Femtosecond-Laser-Based 3D Printing for Tissue Engineering and

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Review

Review: Femtosecond-laser-based 3D Printing for Tissue Engineering and Cell Biology Applications Chee Meng Benjamin Ho, Abhinay Mishra, Kan Hu, Jianing An, Young-Jin Kim, and Yong-Jin Yoon ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00438 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 16, 2017

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ACS Biomaterials Science & Engineering

Review: Femtosecond-laser-based 3D Printing for Tissue Engineering and Cell Biology Applications

Chee Meng Benjamin Ho1,2#, Abhinay Mishra 1,2#, Kan Hu1,2, Jianing An 1, Young-Jin Kim1,2 * and Yong-Jin Yoon 1,2*

1

School of Mechanical & Aerospace Engineering, Nanyang Technological University, 50

Nanyang Avenue, Singapore 639798 2

Singapore Centre for 3D Printing, Nanyang Technological University, 50 Nanyang

Avenue, Singapore 639798 #

These authors have equal contribution.

*Corresponding authors; E-Mail: [email protected], [email protected]

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Abstract

Fabrication of 3D cell scaffolds has gained tremendous attention in recent years due to its applications in tissue engineering and cell biology applications. The success of tissue engineering or cell interactions mainly depends on the fabrication of well-defined microstructures, which ought to be biocompatible for cell proliferation. Femtosecondlaser-based 3D printing is one of the solution candidates that can be used to manufacture 3D tissue scaffolds through computer-aided design (CAD) which can be efficiently engineered to mimic the microenvironment of tissues. UV-based lithography has also been used for constructing the cellular scaffolds but the toxicity of UV light to the cells has prevented its application to the direct patterning of the cells in the scaffold. Although the mask-based lithography has provided a high resolution, it has only enabled 2D patterning not arbitrary 3D printing with design flexibility. Femtosecond-laser-based 3D printing is trending in the area of tissue engineering and cell biology applications due to the formation of well-defined micro- and sub-micrometer structures via visible and near infrared (NIR) femtosecond laser pulses, followed by the fabrication of cell scaffold microstructures with a high precision. Laser direct writing and multi-photon polymerization are being used for fabricating the cell scaffolds, The implication of spatial light modulators in the interference lithography to generate the digital hologram will be the future prospective of mask-based lithography. Polyethylene glycol diacrylate (PEGDA), ormocomp, pentaerythritol tetraacrylate (PETTA) have been fabricated through TPP to generate the cell scaffolds, while SU-8 was used to fabricate the microrobots for targeted drug delivery. Well-designed and precisely fabricated 3D cell scaffolds manufactured by femtosecond-laser-based 3D printing can be potentially used for studying cell migration, matrix invasion and nuclear stiffness to determine stage of 2 ACS Paragon Plus Environment

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cancer and will open broader horizons in the future in tissue engineering and biology applications. Keywords: 3D printing, femtosecond laser, tissue engineering, cell biology, scaffolds.

1. Introduction Tissue engineering is defined as an interdisciplinary field which involves the use of cells and a scaffold/matrix to develop new functional tissues for implantation back to the donor. With the tremendous need for organs and tissue, tissue engineering was developed to fabricate a living replacement for parts of the body1-2. The necessity of tissue engineering has been further demonstrated with the widening gap between supply and demand for transplantable tissues or organs around the world3-4. One vital component of tissue engineering is the use of scaffolds. Scaffolds provide a conducive environment and act as a support for tissue attachment and growth. Scaffolds could also serve as a carrier or template for implanted tissue or delivery of other agents. Currently, there are numerous strategies used to fabricate scaffolds with the different biomaterials available (Figure 1). In a biological environment, the ideal function of a scaffold is to organize the cells into a three -dimensional (3D) architecture and present stimuli that direct the growth and formation of the desired tissue1. This scaffold will also function as a synthetic extracellular matrix (ECM) providing adhesive surfaces for cells to attach to and deposit their own protein to make them more biocompatible. However, vascularization, lack of functional cells, the low mechanical strength of engineered cells, immunologically



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incompatible with host and nutrient limitations are classical issues in the field of tissue engineering5. During the last decade, it is recognized that physical properties of the extracellular environment with other chemical factors, such as growth factors, signaling, and adhesion molecules, have an importance in affecting the cell behavior and development6-7. Topography on the scaffolds in the micro and nano scales, mechanical stiffness and spatial patterning of the ligand are some exemplary physical stimuli used in tissue engineering6-8. However, most cell behavior studies were conducted previously were based on 2D platforms with simple geometries such as grooves and ridges7, 9. This lead to the loss of specific cellular function, changes in cell morphology and cell to cell interactions to their extracellular matrix (ECM) when compared to cell grown in a 3D environment10-12. These days, research on fabrication of 2D or 3D scaffold of polymers such as polycaprolactone (PCL), polyurethane (PU) and polylactic acid (PLA) and their nanocomposites has been carried out for its drug delivery and biomedical application13-25. Although these studies aid in our understanding of cell behavior in 3D matrices, the availability of various fabrication techniques (e.g creation of random pore sizes), the chemical composition of biomaterials and its mechanical properties prevents studying cell behavior in a systematic and quantitative way8. Therefore, a novel 3D cell culture bio-functionalization scaffold and methods have to be developed for proper characterizations of physical stimulus on cell behavior in a 3D environments8. The emergence of new or improved micro and nanofabrication techniques for 3D cell culture systems have become a common trend over the past decade9, 26. Technologies such as electrospinning, molding or the recent rise of 3D printing were deployed in the


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construction of artificial 3D scaffolds. The ability to add material by layers with the use of a single machine, the possibility to create complex geometry scaffolds and the power to control the overall porosity are the key advantages to which 3D printing has become such a popular option. Figure 1 gives a schematic representation of optical fabrications techniques in both micro and nano scales (shown in the 3 smaller circles) and their advantages for tissue engineering and cell biology applications. It is discernible from the figure 1 that these techniques can be used vis-a-vis for fabrication of 3D scaffolds, template for implanting tissues, creating conducive environment for proliferation of cells and improved accuracy on the positioning for the interaction of cells. Nanotechnology can be used to create nanofibers, nanopatterns and controlled-release nanoparticles for applications in tissue engineering to mimic native tissues, such as extracellular fluids, bone marrow, and cardiac tissues5.

