Laser fabrication of 3D scaffolds for tissue engineering
Laser fabrication of 3D scaffolds for tissue engineering
By Dr. Maria Farsari, Researcher at the Institute of the Electronic Structure and Laser, Foundation for Research and Technology-Hellas,
And Professor, Anna Mitraki, Department of Materials Science and Technology,c/o Biology Department University of Crete, and Institute For Electronic Structure and Laser, IESL-FORTH
Tissue Engineering is defined as the technology aiming to ‘apply the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ’ .
One of its research aims is, therefore, to create materials that are biocompatible and capable of integrating living cells. This usually involves three steps: (i) identifying a material suitable for the fabrication of a scaffold for a specific tissue engineering application, (ii) structuring of the scaffold material and (iii) cell seeding into the scaffold for cell culturing in vitro or in vivo.
The choice of scaffold for cell cultivation can greatly influence the attachment, migration and proliferation of cells. Scaffolding materials that are not rejected by the body upon implantation can be metals, ceramics, synthetic polymers and biopolymers, and specific tissue engineering applications demand different materials properties.
Bone replacements need strong and wear resistant materials such as metals and ceramics, while soft tissue replacements can be made of polymeric and biological materials.
In soft tissue engineering, the primary function of the scaffold structure is to provide a micro- and nano-structured 3D environment for the cells to migrate and to proliferate in. The specific properties of the cell scaffold have to be tuned for each tissue engineering application since the 3D environment surrounding the cells provides instructive cues needed to maintain cell phenotype and behaviour.
Once the cells of the engineered tissue have built their own connective tissue, the cell scaffold becomes redundant. Therefore, biomaterials that are bioresorbable and biodegradable on a similar timescale of the production of the extra-cellular matrix of the engineered tissue are preferred.
Micron-sized topography has been shown to play an essential role in determining cell adhesion and surface-bound characteristics influence in this way prominent cellular functions such as survival, proliferation, differentiation, migration, or mediator release.
In particular, 3-dimensional (3D) cell culture offers a more realistic micro- and local-environment where the functional properties of cells can be observed and manipulated. An important factor in the production of working tissue engineering scaffolds is the possibility of a reproducible and controlled method of nanostructuring. A versatile class of the scaffold production techniques which enable the fabrication of tailor made structures directly from computer data via Computer Aided Design / Computer Aided Manufacturing (CAD/CAM) are laser-based solid-free-form (SFF) fabrication techniques [ ].
A number of laser-based SFF techniques have already been implemented and commercialised, including selective laser sintering, stereolithography, and 3D laser nonlinear lithography. These techniques and their application in Tissue Engineering are detailed in the following paragraphs.
Selective Laser Sintering
The Selective Laser Sintering technique employs a laser (usually CO2) to sinter thin layers of powdered materials to form solid three-dimensional objects.
The powder particles will typically have 50 micron diameter. The object is built layer-by-layer from CAD data files. During fabrication, the laser beam is selectively scanned over the powder surface following computer design.
The laser beam heats the powder to melting point and causes the powder particles to melt together to form a solid mass. Subsequent layers are built directly on top of previously sintered layers with new layers of powder being deposited via a roller on top of the previously sintered layer.
As the majority of materials available for selective laser sintering are neither biocompatible nor biodegradable, the application of this technique in Tissue Engineering has remained limited.
Bioceramic scaffolds that can aid the regeneration of hard tissues via laser sintering of Pure biopolymer powders such as polyetheretherketon (PEEK) and HA hydroxyapatite (HA) and physically blended mixtures of PEEK and HA powders have been investigated by Tan et al . Since then, there have been several publications regarding the fabrication of both ceramic and biodesorbable implants .
The term “stereolithography” (SLA) was first used by C. W. Hull in his 1986 patent ‘Apparatus for Production of Three-Dimensional Objects by Stereolithography’. It is defined as a method and apparatus for making solid objects by successively “printing” thin layers of an ultraviolet curable material one on top of the other.
More specifically, SLA is a three-dimensional printing method where a ultra-violet laser is used to photopolymerize a liquid resin and build computer-designed components, one layer at the time. On each layer, the laser beam traces a pattern on the surface of the liquid resin.
The exposure to UV selectively solidifies the resin and makes adhere to the layer below. After the pattern has been ‘laser written’, the SLA’s elevator platform goes down by a single layer. Then, the built section is re-coated with photopolymer using a blade. On this fresh photopolymer, the next layer pattern is laser written on the top of the previous layer. After the completion of the build process, the un-polymerized resin is removed the 3D structure is developed in a suitable solvent.
The first use of SLA in research for Tissue Engineering was reported by Matsuda . In this case, it was a custom made SLA consisting of a moving light pen, an optical fiber which connected to the light pen, a UV light source (Hg-Xe lamp), a fixed sample holder,and a stage controller, and everything was driven by a personal computer.
In the case, 3D scaffolds were fabricated by using an epsilon-caprolactone biodegradable photopolymer. However, due to the limitations of the equipment used, the fabricated structures were small and had low resolution.
The use of a commercially available SLA machine to fabricate biodegradable scaffolds was firstly reported by Cooke et al. in 200310. In this case, the structures were made using a biodegradable resin mixture of diethyl fumarate, poly(propylenefumarate), and a photoinitiator, bisacylphosphine oxide.
Later, Dharwala eta al. reported the rapid prototyping of tissue engineering constructs using photopolymerizable hydrogels 11. In general, however, the use of SLA in tissue engineering applications has been limited, mostly due to the lack high-enough resolution and of availability of materials which are photostructurable, biocompatible and/or biodegradable.
