Femtosecond-Laser-Based 3D Printing for Tissue Engineering ...static.tongtianta.site/paper_pdf/b9221360-8a6d-11e9-b8ad-00163e08bb86.pdfthe future in tissue engineering and biology

Femtosecond-Laser-Based 3D Printing for Tissue Engineering andCell Biology ApplicationsChee Meng Benjamin Ho,†,‡,§ Abhinay Mishra,†,‡,§ Kan Hu,‡,§ Jianing An,‡ Young-Jin Kim,*,‡,§

and Yong-Jin Yoon*,‡,§

‡School of Mechanical & Aerospace Engineering and §Singapore Centre for 3D Printing, Nanyang Technological University, 50Nanyang Avenue, Singapore 639798

ABSTRACT: Fabrication of 3D cell scaffolds has gainedtremendous attention in recent years because of itsapplications in tissue engineering and cell biology applications.The success of tissue engineering or cell interactions mainlydepends on the fabrication of well-defined microstructures,which ought to be biocompatible for cell proliferation.Femtosecond-laser-based 3D printing is one of the solutioncandidates that can be used to manufacture 3D tissue scaffoldsthrough computer-aided design (CAD) which can beefficiently engineered to mimic the microenvironment oftissues. UV-based lithography has also been used forconstructing the cellular scaffolds but the toxicity of UV light to the cells has prevented its application to the direct patterningof the cells in the scaffold. Although the mask-based lithography has provided a high resolution, it has only enabled 2D patterningnot arbitrary 3D printing with design flexibility. Femtosecond-laser-based 3D printing is trending in the area of tissue engineeringand cell biology applications due to the formation of well-defined micro- and submicrometer structures via visible and near-infrared (NIR) femtosecond laser pulses, followed by the fabrication of cell scaffold microstructures with a high precision. Laserdirect writing and multiphoton polymerization are being used for fabricating the cell scaffolds, The implication of spatial lightmodulators in the interference lithography to generate the digital hologram will be the future prospective of mask-basedlithography. Polyethylene glycol diacrylate (PEG-DA), ormocomp, pentaerythritol tetraacrylate (PETTA) have been fabricatedthrough TPP to generate the cell scaffolds, whereas 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 usedfor studying cell migration, matrix invasion and nuclear stiffness to determine stage of cancer and will open broader horizons inthe 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 thatinvolves the use of cells and a scaffold/matrix to develop newfunctional tissues for implantation back to the donor. With thetremendous need for organs and tissue, tissue engineering wasdeveloped to fabricate a living replacement for parts of thebody.1,2 The necessity of tissue engineering has been furtherdemonstrated with the widening gap between supply anddemand for transplantable tissues or organs around theworld.3,4 One vital component of tissue engineering is theuse of scaffolds. Scaffolds provide a conducive environment andact as a support for tissue attachment and growth. Scaffoldscould also serve as a carrier or template for implanted tissue ordelivery of other agents. Currently, there are numerousstrategies used to fabricate scaffolds with the differentbiomaterials available (Figure 1).In a biological environment, the ideal function of a scaffold is

to organize the cells into a three -dimensional (3D) architectureand present stimuli that direct the growth and formation of thedesired tissue.1 This scaffold will also function as a synthetic

extracellular matrix (ECM) providing adhesive surfaces for cellsto attach to and deposit their own protein to make them morebiocompatible. However, vascularization, lack of functionalcells, low mechanical strength of engineered cells, immuno-logical incompatibility with host, and nutrient limitations areclassical issues in the field of tissue engineering.5

During the past decade, it is recognized that physicalproperties of the extracellular environment with other chemicalfactors, such as growth factors, signaling, and adhesionmolecules, have an importance in affecting the cell behaviorand development.6,7 Topography on the scaffolds in the micro-and nanoscales, mechanical stiffness, and spatial patterning ofthe ligand are some exemplary physical stimuli used in tissueengineering.6−8 However, most cell behavior studies wereconducted previously were based on 2D platforms with simplegeometries such as grooves and ridges.7,9 This lead to the loss