Figure 1. Schematic representation of optical micro and nanofabrication techniques for tissue engineering and cell biology applications. Bullets points describes the advantages that these techniques provide in making scaffolds. 5 ACS Paragon Plus Environment


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Nowadays, 3D lithography has emerged as potential fabrication technique, which uses the light to create patterns onto the surface of the substrate coated with photosensitive chemical. Generally, the photoresist mask is essential in mask-based interference lithography, which requires multi-step mask preparation processes. On the contrary, the direct laser writing (DLW) does not require mask so provides high design flexibility without additional processes27. Multi-photon polymerization based on DLW enables to print very small structures with a high printing resolution; the minimum voxel size reaches to sub-100 nm. Therefore, the printing resolution in multi-photon polymerization is not limited by the layer thickness as it has been in stereolithography process. Table 1 shows a comparison of stereolithography, multiphoton polymerization and interference lithography. The resolution of the printed structures through stereolithography is in micron level, while nanometer level to submicron resolution can be printed by the multiphoton polymerization and interference lithography. The major advantage of multiphoton polymerization or optical lithography over interference lithography is that it is not limited to fabricate uniform disseminated periodic patterns but can also fabricate arbitrary free form well designed structures as needed for the biological applications. Without the need of minimum layer thickness, users are able to construct the 3D structure of a better resolution. Many researchers have been considering the use of optical systems over other techniques as it allows users to achieve better resolution with its ability to achieve nanometer size and grants them more control in the fabrication process. In this review, readers will be introduced to the different optical lithography techniques, common terms, and equipment used in the setup of laser lithography and the materials that are compatible with the laser set-up and biological samples. Finally, a


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review of the current state of femtosecond-laser-based 3D printing or 3D laser lithography in the field of fabrication and applications of the 3D scaffolds.

Table 1. A comparison between interference lithography, stereolithography and multiphoton polymerization or 3D optical lithography. The resolution also partly depends on the materials properties.

Technology

Interference lithography

Stereolithography

Multi photon polymerization

Process

Light interference

Layer by layer

Direct laser writing

Energy source

Laser

UV laser

Femtosecond laser

Resolution

Up to 100 nm

Up to 25 µm

Up to 100 nm

Speed

Fast

Fast

Slow

Material

Photocurable

Photocurable

Photocurable

Fabrication advantage

Periodic patterns

Rapid prototyping

Arbitrary free-form structures with precision

2. Interference lithography for periodic structures Interference lithography is considered as a lithographic technique which combines the diffraction and interference phenomenon of light to fabricate multi-dimensional (eg. 1D, 2D, and 3D) periodic structures by using coherent beams. Generally, the inherent periodicity of incident light beams is being utilized to generate periodic structures, while the static spatial orientation of light intensity is also formed by the interference of these beams. The fabricated patterns are generated due to the transfer of emerged intensity distribution to the light sensitive materials such as photoresists or photopolymers, moreover, these structures can be tailored by the combination of different intensity 7 ACS Paragon Plus Environment


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beams28-29. In general, there are two methods of beam configurations which can be currently used to attain 3D interference patterns. 2.1. Phase mask interference lithography Phase-mask-based lithography is the simplest approach to interference lithography. A 3D image is recreated after a coherent beam is exposed to the phase mask, and subsequently, gets transferred to the photoresist to generate interference patterns30. In this lithographic technique, the elastomeric phase mask is configured just above the photoresist volume, followed by the incident light from the top of the phase mask. The structures are then fabricated in the volume of the photoresist by the diffraction of beams. The general fabrication procedure of elastomer phase mask has been depicted in figure 2a, while schematic diagram of phase lithographic setup was shown in figure 2b. The 3D microstructure of fabricated SU-8 by using 2D phase mask has been compiled in figure 2c31.

(a)

(b)

(c)


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Figure 2. Scheme demonstrating the phase mask lithography: a) Fabrication procedure of conformal phase mask; b) Lithography setup; c) Fabricated SU-8 microstructures. Upper left is a Scanning electron microscopy (SEM) image of a PDMS phase with an array of circular holes having diameter of 280 nm and height of 450nm on a 750 nm square lattice. SEM image of 3D microstructure obtained from the mask and theoretical intensity distribution for a 2 × 2 × 2 array of unit cells. Reproduced with permission from ref 31. Copyright 2007 John Wiley and Sons. 2.2. Multi-beam interference lithography Multi-beam interference lithography can be defined as a technique in which 3D pattern is created within the volume of a photoresist by the interference of collimated, coherent laser beams for the fabrication of the targeted structure. In the multi-beam interference lithography setup generally, one laser beam is divided into multiple beams using beam-splitters and then recombined by mirrors to obtain the desired geometry, followed by the controlling of polarization and intensity of the beams by the wave plates and beam splitters. Moreover, the phase of more than four beams cannot be controlled in the free space, is the drawback of this technique. Optical components used in the multibeam interference lithography, optical arrangement, and fabricated SU-8 microstructure are presented in figure 331-33. Diffractive optical elements (DOEs) shown in figure 3b have also been used for multi-beam interference lithography, with designed amplitude and phase distributions. Due to the invention of digital mirror devices, the degree of freedoms and update rate of DOE have been far improved. Precision optics and conversation of polarization are the foremost requirements for the multi-beam interference, as the refraction and polarization changes when the beam is entered inside any photo material.

To overcome this issue, the prism with the same

refractive index can be used for producing interference lithographic patterns as demonstrated by Harb et al. and Jang et al. in figure 3 b-d. There is a limitation of using a



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prism for interference pattern, as every novel structure would need a different prism34-35. In order to solve this issue, the specially-angled prism can be used which can convert the single beam to multiple beams after refraction and these refracted beams can further generate desired interference pattern36-37.

(a)

Optical Component

Functions

Spatial filter

Improve laser beam quality

Mirror

Change the direction of light

Phase plate

Match the path length

Custom prism

Account for index of refraction differences

Beam splitter

Split a beam of light into two components

Wave plate

Alter the polarization state of light

(b)


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(d)

(c)

Figure 3. (a) A table presenting the optical components and their functions in multibeam IL. Reproduced with permission from ref 31.Copyright 2007 John Wiley and Sons; (b) Optical Configuration of typical multibeam interference lithography. Reproduced with permission from ref 33. Copyright 2010 OSA publishing; (c) 6 beam lithographic setup with a prism. Reproduced with permission from ref 31. Copyright 2007 John Wiley and Sons; (d) SEM image of fabricated SU-8 microstructures. Lower left is a computed theoretical intensity distribution for a 2 × 2 × 2 arrays of unit cells. Reproduced with permission from ref 32. Copyright 2007 American Chemical Society.

The foremost advantage of this method, it reduces the aberration due to vibrations, which is the main problem in the previous method and reduces the chance of misalignment of the beam. Nevertheless, the major disadvantage of this procedure is the control of polarization and phase shift of the beam. The emergent polarization depends on incident polarization as well as the prism angles and cannot be varied independently 38. Therefore, this method is well versed for the microstructures in which motif complexity is not as essential for designed patterns.

