Among other biomedical applications, nanoparticles have a vast field of application in regenerative medicine. This article discusses two main approaches, in which either inert particles or magnetic particles are used for tissue engineering. In the first approach, surface augmentation with particles made of different materials stimulates cell proliferation and differentiation through mechano-transduction events. In the second approach, magnetic particles, when introduced into cells to be cultured, permit the cells' defined positioning by the appropriate use of magnets, creating more complex tissue structures than those that are achieved by conventional culture methods.
by Dr José Luis Corchero, Joaquín Seras, Elena García-Fruitós, Esther Vazquez and Antonio Villaverde
In the early 90s the possibility of regenerating damaged human tissues was an emerging issue for scientific discussion. Recent advances in cell biology (particularly the discovery of stem cells), biotechnology and materials sciences have now made this feasible. Regenerative medicine, that is, the tailored, clinically-oriented reconstruction of a damaged tissue, benefits from biocompatible scaffolds on which stem cells can grow and differentiate, either under preliminary ex vivo conditions for further grafting into the injured organ, or as a result of direct in vivo implants.
Scaffolds offer micro-structured 2D or 3D surfaces for cell attachment, differentiation and proliferation, with biomechanical properties that are suitable for supporting novel tissue structures and no toxic effects. However, both preclinical and clinical studies have demonstrated the need for stimulators of cell attachment, as the polymeric materials used for scaffold fabrication inhibit cell colonisation. Growth factors and cell ligands obtained by chemical synthesis or produced by recombinant DNA technologies are therefore used as biologically-active molecules to pattern the scaffold surfaces and thus facilitate cell attachment and expansion. As an alternative but potentially synergistic approach, the topographical manipulation of the nano- and micro-environments on which cells grow as substrate has been revealed as an unusually efficient approach to stimulate cell proliferation on seemingly unsuitable surfaces.
Mechanically-assisted stimulation of cell proliferation
In vivo, cells are in close contact with their environment, which has a profound influence on their behaviour and function by providing different types of signals. Many molecules such as proteins or lipids stimulate cell receptors that trigger signalling pathways at a molecular level, resulting in changes in the cell’s transcriptional pattern. On the other hand, the surrounding topography provides mechanical stimuli, which cells are able to sense through filopodia and to which they respond with a broad range of changes in shape, adhesion, migration and differentiation among others [1].
Two main approaches have been used for surface modification at the micro- and nano-scales, namely top-down and bottom-up [Figure 1]. In the first approach, lithographic techniques have been applied to produce regular topographies at a nanoscale level. Since these modifications are limited by the chemical nature of the substrate used, and the feasible modifications are limited by its chemical composition, the whole strategy is more suitable for analytical than for clinical purposes. However, very interesting data have been generated that permit a better understanding of how cell respond to their surroundings, and allow the design of improved scaffolds and scaffold patterns. For instance, the study of mesenchymal cells (MSCs) growing on polymethylmethacrylate with engineered surfaces has revealed that a certain grade of topographical disorder provides a better environment for MSC differentiation into osteoblasts [2].
Compared to this approach, the bottom-up approach is more flexible regarding the nature of the material to be used as scaffold, since it relies on the deposition of particles made of different materials on the cell culture surface. This modification allows for the selection of different physical and chemical properties in the particles, such as stiffness or hydrophobicity. The bottom-up approach is a more promising strategy for the modification of surfaces or scaffolds. The materials used for topographic modification include polymers, ceramic particles, glass particles, metal particles, carbon nanotubes or cocktails of these [3,4,5]. These materials are often used in combination with functional proteins of the extracellular matrix such as collagen, fibronectin, vitronectin and others [6]. Recently we have shown that bacterial inclusion bodies (IBs), the protein aggregates usually found in recombinant bacteria, provide appropriate mechanical stimuli for mammalian cell proliferation [7]. IBs, which are pseudo-spherical and mechanically stable particles that usually range from 50 to 500 nm, can be tailored for shape and other mechanical properties by the appropriate selection of the genome of the bacteria that produce them [8,9]. Being fully compatible, IBs can be easily produced and isolated from recombinant bacteria by cost-effective procedures and their use as biomaterials is straightforward.
Magnetically-assisted, ex vivo tissue formation
In a different context, magnetic nanoparticles are being used in an increasing number of biomedical applications. The ability to control the location of these particles distally using magnets, and to induce a high concentration in a given tissue or organ, has powerful applications in innovative medicine, including tissue engineering [10].