Received: July 3, 2017Accepted: September 13, 2017Published: September 13, 2017

Review

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of specific cellular function, changes in cell morphology and cellto cell interactions to their extracellular matrix (ECM) whencompared to cell grown in a 3D environment.10−12 These days,research on fabrication of 2D or 3D scaffold of polymers suchas polycaprolactone (PCL), polyurethane (PU) and polylacticacid (PLA) and their nanocomposites has been carried out forits drug delivery and biomedical application.13−25 Althoughthese studies aid in our understanding of cell behavior in 3Dmatrices, the availability of various fabrication techniques (e.gcreation of random pore sizes), the chemical composition ofbiomaterials, and its mechanical properties prevents studyingcell behavior in a systematic and quantitative way.8 Therefore, anovel 3D cell culture biofunctionalization scaffold and methodshave to be developed for proper characterizations of physicalstimulus on cell behavior in a 3D environments.8

The emergence of new or improved micro and nano-fabrication techniques for 3D cell culture systems have becomea common trend over the past decade.9,26 Technologies such aselectrospinning, molding or the recent rise of 3D printing weredeployed in the construction of artificial 3D scaffolds. Theability to add material by layers with the use of a singlemachine, the possibility to create complex geometry scaffoldsand the power to control the overall porosity are the keyadvantages to which 3D printing has become such a popularoption. Figure 1 gives a schematic representation of opticalfabrications techniques in both micro- and nanoscales (shownin the 3 smaller circles) and their advantages for tissueengineering and cell biology applications. It is discernible fromthe Figure 1 that these techniques can be used vis-a-vis forfabrication of 3D scaffolds, template for implanting tissues,creating conducive environment for proliferation of cells andimproved accuracy on the positioning for the interaction ofcells. Nanotechnology can be used to create nanofibers,nanopatterns and controlled-release nanoparticles for applica-tions in tissue engineering to mimic native tissues, such asextracellular fluids, bone marrow, and cardiac tissues.5

Nowadays, 3D lithography has emerged as potentialfabrication technique, which uses the light to create patternsonto the surface of the substrate coated with photosensitivechemical. Generally, the photoresist mask is essential in mask-based interference lithography, which requires multistep mask

preparation processes. On the contrary, the direct laser writing(DLW) does not require mask so provides high designflexibility without additional processes.27 Multiphoton polymer-ization based on DLW enables to print very small structureswith a high printing resolution; the minimum voxel size reachesto sub-100 nm. Therefore, the printing resolution in multi-photon polymerization is not limited by the layer thickness as ithas been in stereolithography process. Table 1 shows a

comparison of stereolithography, multiphoton polymerizationand interference lithography. The resolution of the printedstructures through stereolithography is in micron level, whilenanometer level to submicrometer resolution can be printed bythe multiphoton polymerization and interference lithography.The major advantage of multiphoton polymerization or opticallithography over interference lithography is that it is not limitedto fabricate uniform disseminated periodic patterns but can alsofabricate arbitrary free form well-designed structures as neededfor the biological applications. Without the need of minimumlayer thickness, users are able to construct the 3D structure of abetter resolution. Many researchers have been considering theuse of optical systems over other techniques as it allows users toachieve better resolution with its ability to achieve nanometersize and grants them more control in the fabrication process. Inthis review, readers will be introduced to the different optical

Figure 1. Schematic representation of optical micro and nanofabrication techniques for tissue engineering and cell biology applications. Bulletspoints describes the advantages that these techniques provide in making scaffolds.

Table 1. Comparison between Interference Lithography,Stereolithography, and Multiphoton Polymerization or 3DOptical Lithography; the Resolution Also Partly Depends onthe Materials Properties

technologyinterferencelithography stereolithography

multiphotonpolymerization

process lightinterference

layer by layer direct laser writing

energysource

laser UV laser femtosecond laser

resolution up to 100 nm up to 25 μm up to 100 nmspeed fast fast slowmaterial photocurable photocurable photocurablefabricationadvantage

periodicpatterns

rapidprototyping

arbitrary free-formstructures withprecision

ACS Biomaterials Science & Engineering Review

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lithography techniques, common terms, and equipment used inthe setup of laser lithography and the materials that arecompatible with the laser setup and biological samples. Finally,a review of the current state of femtosecond-laser-based 3Dprinting or 3D laser lithography in the field of fabrication andapplications of the 3D scaffolds.