2.3. Interference lithography by using spatial light modulator Spatial light modulators (SLM) are also being used in interference lithography to create periodic structures. Lasers have been used in wide-ranging applications from medical eye



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surgery and industrial material processing to military targeting and microscopy in research. Most applications necessitate a high beam quality, meaning the beam must have a defined cross section and not diverge too rapidly. The desired beam propagation can be achieved with digital holograms. SLM can be described as a device that is used to modulate the amplitude, phase, or polarization of light beams. An SLM can manipulate an incident beam's phase and thus be used as a variable focal length lens or as a means of manipulating the beam's spatial frequency spectrum.

Figure 4. Optical setup for creating digital hologram (measuring the beam propagation ratio M2) from an unknown beam source (BS). SLM: Spatial light modulator. L: Lens. CCD: Charge-coupled device camera. Reproduced with permission from ref 39. Copyright 2012 OSA publishing.

Schulze et al. have used SLM and manipulated an incident beam's phase in two ways (1) first as a variable focal length lens and (2) second as a means of manipulating the beam's spatial frequency spectrum. Both approaches have enabled the beam propagation ratio (M2) value to be extracted without moving any components and with the potential for real-time measurements39. Using the SLM for method 1 by displaying a spherical lens phase pattern, a camera recorded a beam's curvature and diameter as a


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function of the focal length in a fixed plane behind the hologram. Thus, rather than probing one beam at several planes, several beams were probed at one detection plane. The beam diameter for a variable focus lens yielded M2 reliably and quickly using the analytical formula39. Using the SLM for method 2, an optical field was propagated by Fourier transforming the field at the initial plane, multiplying the transfer function of free space, and then Fourier transforming it using a single lens40-41. The SLM displayed the transfer function of free space, so recording with a camera in a fixed plane behind the SLM yielded an artificially propagated beam. Figure 4 depicted the experimental setup used to measure the M2 from a laser source. The optical setup consists of the beam source, SLM, a CCD camera, and a lens (only in method 2).

Figure 5. Digital holograms for three sample beams using method 1 with a focal length of 400 mm. (a) LG10, (b) LG1±3 (Media 1, Media 2), and (c) LG21. Insets depict resulting measured beam intensities. Reproduced with permission from ref 39. Copyright 2012 OSA publishing.

Schulze et al. have also created different holograms from both methods, by using different Laguerre-Gaussian modes LGpl . (M2 is known to scale with the mode indices p and l according to M2=2p+l+1). The sample beams were produced by displaying the LG mode patterns using a special coding technique42-43. Examples of beams with corresponding hologram patterns were shown in figure 5 which depicts 13 ACS Paragon Plus Environment


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measured and fitted beam diameters as a function of the SLM lens focal length programmed for a Laguerre-Gaussian beam LG21(method 1). Similarly, Lutkenhaus et al. have used four beams with the SLM to fabricate holographic

microstructures44.

The

photoresist

containing

dipentaerythritol

penta/hexaacrylate (DPHPA) as a monomer, rose bengal as a photoinitiator, N-phenyl glycine as a co-initiator and N-vinyl pyrrolidinone as a chain extender; was photosensitized by 532 nm wavelength. The diffracted beams from the phase pattern were assigned and displayed in the SLM. The phases of the interfering beam can be tuned by the gray levels in the pattern and verified by the fabricated structures (Figure 6).

Figure 6. (a) SEM image of fabricated holographic structures in DPHPA. (b) Higher magnification image. Reproduced with permission from ref 44. Copyright 2013 OSA publishing. 3D microstructures have varied application in the field of photonic crystals, biomimetic structures, optical communication, tissue engineering and etc.28-29,

45-50

.

Nowadays, there is a growing demand for three-dimensional (3D), in vitro biological tissue models51. These models are needed for fundamental investigations. For example, to study angiogenesis, tumour cell intra- and extravasations, neuronal growth, or the behaviour of the blood-brain barrier are some areas of investigation. Meanwhile, affordable, reproducible tissue models are needed for drug screening. These applications 14 ACS Paragon Plus Environment

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all call for suitable extracellular scaffolds: microstructures with strictly controlled stiffness or stiffness gradients, pore sizes, and chemistry. SLMs are being used as a programmable phase mask for digitally tunable holographic lithography. In future SLM can be used to produce the desired structure for 3D tissue engineering.

3. 3D lithography 3D lithography is a direct write process in which linear or nonlinear excitation promotes photopolymerization creating a 3D structure. Depending on the type of laser used, either one-photon absorption or multi-photon absorption (multiphoton lithography) can occur. In this section, we will be discussing on the different technologies stereolithography and multiphoton lithography and the materials used for both ablation and polymerization.

3.1. Stereolithography Stereolithography (SLA) is one of the first 3D systems created52. SLA is an additive manufacturing process which makes 3D objects based on a layer-upon-layer deposition arrangement to polymerize photosensitive resin under UV irradiation. There are two main beam delivery systems: (1) scanning or (2) projection. For scanning system, photocurable resin on the surface is exposed in a point-by-point and line by line style while the projection system using a digital micromirror device (DMD) allows a whole layer to be cured at one time. Once the layer has been cured, the stage will move in the z-direction to the next layer to be cured. This process is repeated till the whole 3D part is completed (Figure 7). One of the biggest advantages of SLA is its fabrication speed. Fabrication can be done within a day but this is dependent on the size and complexity of the 3D part.



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SLA also keeps cost low as it a maskless lithography and materials is only consumed when cured. Stereolithography due to its ability to produce high-resolution structures has been used in the construction of scaffold in tissue engineering, the fabrication of microvascular stamps and the examination of the cell to cell interactions53-54. Micron and nanoscale features affect the cell behavior to proliferate and differentiate. As SLA is able to fabricate structures in the micron range and uses UV wavelength, researchers have been looking into other technologies fabricating in the nanoscale range as well as another wavelength that is less toxic to the cells.

(a)

(b)

Figure 7. Schematic illustration of Stereolithography (a) Scanning Stereolithography, and (b) Projection Stereolithography. Reproduced with permission from ref 55. Copyright 2014 American Chemical Society 16 ACS Paragon Plus Environment

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3.2. Laser direct writing and multi-photon polymerization for free-form 3D structures DLW is a process which involves focusing a laser beam onto the material of interest in a specific pattern thus creating either 2D or 3D structures56. These patterns can be created or traced by controlling the laser beam focus point with galvanometric scanners or by moving the sample on a high-resolution XYZ stage with a fixed focal point27. As the laser uses a wide range of wavelength to interact with the material, this versatility allows the user to add (polymerisation), subtract (ablation) or modify different materials without harsh chemicals, preheating of the material and the need for physical contact between the tools and material allowing the user to create true 3D structures27, 5657

. The few advantages have generated interest in using DLW for the fabrication of many

biomedical devices such as stents, prostheses, sensors, drug-delivery devices and tissueengineering scaffold27, 56.