3D laser nonlinear lithography
3D laser nonlinear lithography (NLL) based on multi-photon polymerization (MPP) of photosensitive materials is a direct laser writing technique that allows the fabrication of three-dimensional structures with sub-micron resolution .
The polymerization is based on multi-photon absorption; when the beam of an ultra-fast laser is tightly focused into the volume of a transparent, photosensitive material, the polymerization process can be initiated by non-linear absorption within the focal volume. By moving the laser focus three-dimensionally through the material, 3D structures can be fabricated. The technique has been implemented with a variety of acrylate and epoxy materials and several components and devices have been fabricated such as photonic crystal templates, mechanical devices, and microscopic models (Figure 1).
The unique capability of MPP lies in that it allows the fabrication of computer-designed, fully 3D structures with resolution beyond the diffraction limit. No other competing technology offers these advantages; the classic 3D prototyping techniques described earlier can also produce fully 3D structures, however, they cannot provide resolution better than a few microns.
On the other hand, lithographic techniques with superior resolution, such as e-beam lithography, cannot produce anything more complicated than high-aspect ratio two-dimensional structures.
The basis of multi-photon polymerization is the phenomenon of multi-photon absorption (MPA). There are two types of two-photon absorption: sequential and simultaneous.
In sequential, the absorbing species is excited to a real intermediate state, then, a second photon is absorbed. The presence of the intermediate energy state implies that the material absorbing at this specific wavelength; it will therefore be a surface effect and will follow the Beer-Lambert law. The simultaneous absorption, on which the MPP technique is based, was originally predicted by Maria Göppert- Mayer in 1931 in her doctoral dissertation.
It is defined as ‘an absorption event caused by the collective action of two or more photons, all of which must be present simultaneously to impart enough energy to drive a transition.’
This prediction was not experimentally verified until over 30 years later by Werner Kaiser, when the invention of the laser permitted the first experimental verification of the MPA when two-photon excited fluorescence was detected in a europium-doped crystal.
In simultaneous absorption, there is no real intermediate energy state i.e., the material is transparent at that wavelength. Instead, there is a virtual intermediate energy state and two-photon absorption happens only if another photon arrives within the virtual state lifetime. For this to occur high intensities are required, which can only be provided by a tightly focused femtosecond laser beam. This is illustrated in Figure 2; as it can be seen, the electron transition in this case is caused by two photons of energy hv/2 rather than one of energy hv.
Ti:sapphire lasers are widely used for this purpose. They have two main advantages; firstly they have very short pulses, in the order of a few tens of femtoseconds, so they do not cause thermal damage. Secondly their standard wavelength is 800 nm, which is twice the wavelength of polymerization of a wide range of photopolymers. In addition, most photopolymers are transparent at 800 nm, which allows in-volume focusing of the laser beam with minimal scattering.
When the laser is focused tightly into the material, the photoinitiator used to initiate the polymerization will absorb two photons and produce radicals. As the material response is proportional to the square of the intensity, this will only happen at the focal point, which, combined with the fact that the two-photon transition rate is very small, will provide very high spatial resolution. Theoretically, the highest resolution that can be achieved by a focused laser beam is given by Abbe’s diffraction limit
Equation 1: Abbe’s diffraction limit
where is the laser wavelength and N.A. is the numerical aperture of the focusing objective; this has fuelled the race for ever decreasing wavelengths, such as electron wavelengths and for alternative, non-light patterning techniques such as atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM); however, these techniques only allow surface and not in-volume patterning.
To produce 3D structures with in-volume patterning, and produce photopolymerized voxels smaller than that defined by the diffraction limit, materials with well defined photopolymerization threshold need to be used: As the photoinitiator is excited by the laser process, it produces radicals; these radicals are quenched by oxygen and other quenchers in the system.
Quenching is a competing effect to photopolymerization and is usually considered detrimental to the process. In MPP, however, it can be used to circumvent the diffraction limit and produce structures of very high resolution. This can be done by modifying the light intensity at the focal volume, in a manner so that the light-produced radicals exceed the quenchers and initiate polymerization only at a region where exposure energy is larger than the threshold. In this case the diffraction limit becomes just a measure of the focal spot size and it does not really determine the voxel size.
IESL-FORTH has explored the use of MPP in the construction of both permanent and biodegradable scaffolds. For permanent scaffolds, organic-inorganic hybrid materials were investigated (Figures 3, 4).
Hybrid materials benefit from straight-forward preparation, modification and processing, as well as post-processing chemical and inertness, and good mechanical and chemical stability. Due to their hybrid, organic and inorganic composition, structures made using such materials might provide the appropriate features to help both the mechanical support of the growing cells and the shaping and directionality of the healing of any damage.
Figure 3: 3D scaffolds fabricated using hybrid materials
Figure 4: Confocal images showing the distribution of vinculin (green) and actin (red) in fibroblasts cultured on titanium/silicon (I) and zirconium/silicon (II) and for 3 days. Double stained images are also shown (Ic, IIc). Nuclei were stained (blue) with TO-PRO-3 dye.
For biodegradable scaffolds, polycaprolactone-based materials have been investigated. Such materials have been studied extensively for soft tissue engineering, as not only they are biocompatible and biodegradable, but also they degrade on a similar timescale as tissue formation (Figure 5).
Figure 5: A poly-caprolactone based 3D scaffold (IESL-FORTH)
A different approach has also been proposed by IESL-FORTH , where three dimensional structures have been used as supports for the directed assembly of amyloid peptide fibres. Peptide networks are already investigated as cell supports in the form of injectable hydrogels. The researchers at IESL-FORTH suggest that a combination of larger scaffolds with well-defined biodegradable peptide supports in a “scaffold on scaffold” format could be used as a support to allow the directed growth of several cell types into ordered arrays of functional biological units.
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