2. INTERFERENCE LITHOGRAPHY FOR PERIODICSTRUCTURES

Interference lithography is considered as a lithographictechnique which combines the diffraction and interferencephenomenon of light to fabricate multidimensional (e.g., 1D,2D, and 3D) periodic structures by using coherent beams.Generally, the inherent periodicity of incident light beams isbeing utilized to generate periodic structures, while the staticspatial orientation of light intensity is also formed by theinterference of these beams. The fabricated patterns aregenerated due to the transfer of emerged intensity distributionto the light sensitive materials such as photoresists orphotopolymers, moreover, these structures can be tailored bythe combination of different intensity beams.28,29 In general,there are two methods of beam configurations which can becurrently used to attain 3D interference patterns.2.1. Phase Mask Interference Lithography. Phase-mask-

based lithography is the simplest approach to interferencelithography. A 3D image is recreated after a coherent beam isexposed to the phase mask, and subsequently, gets transferredto the photoresist to generate interference patterns.30 In thislithographic technique, the elastomeric phase mask isconfigured just above the photoresist volume, followed by theincident light from the top of the phase mask. The structuresare then fabricated in the volume of the photoresist by thediffraction of beams. The general fabrication procedure ofelastomer phase mask has been depicted in Figure 2a, whileschematic diagram of phase lithographic setup was shown inFigure 2b. The 3D microstructure of fabricated SU-8 by using2D phase mask has been compiled in Figure 2c.31

2.2. Multibeam Interference Lithography. Multibeaminterference lithography can be defined as a technique in which3D pattern is created within the volume of a photoresist by theinterference of collimated, coherent laser beams for thefabrication of the targeted structure. In the multibeaminterference lithography setup generally, one laser beam isdivided into multiple beams using beam splitters and thenrecombined by mirrors to obtain the desired geometry,followed by the controlling of polarization and intensity ofthe beams by the wave plates and beam splitters. Moreover, thephase of more than four beams cannot be controlled in the freespace, is the drawback of this technique. Optical componentsused in the multibeam interference lithography, opticalarrangement, and fabricated SU-8 microstructure are presentedin Figure 3.31−33 Diffractive optical elements (DOEs) shown inFigure 3b have also been used for multibeam interferencelithography, with designed amplitude and phase distributions.Because of the invention of digital mirror devices, the degree offreedoms and update rate of DOE have been far improved.Precision optics and conversation of polarization are the

foremost requirements for the multibeam interference, as therefraction and polarization changes when the beam is enteredinside any photo material. To overcome this issue, the prismwith the same refractive index can be used for producinginterference lithographic patterns as demonstrated by Harb etal. and Jang et al. in Figure 3b−d. There is a limitation of usinga prism for interference pattern, as every novel structure wouldneed a different prism.34,35 To solve this issue, we can use thespecially angled prism, which can convert the single beam tomultiple beams after refraction and these refracted beams canfurther generate desired interference pattern.36,37

The foremost advantage of this method is that it reduces theaberration due to vibrations, which is the main problem in theprevious method and reduces the chance of misalignment ofthe beam. Nevertheless, the major disadvantage of thisprocedure is the control of polarization and phase shift of thebeam. The emergent polarization depends on incident

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 havingdiameter of 280 nm and height of 450 nm on a 750 nm square lattice. SEM image of 3D microstructure obtained from the mask and theoreticalintensity distribution for a 2 × 2 × 2 array of unit cells. Reproduced with permission from ref 31. Copyright 2007 John Wiley and Sons.



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polarization as well as the prism angles and cannot be variedindependently.38 Therefore, this method is well-versed for themicrostructures in which motif complexity is not as essential fordesigned patterns.2.3. Interference Lithography by Using Spatial Light

