Skip to main content

Tailoring RGD local surface density at the nanoscale toward adult stem cell chondrogenic commitment


Arginine-glycine-aspartic acid (RGD) dendrimer-based nanopatterns on poly(L-lactic acid) were used as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of adult human mesenchymal stem cells. During chondrogenic commitment, active extracellular matrix (ECM) remodeling takes place, playing an instructive role in the differentiation process. Although three-dimensional environments such as pellet or micromass cultures are commonly used for in vitro chondrogenic differentiation, these cultures are rather limited with respect to their ability to interrogate cells in cell–ECM interactions. In the present study, the nanopatterns of the tunable RGD surface density were obtained as a function of the initial dendrimer concentration. The local RGD surface density was quantified through probability contour plots for the minimum interparticle distance, constructed from the corresponding atomic force microscopy images, and correlated with the cell adhesion and differentiation response. The results revealed that the local RGD surface density at the nanoscale acts as a regulator of chondrogenic commitment, and that intermediate adhesiveness of cells to the substrates favors mesenchymal cell condensation and early chondrogenic differentiation.

This is a preview of subscription content, access via your institution.


  1. [1]

    Jiang, F.; Hörber, H.; Howard, J.; Müller, D. J. Assembly of collagen into microribbons: Effects of pH and electrolytes. J. Struct. Biol. 2004, 148, 268–278.

    Article  Google Scholar 

  2. [2]

    Smith, M. L.; Gourdon, D.; Little, W. C.; Kubow, K. E.; Eguiluz, R. A.; Luna-Morris, S.; Vogel, V. Force-induced unfolding of fibronectin in the extracellular matrix of living cells. PLoS Biol. 2007, 5, e268.

    Article  Google Scholar 

  3. [3]

    Little, W. C.; Smith, M. L.; Ebneter, U.; Vogel, V. Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage. Matrix Biol. 2008, 27, 451–461.

    Article  Google Scholar 

  4. [4]

    Christman, K. L.; Enriquez-Rios, V. D.; Maynard, H. D. Nanopatterning proteins and peptides. Soft Matter 2006, 2, 928–939.

    Article  Google Scholar 

  5. [5]

    Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27, 3044–3063.

    Article  Google Scholar 

  6. [6]

    Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blümmel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. Activation of integrin function by nanopatterned adhesive interfaces. ChemPhysChem 2004, 5, 383–388.

    Article  Google Scholar 

  7. [7]

    Cavalcanti-Adam, E. A.; Micoulet, A.; Blümmel, J.; Auernheimer, J.; Kessler, H.; Spatz, J. P. Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly. Eur. J. Cell. Biol. 2006, 85, 219–224.

    Article  Google Scholar 

  8. [8]

    Arnold, M.; Schwieder, M.; Blümmel, J.; Cavalcanti-Adam, E. A.; López-Garcia, M.; Kessler, H.; Geiger, B.; Spatz, J. P. Cell interactions with hierarchically structured nano-patterned adhesive surfaces. Soft Matter 2009, 5, 72–77.

    Article  Google Scholar 

  9. [9]

    Cavalcanti-Adam, E. A.; Volberg, T.; Micoulet, A.; Kessler, H.; Geiger, B.; Spatz, J. P. Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands. Biophys. J. 2007, 92, 2964–2974.

    Article  Google Scholar 

  10. [10]

    Medda, R.; Helth, A.; Herre, P.; Pohl, D.; Rellinghaus, B.; Perschmann, N.; Neubauer, S.; Keßsler, H.; Oswald, S.; Eckert, J. et al. Investigation of early cell–surface interactions of human mesenchymal stem cells on nanopatterned ß-type titanium-niobium alloy surfaces. Interface Focus 2014, 4, 20130046.

    Google Scholar 

  11. [11]

    Wang, X.; Yan, C.; Ye, K.; He, Y.; Li, Z. H.; Ding, J. D. Effect of RGD nanospacing on differentiation of stem cells. Biomaterials 2013, 34, 2865–2874.

    Article  Google Scholar 

  12. [12]

    Wang, X.; Ye, K.; Li, Z. H.; Yan, C.; Ding, J. D. Adhesion, proliferation, and differentiation of mesenchymal stem cells on RGD nanopatterns of varied nanospacings. Organogenesis 2013, 9, 280–286.