3.2.1. System configuration A typical laser setup shown in figure 8 (a) and (b) involves: (1) Laser source, (2) beam/sample motion system, (3) beam focusing optics, (4) beam intensity control and beam shutter and (5) control software27. 1. Laser source – The laser provides the spatially coherent energy needed for processing of material. Ultra-short pulse laser (ie. Ti: sapphire lasers, Ytterbiumdoped laser or frequency doubled Er-doped fiber), especially in the femtosecond range, allows for high energies to be emitted in longer wavelengths, leading to nonlinear optical processes such as two-photon absorption (TPA). Some key



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factors to consider when choosing a laser source includes laser wavelengths provided for absorption by the material, pulse durations, and energy, repetition rates, laser exposure time, average and peak powers. 2. Motion systems – Galvanometric scanners (movable mirrors) for moving the laser beam or moving the sample on high-resolution XYZ stages (resolution approximate several nm to µm) can be used with the laser to “write” or create the 3D structures. There can be various combinations of the motion stages for realizing 3D motion. The representative examples are three-axis PZT flexure stage, three-axis motorized stage, 2D galvano-scanner with a height control servo; all these scanning systems are x-, y-, z- Cartesian coordinate. For central symmetric structures, r-, theta-, z- scanning systems with one rotational, two translational stages can be also used for the 3D motion. 3. Focusing optics – A consistently focused laser beam is required for maintaining the high intensities of the laser. Usually, a standard microscope objective lens is used to focus the laser. The spot size of the laser is determined by altering the numerical aperture (N.A) of the focusing optics. Immersion oil lens may be used when N.A of the objective is higher than 1. 4. Beam intensity control – The beam intensity and exposure onto the sample can be controlled by 2 types of systems. Beam on-off control can be achieved by using a fast mechanical shutter or an electro/acousto-optic modulator while beam intensity control can be achieved using neutral density filters, a variable attenuator or a combination of a polarizer and a waveplate.


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5. Control/monitoring software – A central computer with different integrated programs is used to allow synchronization for both optical and mechanical accessories. Charge-coupled devices (CCDs) with different objectives lens can be used for capturing images and monitoring processes in real time. For two-photon polymerization (TPP), two representative lasers are crystal-based mode-locked Ti:Sapphire femtosecond lasers and frequencydoubled Er-doped fiber femtosecond lasers at the near-infrared central wavelength around 800 nm. Thanks to the TPP process, the photo-sensitive polymers having the absorption wavelength shorter than 400 nm can be efficiently polymerized. The optimal laser parameters differ depending on the target materials but the general conditions can be summarized as following. The pulse duration is less than 300 fs, the repetition rate is less than 100 MHz, the power is less than 200 mW, and the magnification of the objective lens for the printing is less than 60X. In the writing of the waveguide inside the transparent material, much higher peak power is required because it is based on material damage by multi-photon absorption phenomena. Generally, lower repetition rate high-power amplifiers, like Ti:Sapphire regenerative amplifiers or multi-pass amplifiers are requested for the efficient manufacturing of the waveguides.



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(a)

(b)

Figure 8. (a) Schematic demonstration of a laser set up for TPP. Reproduced with permission from ref 58. Copyright 2010 The American Society of Mechanical Engineers. (b) Schematic diagram of femtosecond laser waveguide writing set up. VA: variable attenuator, MS: mechanical shutter, SHG: simple harmonic generator (optional), PC:


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polarization controller, BSO: beam shaping optics (optional), MO: microscope objective. Reproduced with permission from ref 59. Copyright 2011 John Wiley and Sons. 3.2.2. Non-thermal laser ablation (single photon) Femtosecond laser ablation as first demonstrated on polymethylmethacrylate in 198760-61. Over the next 30 years, precision and resolution of the ablation area have improved from the microscale to the nanoscale62. Laser ablation is a process using optical energy (photon) from the laser to remove material from a solid. Large numbers of an electron in the solid are being ionized by the laser resulting in phase or structural modifications. Depending on the amount of energy absorbed and the depth of absorption permanent change in the refractive index or even a void in the material can occur. The spot sizes of the laser and absorption wavelength of the material are some factors in determining the resolution of the ablation. One point to take note as laser ablation is performed by removing a small amount of material; it cannot take place at locations where the laser path is obstructed. Power intensity and pulse duration play a huge role in how the laser beam interacts with the material. The high energies and short pulses are desired as it reduced thermal effects around the spot. Generally, the heat diffusion to the surrounding materials takes several tens of picoseconds. Therefore, the shorter pulses than this heat diffusion will minimize the unexpected thermal effects to surrounding base materials. The high energy is required to initiate photo-chemical or photo-thermal effects to the materials. Especially, in micro/nano 3D printing based on TPP, high-level pulse energy is the prerequisite for starting nonlinear two-photon absorption (TPA) process56. To create interior geometries and structures within a bulk material an ultrafast pulse laser is used. CW laser ablation occurs when the materials adsorb and convert the light energy to heat 21 ACS Paragon Plus Environment


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energy causing the material to melt leaving behind a huge heat-affected zone (HAZ) (Figure 9). However, for ultra-fast pulses (picosecond and femtosecond ones), due to the ultrashort pulse width, there cannot be efficient heat conduction to the surrounding lattice structure which enables a well-defined clean manufacturing with minimal physical damage to the nearby materials63-64. Other advantages include micro-sized structure creation, no collateral damage to the surroundings, clean process look, no material property change, and capability for transparent material sub-surface engraving. TPA with Femtosecond lasers may be used for ablation of materials that exhibit poor absorption such as fused quartz and various glasses62.

(a)

(b)


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Figure 9. (a) Laser material interactions between different types of laser. (b) Holes drilled in 100 mm thick steel foils by ablation using different laser pulses. On the left using fs laser while on the right using a nanosecond (ns) laser. Reproduced with permission from ref 64. Copyright 2014 Nature Publishing Group. 3.2.3. Multi-photon laser polymerization (multiphoton) Polymerisation is defined as a process where a large volume of small molecules is joined together to formed a big molecule. The process of two-photon polymerization (TPP) is like the SLA process. A laser is used to excite photoinitiator molecules, creating free radicals causing chemical reactions between photoinitiator molecules and monomers/oligomers within a transparent resin. The key difference between the SLA and the TPP process is the use of two-photon absorption (TPA) for excitation of photoinitiator molecules shown in figure 10. To achieve TPA, a femtosecond laser is utilized65-66. A virtual state is created where two-photon of same or different frequency are absorbed by the photoinitiator (Figure 10). This enables longer wavelength to possess a similar electronic excitation like a single photon which requires much higher energy65-66. This nonlinearity of TPA not only excites the photoinitiator molecules with less energy but material crosslinking can occur within the immediate vicinity of the focal volume67, leading to significantly higher resolutions than other direct write techniques (up to 100 nm)67. Ultrashort pulsed lasers such as femtosecond lasers are generally used for multiphoton processes, as they are based on second or third order non-linear optical phenomena which are much more efficient at higher intensities. TPP provides several advantages over conventional processes especially in the fabrication of small medical devices. There is a wide range of near infrared transparent materials such as GelMA and PEG-DA which are biocompatible and suitable for tissue engineering68. The cost should 23 ACS Paragon Plus Environment


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be reduced as no cleanroom facilities and specialized equipment besides the laser are required

69

as compared to other fabrication techniques such as deep reactive ion etching

and lithography70. This flexibility in the future may lead to fabrication of a medical device within close proximity of an operating room or another clinical site.

