Tissue engineering focuses on developing methods for tailor-made, living-issue ‘spare parts’ for damaged or diseased organs. To engineer complex 3D tissue from cells, it is critical to produce and use the appropriate scaffold.1 Cells placed on a flat surface typically grow in a monolayer. Likewise, 3D cell cultures can only be grown in a 3D microenvironment or tissue scaffold.
To produce suitable cell-culture conditions, we took instructive cues from nature. In live tissue, the nanoscale chemical, structural, and mechanical environment for cells is provided by their connective tissue, known as the extracellular matrix (ECM).2 A large effort in tissue engineering focuses on producing biomaterials that reproduce the ECM's structure for a given tissue type. We are still far away from exactly mimicking the ECM with a cell scaffold, but important advances are providing an increasingly detailed substitute.
3D tissue scaffolds have been produced as random or ordered microstructured networks. Random 3D networks are made by solvent casting with particulate leaching, electrospinning, or phase separation combined with freeze-drying/critical-point drying.3 Solid free-form (SFF) fabrication techniques make user-controlled 3D structures directly from data using computer aided design/manufacturing software. Random networks, when compared to user-defined structures, are generally easier to produce in bulk, but their microstructure and physical properties are more difficult to control, analyze, and interpret. Studies based on scaffolds formed from random 3D networks dominate current research because of their ease of manufacture compared to fabricating ordered networks.
SFF manufacture, by comparison, is suited to exploring the relationship between 3D topology and cell growth. More specifically, it requires optimizing and balancing the scaffold's strut geometrical parameters with its hole geometries.4 The struts ensure mechanical stability of the scaffold, while the holes allow for mass transport to aid cell/nutrient delivery and tissue generation. Understanding this complex balance for a given tissue will allow scaffold construction. Other researchers5 have grown directionally aligned heart muscle that mimics natural heart tissue using a scaffold with an ordered, accordion-like honeycomb microstructure.
(a) Microstereolithography (μSL) setup. (b) and (c) Side/top views of structure produced by μSL (scale bar: 10μm).6
We aim to build high-resolution structures (0.1–10μm feature size) in biodegradable and biocompatible polymers to be used as tissue-engineering scaffolds. We use polymers such as polycaprolactone, polylactide, and trimethylcarbonate. These materials can be produced in a photocurable form using a well-established synthesis method.7 Using these polymers ensures that the scaffold material will provide initial rigidity to an engineered tissue while it builds its own ECM, and that the engineered scaffold biodegrades and is resorbed by the tissue.
These photocurable polymers are structured with microstereolithography, which enables SFF prototyping of 3D objects with photopolymerization. In this setup, a focused light source is scanned through a liquid photocurable material: see Figure 1(a). The exposed regions photocure, while unexposed material remains liquid and can be washed away, resulting in a 3D pattern. With colleagues in Greece and Germany, we have explored a novel route to 3D photostructuring6 by exploiting two-photon polymerization (2PP) using a femtosecond IR laser (~800nm). Notably, this allows production of user-defined submicrometer 3D constructs. In an initial study, we showed that polycaprolactone-based polymer scaffolds of 3–4μm resolution can be built by 2PP: see Figures 1(b and 1(c).6 The methodologies enable production of high-resolution user-defined structures of biocompatible/degradable polymers. We are currently further exploring both the structuring of these polymers and cell growth on produced 3D cell scaffolds. As a second stage, we plan to produce well-defined structures for specific tissue-engineering applications such as nerve conduits. The method also enables us to investigate the relationship between scaffold structure and cell growth/stem-cell differentiation.
The author was supported by an Engineering and Physical Sciences Research Council Life Sciences Interface fellowship (grant number EP/C532066/1). The author also thanks his collaborators Erol A. Hasan, Arune Gaidukeviciute, Demetra S. Achilleos, Anthie Ranella, Carsten Reinhardt, Aleksandr Ovsianikov, Xiao Shizhou, Costas Fotakis, Maria Vamvakaki, Boris N. Chichkov, and Maria Farsari.
Engineering Materials Department
University of Sheffield
Frederik Claeyssens is a lecturer in biomaterials. He joined his current department in 2008 from the University of Bristol's Chemistry Department in the UK, where he was a research fellow at the Life Sciences Interface. He holds his first degree in chemistry from Ghent University in Belgium and his PhD in chemistry from the University of Bristol. He was a postdoctoral research associate at the Universities of Birmingham (UK) and Bristol.
6. F. Claeyssens, E. A. Hasan, A. Gaidukeviciute, D. S. Achilleos, A. Ranella, C. Reinhardt, A. Ovsianikov, X. Shizhou, C. Fotakis, M. Vamvakaki, B. N. Chichkov, M. Farsari, Three-dimensional biodegradable structures fabricated by two-photon polymerization, Langmuir 25, pp. 3219-3223, 2009.