    Article  Google Scholar 

  13. [13]

    Li, Z. H.; Cao, B.; Wang, X.; Ye, K.; Li, S. Y.; Ding, J. D. Effects of RGD nanospacing on chondrogenic differentiation of mesenchymal stem cells. J. Mater. Chem. B 2015, 3, 5197–5209.

    Article  Google Scholar 

  14. [14]

    Wang, X.; Li, S. Y.; Yan, C.; Liu, P.; Ding, J. D. Fabrication of RGD micro/nanopattern and corresponding study of stem cell differentiation. Nano Lett. 2015, 15, 1457–1467.

    Article  Google Scholar 

  15. [15]

    Stephanopoulos, N.; Freeman, R.; North, H. A.; Sur, S.; Jeong, S. J.; Tantakitti, F.; Kessler, J. A.; Stupp, S. I. Bioactive DNA-peptide nanotubes enhance the differentiation of neural stem cells into neurons. Nano Lett. 2015, 15, 603–609.

    Article  Google Scholar 

  16. [16]

    Rolland, O.; Turrin, C. O.; Caminade, A. M.; Majoral, J. P. Dendrimers and nanomedicine: Multivalency in action. New J. Chem. 2009, 33, 1809–1824.

    Article  Google Scholar 

  17. [17]

    Saovapakhiran, A.; D’Emanuele, A.; Attwood, D.; Penny, J. Surface modification of PAMAM dendrimers modulates the mechanism of cellular internalization. Bioconjug. Chem. 2009, 20, 693–701.

    Article  Google Scholar 

  18. [18]

    Albertazzi, L.; Fernandez-Villamarin, M.; Riguera, R.; Fernandez-Megia, E. Peripheral functionalization of dendrimers regulates internalization and intracellular trafficking in living cells. Bioconjug. Chem. 2012, 23, 1059–1068.

    Article  Google Scholar 

  19. [19]

    Mikhail, A. S.; Jones, K. S.; Sheardown, H. Dendrimergrafted cell adhesion peptide-modified PDMS. Biotechnol. Prog. 2008, 24, 938–944.

    Article  Google Scholar 

  20. [20]

    Kino-oka, M.; Kim, J.; Kurisaka, K.; Kim, M. H. Preferential growth of skeletal myoblasts and fibroblasts in co-culture on a dendrimer-immobilized surface. J. Biosci. Bioeng. 2013, 115, 96–99.

    Article  Google Scholar 

  21. [21]

    Lomba, M.; Oriol, L.; Sánchez-Somolinos, C.; Grazú, V.; Moros, M.; Serrano, J. L.; Martínez De la Fuente, J. Cell adhesion on surface patterns generated by the photocrosslinking of hyperbranched polyesters with a trisdiazonium salt. React. Funct. Polym. 2013, 73, 499–507.

    Article  Google Scholar 

  22. [22]

    Kim, M. H.; Kino-oka, M.; Morinaga, Y.; Sawada, Y.; Kawase, M.; Yagi, K.; Taya, M. Morphological regulation and aggregate formation of rabbit chondrocytes on dendrimerimmobilized surfaces with D-glucose display. J. Biosci. Bioeng. 2009, 107, 196–205.

    Article  Google Scholar 

  23. [23]

    Kim, M. H.; Kino-oka, M.; Kawase, M.; Yagi, K.; Taya, M. Synergistic effect of D-glucose and epidermal growth factor display on dynamic behaviors of human epithelial cells. J. Biosci. Bioeng. 2007, 104, 428–431.

    Article  Google Scholar 

  24. [24]

    Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. Cell adhesion and motility depend on nanoscale RGD clustering. J. Cell Sci. 2000, 113, 1677–1686.

    Google Scholar 

  25. [25]

    Lagunas, A.; Castaño, A. G.; Artés, J. M.; Vida, Y.; Collado, D.; Pérez-Inestrosa, E.; Gorostiza, P.; Claros, S.; Andrades, J. A.; Samitier, J. Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycineaspartic acid (RGD) density effects on cell adhesion. Nano Res. 2014, 7, 399–409.

    Article  Google Scholar 

  26. [26]

    Singh, P.; Schwarzbauer, J. E. Fibronectin and stem cell differentiation—Lessons from chondrogenesis. J. Cell Sci. 2012, 125, 3703–3712.