Magnetic nanoparticles contain a magnetic core (usually composed of magnetite Fe3O4 or maghemite γ-Fe2O3) that confers the unique feature of reacting to magnetic forces. This core is usually coated with a polymeric layer that minimises hydrophobic interactions, enhancing colloid dispersion and biocompatibility. Procedures to produce coated magnetic particles have been summarised recently [11,12]. An emerging tissue engineering strategy, namely magnetic force-based tissue engineering (Mag-TE), employs cells that have been magnetically labelled with magnetite cationic liposomes (MCLs) [Figure 2]. Such MCL-labelled cells can be manipulated and organised by magnetic force, and maintain their functionality (indicating that MCLs are not toxic). In the Mag-TE approach, a magnet is applied under the culture plate, attracting and accumulating magnetically-labelled cells. This allows populations of MCL-labelled cells to be sequentially driven to the surface to create 2D patterned or even 3D multilayered structures. This approach has already been tested with several cell lines, including human umbilical vein endothelial cells [13], retinal pigment epithelial cells [14], keratinocytes [15], mesenchymal stem cells [16] and cardiomyocytes [17], with promising results.
In addition Mag-TE allows the in vitro fabrication and harvesting of cell sheets and heterotypic, layered co-cultures containing different cell lines, providing a proof-of-principle for the applicability of this approach for generating complex heterogeneous tissues [18]. Moreover, tubular structures (for example, urinary tissue formed by urothelial cells or vascular tissues consisting of endothelial cells, smooth muscle cells, and fibroblasts) can also be created using the Mag-TE protocol. In this approach, magnetically-labelled cells form a cell sheet onto which a cylindrical magnet is rolled and then removed after the tubular structure has been formed [19]. A variation of Mag-TE, called ‘Mag-seeding’, facilitates cell seeding into the deep internal space of scaffolds resulting in higher scaffold-seeding efficiencies [20].
Concluding remarks
Different types of nano- and micro-particles have been explored to mechanically stimulate mammalian cell adhesion, proliferation and differentiation upon manipulation of scaffold surfaces. A certain extent of topographical disorder greatly facilitates cellular responses to the induced mechanical stimuli. The labelling of mammalian cells with magnetic nanoparticles allows the fabrication of complex tissues that are not achievable by conventional cell culture, such as 2D and 3D cell layers, tubular tissues, or ordered 3D assemblies consisting of several cell types.
References
1. Dalby MJ. Int J Nanomedicine 2007; 2(3): 373-381.
2. Dalby MJ et al. Nat Mater 2007; 6(12): 997-1003.
3. Liu A et al. Acta Biomater 2008; 4(4): 1005-1015.
4. Ito A et al. Biomaterials 2005; 26(31): 6185-6193.
5. Gu HY et al. Biomaterials 2004; 25(17): 3445-3451.
6. Ma Z et al. Colloids Surf.B Biointerfaces 2007; 60(2): 137-157.
7. Elena Garcia-Fruitós et al. Advanced Materials 2009; 21: 1-5.
8. Diez-Gil C et al. Biomaterials 2010.
9. Garcia-Fruitos E et al. Nanotechnology 2010; 21(20); 205.
10. Corchero JL, Villaverde A. Trends Biotechnol 2009; 27(8): 468-476.
11. Laurent S et al. Chem Rev 2008; 108(6): 2064-2110.
12. Lu AH et al. Angew Chem Int Ed Engl 2007; 46(8): 1222-1244.
13. Akiyama H et al. Biomed Microdevices 2009; 11(4): 713-721.
14. Ito A et al. Tissue Eng 2005; 11(3-4): 489-496.
15. Ito A et al. Tissue Eng 2004; 10(5-6): 873-880.
16. Shimizu K et al. J Biomed Mater Res B Appl Biomater 2007; 82(2): 471-480.
17. Shimizu K et al. Biotechnol Bioeng 2007; 96(4): 803-809.
18. Ito A et al. J Biosci Bioeng 2007; 104(5): 371-378.
19. Ito A et al. Tissue Eng 2005; 11(9-10):
1553-1561.
20. Shimizu K et al. J Biomed Mater Res B Appl Biomater 2006; 77(2): 265-272
Acknowledgments:
We appreciate the financial support received from MICINN (BIO2007-61194 and ACI2009-0919), AGAUR (2009SGR-108) and CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN, Spain), an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. AV has been distinguished with an ICREA ACADEMIA award.
The authors
José Luis Corchero, Joaquín Seras,
Elena García-Fruitós, Esther Vazquez
and Antonio Villaverde*
CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, 08193
Barcelona, Spain, and Institut de
Biotecnologia i de Biomedicina and Departament de Genètica i de
Microbiologia, Universitat Autònoma
de Barcelona, 08193 Bellaterra
(Cerdanyola del Vallès),
Barcelona,
Spain
* corresponding author