(a)

(b)

(c)

Figure 10. (a) Mechanism of simultaneous excitation by TPA. (b) and (c) Schematic representation of two methods for increasing the TPA probability in which density of photon is increased by (b) spatial compression using high numerical aperture (NA) objectives, (c) temporal compression using ultrafast lasers. Reproduced with permission from ref 71. Copyright 2013 Intech Open. 3.2.4 Higher degree of freedom; by combining laser lithography and ablative laser machining By tuning different wavelength provided by the laser, subtractive fabrication i.e. ablation or selective etching and additive fabrication i.e. TPP can be achieved together with just one laser set up. Sugioka and team were able to combine both methods thus


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creating a hybrid femtosecond laser microfabrication to achieve true 3D glass/polymer composite biochips with multiscale features and high performance72 (Figure 11). Biochips or lab on the chip is “miniaturized laboratory” where multiple biological reactions can be detected, separated and analysis within one chip in a highly efficient and sensitive way. Additional advantages include low material consumption, low cost, and compactness. In order for these different features to be fabricated on the biochip, a variety of methods such as photolithography73, soft lithography74-75, and nanoimprint lithography76 have been used while addition post-processing may be required as well. When comparing the hybrid technique to the methods mention above, three advantages can be observed. (1) True 3D microfluidic structures can be fabricated without complications such as stacking and bonding/sealing with other substrates. (2) Maskless and free-form technology allows for a more biomimetic environment for cell culture77-79. (3) Additional polymer microstructures which provide additional functionality can be integrated after the chip had been made. In order to fabricate the biochip using the hybrid technique, two main steps are involved. For creating the 3D hollow microchannels, TPA was achieved using Femtosecond (fs) laser on the photosensitive Foturan glass. Surface smoothness is improved by thermal annealing. Next 3D polymer microstructures were fabricated by TPP for chip functionalization. This hybrid method allows the user to create biochips with different functionalities with better speed and greater flexibility80.



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Figure 11. (a) Scheme presenting the fabrication process of 3D ship-in-a-bottle biochip by hybrid fs laser microprocessing. It proceeds with fs laser scanning followed by 1st annealing, HF etching, 2nd annealing, polymer coating, TPP, and development. (b–e) Images of 3D Y-shaped microchannel (b) laser scanning and the 1st annealing, (c) HF etching, (d), 2nd annealing (e) 3D polymer microstructure by TPP (f) SEM images demonstrating 45° tilted and top view of 3D microchannels. Six types of shapes with varying sizes from 250–280 µm (rectangle, round, elliptical, pentagram, triangle and hexagon,) were fabricated. Reproduced with permission from ref 72. Copyright 2014 John Wiley and Sons.


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3.3. Materials for laser direct writing and multi-photon polymerization In order to create internal 3D structures, materials must not be able to have any linear absorption at the wavelength of the femtosecond laser. The use of femtosecond laser have advantages over another laser such as (1) the nonlinear nature of the absorption confines any induced changes to the focal volume and (2) the absorption process is independent of the material, enabling optical devices to be fabricated in compound substrates of different materials. There are two main materials used for 3D fabrication of scaffolds (1) Glass and (2) Photopolymers. Glass Glass is one of the preferred choices for making microfluidics for biological application. It is chosen due to its low background fluorescence, high strength, and thermoconductivity81. The excellent inertness of glass makes it biocompatible with low nonspecific absorption. However, it is not gas permeable making less feasible for long term cell studies. Traditional glass microfluidic chips are created by etching into the glass by wet or dry methods with the formation of fluidic features such as valves and pump require bonding or hybrid layer attachment to enclose81. With the used of a femtosecond laser, whole microfluidics chips can be fabricated, even with micro components such as micropumps and microvalves82. Table 2 shows the summary of materials and laser used.

Table 2: Summary of materials and lasers used 27 ACS Paragon Plus Environment


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Type of Glass

Laser Source

Part of the Chips/resolution

Application

Ref

Photostructurable glass (FOTURAN)

Ytterbium-Doped Femtosecond Fiber Lasers with a pulse of 150fs, wavelength of 775 nm wavelength, and repetition rate of 1 kHz

MicroChannels up to 150 µm

“Nanoaquariums” for the studies of motility of bacteria ( Euglena gracilis

83

Photostructurable glass (FOTURAN)

Ytterbium-Doped Femtosecond Fiber Lasers with a pulse of 360fs, wavelength of 1045nm and repetition rate of 200kHz

MicroChannels up to 100 µm

“Nanoaquariums” for the studies of motility of bacteria (Phormidium)

84

Fused silica substrate

Ytterbium-Doped Femtosecond Fiber Lasers with a pulse duration of 460fs, wavelength of 1047nm and repetition rate of 500kHz

MicroChannels up to 100µm

Mammalian Cell sorting

85

FoturanTM glass

Laser with a pulse duration of 150 fs and wavelength of 775 nm

Microplate acting as a Microvalve in a microreactor up to 1mm

Control the flow direction of fluids in the microreactor.

86

Porous glass and fused silica

Laser with a pulse duration of 340 fs, wavelength of 1,045 nm wavelength, and repetition rate of 200 kHz

Micropump, micromixer, nanograting up to 40nm

Control the flow direction and mixing of fluids in the fluidic chip

87

Silica glass

Ti: sapphire laser with 20Hz laser pulses and wavelength of 800 nm

The optical rotator of different shapes up to 12 µm

Acting either as a pump or mixer

88

Silica glass

Ti: sapphire laser with a pulse duration of 120fs, wavelength of 800nm and a repetition rate 1–100 kHz

3D multilayer microfluidic chips up to 150 µm

Increasing the flexibility and complexity of chip

89

Silica glass

Ti: sapphire laser with a the pulse duration of ~100 fs, wavelength of 800 nm and a repetition rate of 250 kHz

Nanofluidic channels up to 50nm periodic nanograting

DNA analysis, e.g. stretching of DNA molecule

90


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Photopolymer Photopolymers are polymer materials which physical or chemical properties undergo a change when exposed directly or indirectly with light91. It comprises of a composite material containing at least two basic components: (1) a polymerizable material possessing polymerizable functional groups to form the polymeric structure backbone, and (2) a photoinitiator to provide the active species after absorbing the laser energy for the polymerization27,

91

. To date, a large combination of polymeric materials and

photoinitiator combinations have been used biological applications. Most materials are in the form of negative photoresists such as acrylate materials, hybrid materials, and hydrogels92. In the following sections, we will discuss briefly on the photoinitiators and hydrogels for bioapplications.

