  Google Scholar 

  39. Baker FS, et al. Secondary electron imaging at gas pressures in excess of 1 KPa. Appl Phys Lett. 2007;91:053122/1–3.

    ADS  Google Scholar 

  40. Iliescu M, et al. Ultrastructure of hybrid chitosan-glycerol phosphate blood clots by environmental scanning electron microscopy. Microsc Res Tech. 2008;71:236–47.

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Gerardo Marino.

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Marino, G., Rosso, F., Cafiero, G. et al. β-Tricalcium phosphate 3D scaffold promote alone osteogenic differentiation of human adipose stem cells: in vitro study. J Mater Sci: Mater Med 21, 353–363 (2010). https://doi.org/10.1007/s10856-009-3840-z

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  • DOI: https://doi.org/10.1007/s10856-009-3840-z

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