    Article  Google Scholar 

  27. [27]

    Griffin, M. F.; Butler, P. E.; Seifalian, A. M.; Kalaskar, D. M. Control of stem cell fate by engineering their micro and nanoenvironment. World J. Stem Cells 2015, 7, 37–50.

    Article  Google Scholar 

  28. [28]

    Horcas, I.; Fernández, R.; Gómez-Rodríguez, J. M.; Colchero, J.; Gómez-Herrero, J.; Baro, A. M. WSXM: A software for scanning probe microscopy and a tool for nanotechnology. Rev. Sci. Instrum. 2007, 78, 13705–13713.

    Article  Google Scholar 

  29. [29]

    Magalhães, J.; Lebourg, M., Deplaine, H.; Gómez-Ribelles, J. L.; Blanco, F. J. Effect of the physicochemical properties of pure or chitosan-coated poly(L-lactic acid) scaffolds on the chondrogenic differentiation of mesenchymal stem cells from osteoarthritic patients. Tissue Eng. Part A 2015, 21, 716–728.

    Article  Google Scholar 

  30. [30]

    Boas, U.; Heegaard, P. M. H. Dendrimers in drug research. Chem. Soc. Rev. 2004, 33, 43–63.

    Article  Google Scholar 

  31. [31]

    Mager, D. M.; LaPointe, V.; Stevens, M. M. Exploring and exploiting chemistry at the cell surface. Nat. Chem. 2011, 3, 582–589.

    Article  Google Scholar 

  32. [32]

    Zelzer, M.; Majani, R.; Bradley, J. W.; Rose, F. R. A. J.; Davies, M. C.; Alexander, M. R. Investigation of cell–surface interactions using chemical gradients formed from plasma polymers. Biomaterials 2008, 29, 172–184.

    Article  Google Scholar 

  33. [33]

    Huang, J. H.; Gräter, S. V.; Corbellini, F.; Rinck, S.; Bock, E.; Kemkemer, R.; Kessler, H.; Ding, J. D.; Spatz, J. P. Impact of order and disorder in RGD nanopatterns on cell adhesion. Nano Lett. 2009, 9, 1111–1116.

    Article  Google Scholar 

  34. [34]

    Chen, S.; Lewallen, M.; Xie, T. Adhesion in the stem cell niche: Biological roles and regulation. Development 2013, 140, 255–265.

    Article  Google Scholar 

  35. [35]

    Rojas-Ríos, P.; González-Reyes, A. The plasticity of stem cell niches: A general property behind tissue homeostasis and repair. Stem Cells 2014, 32, 852–859.

    Article  Google Scholar 

  36. [36]

    Bobick, B. E.; Chen, F. H.; Le, A. M.; Tuan, R. S. Regulation of the chondrogenic phenotype in culture. Birth Defects Res. C Embryo Today 2009, 87, 351–371.

    Article  Google Scholar 

  37. [37]

    Zhu, Y. B.; Gao, C. Y.; Liu, X. Y.; He, T.; Shen, J. C. Immobilization of biomacromolecules onto aminolyzed poly(L-lactic acid) toward acceleration of endothelium regeneration. Tissue Eng. 2004, 10, 53–61.

    Article  Google Scholar 

  38. [38]

    Takiewicz, W. I.; Seras-Franzoso, J.; García-Fruitós, E.; Vazquez, E.; Ventosa, N.; Peebo, K.; Ratera, I.; Villaverde, A.; Veciana, J. Two-dimensional microscale engineering of protein-based nanoparticles for cell guidance. ACS Nano 2013, 7, 4774–4784.

    Article  Google Scholar 

  39. [39]

    Healy, C.; Uwanogho, D.; Sharpe, P. T. Regulation and role of Sox9 in cartilage formation. Dev. Dyn. 1999, 215, 69–78.

    Article  Google Scholar 

  40. [40]

    Kumar, D.; Lassar, A. B. The transcriptional activity of Sox9 in chondrocytes is regulated by RhoA signaling and actin polymerization. Mol. Cell. Biol. 2009, 29, 4262–4273.