Modulator. Spatial light modulators (SLM) are also beingused in interference lithography to create periodic structures.Lasers have been used in wide-ranging applications frommedical eye surgery and industrial material processing tomilitary targeting and microscopy in research. Most applica-tions necessitate a high beam quality, meaning the beam musthave a defined cross-section and not diverge too rapidly. Thedesired beam propagation can be achieved with digitalholograms. SLM can be described as a device that is used tomodulate the amplitude, phase, or polarization of light beams.An SLM can manipulate an incident beam’s phase and thus beused as a variable focal length lens or as a means ofmanipulating the beam’s spatial frequency spectrum.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 spatialfrequency spectrum. Both approaches have enabled the beampropagation ratio (M2) value to be extracted without movingany components and with the potential for real-time measure-ments.39 Using the SLM for method 1 by displaying a sphericallens phase pattern, a camera recorded a beam’s curvature anddiameter as a function of the focal length in a fixed planebehind the hologram. Thus, rather than probing one beam atseveral planes, several beams were probed at one detectionplane. The beam diameter for a variable focus lens yielded M2

reliably and quickly using the analytical formula.39 Using theSLM for method 2, an optical field was propagated by Fouriertransforming the field at the initial plane, multiplying thetransfer function of free space, and then Fourier transforming itusing a single lens.40,41 The SLM displayed the transfer functionof free space, so recording with a camera in a fixed plane behindthe SLM yielded an artificially propagated beam. Figure 4depicted the experimental setup used to measure the M2 from alaser source. The optical setup consists of the beam source,SLM, a CCD camera, and a lens (only in method 2).

Schulze et al. have also created different holograms fromboth methods, by using different Laguerre-Gaussian modesLGpl. (M

2 is known to scale with the mode indices p and laccording to M2 = 2p + l + 1). The sample beams wereproduced by displaying the LG mode patterns using a specialcoding technique.42,43 Examples of beams with correspondinghologram patterns are shown in Figure 5, which depictsmeasured and fitted beam diameters as a function of the SLMlens focal length programmed for a Laguerre-Gaussian beamLG21 (method 1).

Similarly, Lutkenhaus et al. have used four beams with theSLM to fabricate holographic microstructures.44 The photo-resist 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 achain extender; was photosensitized by 532 nm wavelength.

Figure 3. (a) Table presenting the optical components and theirfunctions in multibeam IL. Reproduced with permission from ref 31.Copyright 2007 John Wiley and Sons. (b) Optical configuration oftypical multibeam interference lithography. Reproduced withpermission from ref 33. Copyright 2010 OSA Publishing. (c) Sixbeam lithographic setup with a prism. Reproduced with permissionfrom ref 31. Copyright 2007 John Wiley and Sons. (d) SEM image offabricated SU-8 microstructures. Lower left is a computed theoreticalintensity distribution for a 2 × 2 × 2 arrays of unit cells. Reproducedwith permission from ref 32. Copyright 2007 American ChemicalSociety.

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

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



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The diffracted beams from the phase pattern were assigned anddisplayed in the SLM. The phases of the interfering beam canbe tuned by the gray levels in the pattern and verified by thefabricated structures (Figure 6).

3D microstructures have varied application in the field ofphotonic crystals, biomimetic structures, optical communica-tion, tissue engineering, etc.28,29,45−50 Nowadays, there is agrowing demand for three-dimensional (3D), in vitro biologicaltissue models.51 These models are needed for fundamentalinvestigations. For example, angiogenesis, tumor cell intra- andextravasations, neuronal growth, or the behavior of the blood−brain barrier are some areas of investigation. Meanwhile,affordable, reproducible tissue models are needed for drugscreening. These applications all call for suitable extracellularscaffolds: microstructures with strictly controlled stiffness orstiffness gradients, pore sizes, and chemistry. SLMs are beingused as a programmable phase mask for digitally tunableholographic lithography. In the future, SLM can be used toproduce the desired structure for 3D tissue engineering.

3. 3D LITHOGRAPHY3D lithography is a direct write process in which linear ornonlinear excitation promotes photopolymerization creating a3D structure. Depending on the type of laser used, either one-photon absorption or multiphoton absorption (multiphotonlithography) can occur. In this section, we will be discussing onthe different technologies stereolithography and multiphoton

lithography and the materials used for both ablation andpolymerization.