Photoinitiators There are two main classes of a photoinitiator based on the nature of active species they generate: (1) radical photoinitiators and (2) cationic photoinitiators. Radical photoinitiators generate free radicals which initiates the polymerization of acrylates or vinyl ethers while cationic initiators produce cations, which are used for the polymerization of epoxides or vinyl ethers27. Nowadays, efforts are being made to synthesize fast and efficient photoinitiators specifically for multi-photon applications93. Moreover, there are lots of efforts to synthesize biocompatible photoinitiators, explicitly for bio-applications. Classic dyes such as Bengal Rose, Eosin, Nile Red, biomolecules such as Flavin mononucleotide are some of the more common natural biocompatible photoinitiator available27.



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Material

Natural or Synthetic

Photo-initiator

Laser source

Cells

Application

Ref

Bovine Serum Albumin (BSA) 1.5 × 10-4 M, or 1% w/w fibrinogen

Natural

Rose Bengal

Titanium: sapphire femtosecond laser (100 fs, 76 MHz, 700 – 1000nm)

Mouse-lymphocytic leukemia cells (L1210 cell), fibroblast, neurons

Drug delivery, devices of cell sorting, cell encapsulation, and tissue engineering

94-95

Trimethylol-propane triacrylate (TMPTA)

Synthetic

Rose bengal and the co-initiator triethanolamine (TEA) (0.1 M)


L1210 cell, fibroblast neurons

Drug delivery, devices of cell sorting, cell encapsulation, and tissue engineering

94-95

Collagen (type I, II, IV)

Natural

Modified benzophenone dimer (BPD)

Titanium: sapphire femtosecond laser with pulse energies at the 500 pJ/pulse.

Primary human dermal fibroblasts

Tissue scaffold

96

Collagen type I

Natural

Rose bengal amine derivative.


-

Drug delivery or tissue scaffold

97

BSA

Natural

Methylene blue (1.2– 3 mM) or flavin adenine dinucleotide (FAD; 5 mM)

Titanium: sapphire laser (76kHz, 730–740 nm.)

E.coli

Cell studies

98-99

BSA

Natural

Flavin adenine dinucleotide disodium salt hydrate(1-4 mM)

Laser (790 m), entering the microscope were 30-60 mW.

Neuroblastomaglioma cells (NG10815)

Tissue scaffold

100

(1 × 10-4 M)


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PEG-DA (MW 742)

Synthetic

2 wt% (Irgacure 369, Ciba)

Ti:sapphire oscillator (120 fs, 80 MHz, 780 nm)

Ovine endothelial cells (ECs)

Tissue scaffold

101

2-hydroxyethyl methacrylate (HEMA) and PEG-DA

Synthetic

2,2-dimethoxy-2phenyl acetophenone (Irgacure 651)/ 2photon sensitive chromophore (AF240). P127 act as a surfactant

Titanium: sapphire laser operating mode-locked (~80 MHz, ~200 fs) at a nearinfrared wavelength of 780 nm was used for fabrication

-

Tissue engineering

102

Hyaluronic acid-glycidyl methacrylate (HAGM) or HA– PEG-DA

Natural

Irgacure 2959

Ytterbium femtosecond laser pulses (250 fs, 21 MHz, 520 nm)

Human dermal HFF1 fibroblasts and human osteoblast-like cell line MG-63

Tissue engineering

103

Gelatin-methacrylamide (GELMA)

Natural

Irgacure 2959

Femtosecond laser emitting at around 515 nm

Primary adiposederived stem cell (ASC)/ Porcine Mesenchymal Stem Cells

Tissue engineering scaffold

104-105

Vinyl ester derivative of gelatin hydrolysate

Natural

WSPI ,4- bis(4-(N,Nbis(6-(N,N,Ntrimethylammonium) hexyl)amino)-styryl)2,5dimethoxybenzene tetraiodide

Ti: sapphire laser system (80fs, 75MHz, 800 nm)

Human osteosarcoma cell line MG63

Cell studies

106

PEG-DA

Synthetic

G2CK, E2CK, P2CK

Ti: sapphire laser (100fs, 80MHz, 780nm)

MG63 osteosarcoma cells and outgrowth endothelial cells

Tissue engineering

107



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(OEC) GELMA

Natural

G2Ck and P2CK

Ti: sapphire femtosecond laser (80fs, 75MHz, 800nm)

MG63 osteosarcoma cells

Tissue engineering

108

PEG-DA (302MW and 742mw)

Synthetic

Michler’s ketone (4,4′bis(diethylamino)ben zophenone) / irgacure 2959 and 369

Ti: sapphire femtosecond laser pulses (120 fs, 80 MHz, 780 nm)

L929 mouse fibroblasts

Cell studies and tissue engineering

109

PEG-DA, 700 Da

Synthetic

WISP

A pulsed, laser beam of a Ti: sapphire laser (100fs, 73 MHz, 810 nm)

Caenorhabditis elegans

Scaffold

110

A combination of Synthetic Acrylamide (AAm), N,N′methylenebis(acrylamide)(MB AAm), Acrylamide (AAm), N, N′-methylenebis(acrylamide) (MBAAm)

Benzil and 2-benyl-2(dimethylamino)-4′morpholinobutyrophe none

Ti: Sapphire laser (80 fs 780 nm, 82 MHz)

-

Biomedical applications (microactuators and micro manipulators)

111

PEG-DA, 700 Da

Synthetic

2,7-bis(2-(4pentaneoxy-phenyl)vinyl)anthraquinone (N) with a C2vsymmetrical structure and 2hydroxypropyl-βcyclodextrin

Ti: sapphire femtosecond laser beam (120 fs, 80 MHz, 780 nm)

-

Scaffold

112

Matrix metalloproteinasesensitive peptide (GGPQGIWGQGK,

Synthetic

2,2-dimethoxy-2A laser tuned to 720 nm with phenyl- acetophenone a scan speed of 25 µsec/pixel and an intensity of 60

Human umbilical vein endothelial cells

Scaffold/cell studies

113


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abbreviated PQ) into the backbone of PEG-DA derivative

(DMAP)

mW/µm2 was then used to excite

(HUVECs,

AKRE (an acrylate based custom made photopolymer AKRE37 consisting of tris (2hydroxy ethyl) isocyanurate triacrylate and 4,4’-bis(dimethyl amino) benzophenone), ORMOSIL (hybrid organic– inorganic SZ2080 material) ORMOCER and biodegradable PEG-DA (MW 258)

Synthetic and natural

-

Ti: sapphire laser with average output power of 500 mW (80fs, 80MHz, 800 nm)

Primary stem cell culture derived from adult rabbit muscle

Scaffold

114

PEG-DA, MW 700

Synthetic

1% (w/v) of the photoinitiator Irgacure 819

800 nm Ti: sapphire femtosecond laser

-

Tissue scaffold

115

PEG-DA MW 700

Synthetic

Lithium phenyl2,4,6-trimethyl benzoylphosphinate (LAP)

Ti: sapphire femtosecond laser (80MHz, 800nm) with a maximum power of 350 mW.