    Article  Google Scholar 

  41. [41]

    Bang, O. S.; Kim, E. J.; Chung, J. G.; Lee, S. R.; Park, T. K.; Kang, S. S. Association of focal adhesion kinase with fibronectin and paxillin is required for precartilage condensation of chick mesenchymal cells. Biochem. Biophys. Res. Commun. 2000, 278, 522–529.

    Article  Google Scholar 

  42. [42]

    DeLise, A. M.; Fisher, L.; Tuan, R. S. Cellular interactions and signaling in cartilage development. Osteoarthritis Cartilage 2000, 8, 309–334.

    Article  Google Scholar 

  43. [43]

    Palecek, S. P.; Loftus, J. C.; Ginsberg, M. H.; Lauffenburger, D. A.; Horwitz, A. F. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 1997, 385, 537–540.

    Article  Google Scholar 

  44. [44]

    Woods, A.; Wang, G. Y.; Beier, F. RhoA/ROCK signaling regulates Sox9 expression and actin organization during chondrogenesis. J. Biol. Chem. 2005, 280, 11626–11634.

    Article  Google Scholar 

  45. [45]

    Kosher, R. A.; Kulyk, W. M.; Gay, S. W. Collagen gene expression during limb cartilage differentiation. J. Cell Biol. 1986, 102, 1151–1156.

    Article  Google Scholar 

  46. [46]

    Lim, Y. B.; Kang, S. S.; Park, T. K.; Lee, Y. S.; Chun, J. S.; Sonn, J. K. Disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating protein kinase C-a signaling. Biochem. Biophys. Res. Commun. 2000, 273, 609–613.

    Article  Google Scholar 

  47. [47]

    Lim, Y. B.; Kang, S. S.; An, W. G.; Lee, Y. S.; Chun, J. S.; Sonn, J. K. Chondrogenesis induced by actin cytoskeleton disruption is regulated via protein kinase C-dependent p38 mitogen-activated protein kinase signaling. J. Cell. Biochem. 2003, 88, 713–718.

    Article  Google Scholar 

  48. [48]

    Murphy-Ulrich, J. E. The de-adhesive activity of matricellular proteins: Is intermediate cell adhesion an adaptive state? J. Clin. Invest. 2001, 107, 785–790.

    Article  Google Scholar 

  49. [49]

    Bi, W. M.; Huang, W. D.; Whitworth, D. J.; Deng, J. M.; Zhang, Z. P.; Behringer, R. R.; de Crombrugghe, B. Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc. Natl. Acad. Sci. USA 2001, 98, 6698–6703.

    Article  Google Scholar 

  50. [50]

    Chen, S.; Fu, P. L.; Cong, R. J.; Wu, H. S.; Pei, M. Strategies to minimize hypertrophy in cartilage engineering and regeneration. Genes Dis. 2015, 2, 76–95.

    Article  Google Scholar 

  51. [51]

    Chen, W.-H.; Lai, M.-T.; Wu, A. T. H.; Wu, C.-C.; Gelovani, J. G.; Lin, C.-T.; Hung, S.-C.; Chiu, W.-T.; Deng, W.-P. In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes Arthritis Rheum. 2009, 60, 450–459.

    Article  Google Scholar 

Download references


Authors acknowledge Prof. Pau Gorostiza for its help in STM experiments, Albert G. Castaño for his help in d min quantification, and Dr. David Caballero for his support in microscopy video recording. This work was supported by Networking Biomedical Research Center (CIBER), Spain. CIBER is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and the Instituto de Salud Carlos III, with the support of the European Regional Development Fund. This work has been financially supported by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (2014 SGR 1442). This work was funded by the project OLIGOCODES (No. MAT2012-38573-C02) and by the project CTQ2013-41339-P, awarded by the Spanish Ministry of Economy and Competitiveness. C. R. P. acknowledges financial support from the Spanish Ministry of Economy and Competitiveness grant (No. IFI15/00151).

Author information



Corresponding author

Correspondence to Anna Lagunas.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lagunas, A., Tsintzou, I., Vida, Y. et al. Tailoring RGD local surface density at the nanoscale toward adult stem cell chondrogenic commitment. Nano Res. 10, 1959–1971 (2017).

Download citation


  • dendrimer
  • arginine-glycine-aspartic acid (RGD)
  • nanopattern
  • human mesenchymal stem cells (hMSCs)
  • chondrogenesis