3.1. Stereolithography. Stereolithography (SLA) is one ofthe first 3D systems created.52 SLA is an additive manufacturingprocess that makes 3D objects based on a layer-upon-layerdeposition arrangement to polymerize photosensitive resinunder UV irradiation. There are two main beam deliverysystems: (1) scanning or (2) projection. For scanning system,photocurable resin on the surface is exposed in a point-by-pointand line by line style while the projection system using a digitalmicromirror device (DMD) allows a whole layer to be cured atone time. Once the layer has been cured, the stage will move inthe z-direction to the next layer to be cured. This process isrepeated until the whole 3D part is completed (Figure 7). Oneof the biggest advantages of SLA is its fabrication speed.Fabrication can be done within a day but this is dependent onthe size and complexity of the 3D part. SLA also keeps cost lowas it a maskless lithography and materials is only consumedwhen cured. Stereolithography due to its ability to producehigh-resolution structures has been used in the construction ofscaffold in tissue engineering, the fabrication of microvascularstamps and the examination of the cell-to-cell interactions.53,54

Micron and nanoscale features affect the cell behavior toproliferate and differentiate. As SLA is able to fabricatestructures in the micron range and uses UV wavelength,researchers have been looking into other technologiesfabricating in the nanoscale range as well as another wavelengththat is less toxic to the cells.

3.2. Laser Direct Writing and Multiphoton Polymer-ization for Free-Form 3D Structures. DLW is a process thatinvolves focusing a laser beam onto the material of interest in aspecific pattern thus creating either 2D or 3D structures.56

These patterns can be created or traced by controlling the laserbeam focus point with galvanometric scanners or by moving thesample on a high-resolution XYZ stage with a fixed focalpoint.27 As the laser uses a wide range of wavelength to interactwith the material, this versatility allows the user to add(polymerization), subtract (ablation), or modify differentmaterials without harsh chemicals, preheating of the material,and the need for physical contact between the tools andmaterial allowing the user to create true 3D structures.27,56,57

Figure 6. (a) SEM image of fabricated holographic structures inDPHPA. (b) Higher magnification image. Reproduced withpermission from ref 44. Copyright 2013 OSA Publishing.

Figure 7. Schematic illustration of Stereolithography (a) Scanning Stereolithography, and (b) Projection Stereolithography. Reproduced withpermission from ref. 55. Copyright 2014 American Chemical Society.



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The few advantages have generated interest in using DLW forthe fabrication of many biomedical devices such as stents,prostheses, sensors, drug-delivery devices, and tissue-engineer-ing scaffold.27,56

3.2.1. System Configuration. A typical laser setup shown inFigure 8a, b involves: (1) laser source, (2) beam/samplemotion system, (3) beam focusing optics, (4) beam intensitycontrol and beam shutter, and (5) control software.27

(1) Laser source. The laser provides the spatially coherentenergy needed for processing of material. Ultrashortpulse laser (ie. Ti: sapphire lasers, ytterbium-doped laseror frequency doubled Er-doped fiber), especially in thefemtosecond range, allows for high energies to beemitted in longer wavelengths, leading to nonlinearoptical processes such as two-photon absorption (TPA).Some key factors to consider when choosing a lasersource includes laser wavelengths provided for absorp-tion by the material, pulse durations, and energy,repetition rates, laser exposure time, and average andpeak powers.

(2) Motion systems. Galvanometric scanners (movablemirrors) for moving the laser beam or moving thesample on high-resolution XYZ stages (resolutionapproximate several nm to μm) can be used with thelaser to “write” or create the 3D structures. There can bevarious combinations of the motion stages for realizing3D motion. The representative examples are three-axisPZT flexure stage, three-axis motorized stage, 2Dgalvano-scanner with a height control servo; all thesescanning systems are x, y, z Cartesian coordinates. Forcentral symmetric structures, r, θ, z scanning systemswith one rotational and two translational stages can alsobe used for the 3D motion.

(3) Focusing optics. A consistently focused laser beam isrequired for maintaining the high intensities of the laser.Usually, a standard microscope objective lens is used tofocus the laser. The spot size of the laser is determinedby altering the numerical aperture (N.A) of the focusingoptics. Immersion oil lens may be used when the N.A. ofthe objective is higher than 1.

Figure 8. (a) Schematic demonstration of a laser set up for TPP. Reproduced with permission from ref 58. Copyright 2010 The American Society ofMechanical Engineers. (b) Schematic diagram of femtosecond laser waveguide writing set up. VA, variable attenuator; MS, mechanical shutter; SHG,simple harmonic generator (optional); PC, polarization controller; BSO, beam shaping optics (optional); MO, microscope objective. Reproducedwith permission from ref 59. Copyright 2011 John Wiley and Sons.