Anchorage-dependent Cell studies embryonic fibroblast 10T1/2 cell line

116

Fibrinogen, fibronectin, lectin Con A, BSA

Natural

Rose bengal

Ti: sapphire laser (100fs, 76 MHz, and 800nm)

-

Scaffolds and ECMs for tissue engineering.

117

BSA only (Sigma, 10 mg/mL) and BSA (50 mg/mL)/laminin (Millipore, 0.5 mg/mL) mixture

Natural

Rose Bengal (1 mM)

Ti: sapphire laser are approximately 5 mW, or about 100 pJ per pulse.

H9 embryonic stem cell-derived human mesenchymal stem cells (MSCs)

Scaffolds

118



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Hydrogels Hydrogels are materials capable of retaining water in it 3D polymeric network. Most common hydrogels possess only 0.5– 20 wt% of dry polymer mass with the remaining part consisting of water which provides a high resemblance to the extracellular matrix and providing good biocompatibility with living organisms. In addition, the soft and elastic properties minimize irritation and damage to surrounding tissues. For the preparation of hydrogels, well-designed polymeric chemical structure and architecture is essential. Depending on their origin, the polymeric compositions are classified into natural polymer hydrogels (e.g. collagen, dextran, fibrin, chitosan) synthetic polymer hydrogels (e.g. poly(ethylene glycol) derivatives) and their combination (e.g. collagenacrylate, alginate-acrylate). Depending on the biological applications each type of hydrogels has its advantages and disadvantages. For example natural hydrogels are more suited for supporting cellular activates while being easily degraded with water-soluble enzymes, however, they are more susceptible to pathogens, causing an immune response, batch variation, and low mechanical property. On the other hand, synthetic hydrogels can be precisely controlled and adjustable for a wide range of applications with better mechanical strength but low biodegradability (degradation via hydrolysis, solubilisation, and mechanical erosion), inherent bioactive properties and possibly toxic substances released are some of its disadvantages.


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4. Applications in cell biology Femtosecond-laser-based 3D printing of biomaterials and cells scaffolds can be potentially used for various biomedical applications, from drug delivery and diagnostic testing to in vitro tissue engineering and regeneration. 4.1 3D cell cultures Klein et al. have fabricated polymer composite microstructures of with distinct protein-binding properties as shown in figure 12. Ormocomp was being used as a biocompatible photoresist for the protein-binding cubes in the scaffolds119. Proteinrepelling frameworks were generated by using photoresist composed of PEG-DA and 3% (w/w) Irgacure 369 as a photoinitiator, while PETTA was used as cross-linker to enhance the mechanical properties of these 3D scaffolds. Various Concentration of PETTA (0, 4.8, 9.1, 33.3, 100% (w/w)) in PEG-DA were prepared and 2D patterns were fabricated by DLW. Moreover, Ormocomp squares (40 µm × 40 µm) were embedded in a PEG-DA with increasing PETTA concentrations.

Figure 12: SEM pictures demonstrating the 3D fabrication of polymer composites scaffolds by DLW. (a) 3D microstructures containing PEG-DA - 4.8% PETTA were polymerized, followed by the developing. (b) Photoresist ormocomp cubes (one cube was shown in red colour for demonstration) were attached to the PEG-DA beams with 35 ACS Paragon Plus Environment


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precision. (c) An Ormocomp cube (red colour) in high magnification. Reproduced with permission from ref 119. Copyright 2011 John Wiley and Sons.

PEG-DA - 4.8% PETTA pillars having ~ 7 µm diameter and ~23 µm height interconnected by 1 µm diameter beams having 10 µm length (Figure12 a) were fabricated by DLW. Subsequently, the scaffolds were developed by isopropyl alcohol (IPA) and casted with the ormocomp (second photoresist). Ormocomp cubes having the dimensions of 2.5 µm were adhered in the middle of PEG-DA beams by polymerization; after the development of first microstructures (Figure 12 b,c). Spatially controlled 3D microstructures containing protein binding ormocomp cubes and protein repellent PEGDA scaffold has been fabricated by DLW. As predicted upon the cultivation of fibronectin, ECM protein preferentially binds to the ormocomp part, followed by the culture of fibroblasts on the scaffolds. Interestingly Klein et al. have found that cells were adhered to ormocomp or connected through the several cubes and studied via confocal microscopy (Figure 13 a). 3D growth pattern of cells within the interconnected can be observed in figure 13 b, c. Immunostaining by actin has demonstrated the periphery of the adhered cells while cell contact sites were majorly and the ormocomp cubes not on PEG-DA part was revealed by paxillin staining (Figure 13 d-f). It was revealed that the cells cultured in these scaffolds specifically form cell adhesion sites in the ECM functionalized sections. This research has shown the future possibilities for systematic studies of spatial ligand distributions and stiffness of the scaffold on the cell behavior in 3D environments.


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Figure 13. Confocal images showing the cell growth in 3D scaffolds. Primary chicken fibroblasts were cultivated in the scaffolds and immunostained for fibronectin (red), factin (green), and paxillin (yellow). (a) Top view of a image stack illustrating that the cells were adhering to the fibronectin-positive ormocomp cubes, (b) Top view showing cells adhered to the 4 ormocomp cubes, (c) 3D reconstruction of image stack showing a single cell adhering to ormocomp cubes in different heights, (d–f) Single confocal sections of a cell adhering to seven ormocomp cubes. (d) f-actin fibers, (e) paxillin immunofluorescence, and (f) overlay image. Reproduced with permission from ref 119. Copyright 2011 John Wiley and Sons.



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4.2 Micro molding of biological scaffolds Koroleva et al. has generated TPP scaffolds and replicated it by using micro molding120. These microstructures were fabricated via TPP on a glass substrate and a negative mold of PDMS was prepared by polymerizing it at 100 °C for 1 h. PDMS was separated by using the piezo stage and the mold was covered by photosensitive PLA polymer. The desired biocompatible PLA microstructures were polymerized by the UV light and separated by using piezo stage. The well-defined hexagonal hollow cylinders having height, wall thickness and diameter of 300 µm, 20 µm and 100 µm were obtained. The photocured material can be easily recovered from the PDMS mold and can be used for subsequent replicated batches of the PLA. Figure 14 shows the SEM image of the TPP scaffold and replicated scaffolds.

Figure 14: SEM images of the microstructures fabricated by (left) TPP and (right) microreplication. Reproduced with permission from ref 120. Copyright 2013 IOP publishing.


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Koroleva et al. seeded primary Schwann cells on the micro-replicated 3D scaffolds for 7 days and observed it by SEM120. It was observed that the Schwann cells adhered to the PLA 3D microstructures in spindle-like and flat cell morphologies. The spindle-like cells adhered on the 3D structures (Figure 15(a)) while, flat morphology cells bounded between the cells and the walls of the PLA scaffold (Figure 15(b)) and indicating the cell growth.

(a)

(b)

Figure 15. (a) SEM images of primary Schwann cells on 3D scaffolds (a) magnification 1750×, scale bar 50 µm and (b) magnification 1250X, scale bar 100 µm. Arrows indicate spindle-like morphology cells: (a) bridging structural features (b) flat morphology cells lining the intra-luminal walls. Reproduced with permission from ref 120. Copyright 2013 IOP publishing.

4.3 Cell-matrix invasion Cell-matrix invasion is an important part of the pathological process which includes leukocyte extravasation and cancer cell metastasis. In recent years cell matrix invasion was studied by the migration of cells within the polymer scaffolds which plays 39 ACS Paragon Plus Environment


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the role of ECM. In order to study the cell matrix invasion, Greiner et al. have fabricated the well-designed polymer cell scaffolds DLW as shown in figure 16. 3D microstructures of biocompatible polymer PETTA with different mesh sizes (2,5 and 10 µm) were fabricated. Additionally, fibronectin was coated to enhance the cell adhesion and matrix invasion121.

Figure 16. Microporous 3D cell culture scaffolds produced by DLW. SEM images of 3D PETTA scaffolds with mesh sizes of 2 µm, 5 µm, and 10 µm fabricated by DLW. Reproduced with permission from ref 121. Copyright 2014 Elsevier.


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It is well-known fact that cells change their morphology while migrating through the pores of ECM during embryogenesis, wound healing, or metastasis. Greiner et al. have studied the effect of nucleus stiffness on the scaffold invasion. MEF WT and MEF lmna KO cells were cultured on the scaffolds with 10 µm pores for 24 h and were fixed and labeled with DRAQ5 to visualize the nucleus of the cells. It was interesting to observe that both cells have different invasive behaviour (Figure 17A and B). 55% of the MEF WT cells were able to enter in the scaffold, while 80% of MEF lmna KO cells were partial or fully embedded in the scaffold (Figure 17C). MEF lmna Ko cells have a large percentage of cell matrix invasion as compared to MEF WT cells. The nucleus of MEF lmna KO cells was smaller than the MEF WT cells, which deforms its morphology during cell migration. Moreover, additional AFM and invasion studies have also confirmed the link between nucleus stiffness and scaffold population. Thus 3D printed scaffolds with predefined pore sizes can be used for cell migration and invasion studies with different ECM molecules. Moreover, 3D printed scaffolds can be potentially used for screening and determining cancer stages based on the nuclear mechanics and matrix invasion.



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Figure 17. Fibroblast invasion into 3D scaffolds on glass. (A, B) Top and side view 3D reconstructions of wild-type mouse embryonic fibroblasts (MEF WT) and lmna knockout MEFs(MEF lmna KO) on 3D scaffolds with a mesh size of 10 µm. The cell nuclei of MEF WT and MEF lmna KO either localized on top of the 3D scaffold, or they were partially or fully embedded within the 3D structure (nuclei ¼ red, F-actin ¼ green, 3D scaffold ¼ white). (C) Significantly more cell nuclei were partially or fully embedded in the KO cell line than for WT cells (chi-square test; *, p 72 cells from 5


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independent experiments) Reproduced with permission from ref121. Copyright 2014 Elsevier.

4.4. Targeted cell delivery Kim et al. have demonstrated multifunctional microrobots for targeted cell delivery using 3D laser lithography122. Ni and Ti layers were coated fabricated microrobots to induce the magnetism and biocompatibility, respectively, followed by the coating of Poly- L -lysine (PLL) before cell culture HEK 293 cells were used for the cell culture on the fabricated microstructures and were affixed using paraformaldehyde solution. SEM imaging was performed after 96 h. Figure 18 showed SEM and confocal microscopy images of the microrobots. Interestingly filopodia formation was observed during cell migration, as shown in figure 18b, which was the indication of the cell interaction with microrobots. There were no signs of cytotoxicity and biocompatibility analysis was confirmed by the cell adherence, migration, and proliferation of the structures. The movement of microrobots was demonstrated by the application of the external magnetic field, which can be used for in-vivo for the cell transportation, gene delivery, and drug delivery applications.



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Figure 18. (a) SEM image of a hexahedral microrobot after cell culture and (b) high magnification SEM image showing filopodia.Confocal microscope images of the (c) hexahedral and (d) cylindrical microrobots after staining of the cells. Reproduced with permission from ref 122. Copyright 2014 John Wiley and Sons.

5. Conclusion and Future Prospects This Review showed the recent trends of cell biology applications enabled by ultra-short femtosecond laser pulses, followed by the latest lithography studies and system developments. DLW and multi-photon polymerization have gained a lot of attention due to a higher degree of freedom for generating free-form 3D structures precisely by using visible and NIR wavelength. As introduced, in the recent studies, there has been much more focus on the lithography techniques based on ultra-short pulse lasers rather than using traditional UV- or mask-based lithography for biological applications. In addition, femto-second laser based 3D printing can be used for cell-to-cell interaction studies in the confined micro-environment, cell property measurements, bio MEMs and 44 ACS Paragon Plus Environment

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microfluidics, in near future. DLW of multiple cells in Femtosecond-laser-based 3D lithography has emerged as a powerful tool and will expand its own leadership in the field of tissue engineering and cell biology. Acknowledgements This study was financially supported by Singapore National Research Foundation (NRFNRFF2015-02), Singapore Ministry of Education (MOE) under its Tier 1 Grant (RG85/15, RG35/12, and RGC4/13) and the Singapore Centre for 3D Printing. It was also supported based on research collaboration agreement by Panasonic Factory Solutions Asia Pacific (PFSAP) and Singapore Centre for 3D Printing (RCA-15/027).

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Table of Contents (TOC)

Review: Femtosecond-laser-based 3D printing for Tissue Engineering and Cell Biology Applications #

#

Chee Meng Benjamin Ho 1, 2 , Abhinay Mishra 1,2 , Kan Hu1,2, Jianing An 1, Young-Jin Kim1,2 * and Yong-Jin Yoon 1,2* 1

School of Mechanical and Aerospace Engineering, Nanyang Technological University (NTU), 50 Nanyang Avenue, 639798 Singapore 2 Singapore Centre for 3D Printing, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798


Femtosecond-Laser-Based 3D Printing for Tissue Engineering and

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