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(4) Beam intensity control. The beam intensity and exposureonto the sample can be controlled by 2 types of systems.Beam on−off control can be achieved by using a fastmechanical shutter or an electro/acousto-optic modu-lator while beam intensity control can be achieved usingneutral density filters, a variable attenuator, or acombination of a polarizer and a waveplate.

(5) Control/monitoring software. A central computer withdifferent integrated programs is used to allow synchro-nization for both optical and mechanical accessories.Charge-coupled devices (CCDs) with different objectiveslens can be used for capturing images and monitoringprocesses in real time.

For two-photon polymerization (TPP), two representativelasers are crystal-based mode-locked Ti:sapphire femtosecondlasers and frequency-doubled Er-doped fiber femtosecondlasers at the near-infrared central wavelength around 800 nm.Thanks to the TPP process, the photosensitive polymers havingthe absorption wavelength shorter than 400 nm can beefficiently polymerized. The optimal laser parameters differdepending on the target materials but the general conditionscan be summarized as following. The pulse duration is less than300 fs, the repetition rate is less than 100 MHz, the power isless than 200 mW, and the magnification of the objective lensfor the printing is less than 60×. In the writing of the waveguideinside the transparent material, much higher peak power isrequired because it is based on material damage by multiphotonabsorption phenomena. Generally, lower repetition rate high-power amplifiers, like Ti:sapphire regenerative amplifiers ormultipass amplifiers are requested for the efficient manufactur-ing of the waveguides.3.2.2. Nonthermal Laser Ablation (Single Photon). Femto-

second laser ablation as first demonstrated on poly(methylmethacrylate) in 1987.60,61 Over the next 30 years, precisionand resolution of the ablation area have improved from themicroscale to the nanoscale.62 Laser ablation is a process usingoptical energy (photon) from the laser to remove material froma solid. Large numbers of an electron in the solid are beingionized by the laser resulting in phase or structuralmodifications. Depending on the amount of energy absorbedand the depth of absorption permanent change in the refractiveindex or even a void in the material can occur. The spot sizes ofthe laser and absorption wavelength of the material are somefactors in determining the resolution of the ablation. One pointto take note as laser ablation is performed by removing a smallamount of material; it cannot take place at locations where thelaser path is obstructed.Power intensity and pulse duration play a huge role in how

the laser beam interacts with the material. The high energiesand short pulses are desired as it reduced thermal effectsaround the spot. Generally, the heat diffusion to thesurrounding materials takes several tens of picoseconds.Therefore, the shorter pulses than this heat diffusion willminimize the unexpected thermal effects to surrounding basematerials. The high energy is required to initiate photochemicalor photothermal effects to the materials. Especially, in micro/nano 3D printing based on TPP, high-level pulse energy is theprerequisite for starting nonlinear two-photon absorption(TPA) process.56 To create interior geometries and structureswithin a bulk material, we used an ultrafast pulse laser. CWlaser ablation occurs when the materials adsorb and convert thelight energy to heat energy causing the material to melt leaving

behind a huge heat-affected zone (HAZ) (Figure 9). However,for ultrafast pulses (picosecond and femtosecond ones), due to

the ultrashort pulse width, there cannot be efficient heatconduction to the surrounding lattice structure which enables awell-defined clean manufacturing with minimal physical damageto the nearby materials.63,64 Other advantages include micro-sized structure creation, no collateral damage to thesurroundings, clean process look, no material property change,and capability for transparent material subsurface engraving.TPA with Femtosecond lasers may be used for ablation ofmaterials that exhibit poor absorption such as fused quartz andvarious glasses.62

3.2.3. Multiphoton Laser Polymerization (Multiphoton).Polymerization is defined as a process where a large volume ofsmall molecules is joined together to formed a big molecule.The process of two-photon polymerization (TPP) is like theSLA process. A laser is used to excite photoinitiator molecules,creating free radicals causing chemical reactions betweenphotoinitiator molecules and monomers/oligomers within atransparent resin. The key difference between the SLA and theTPP process is the use of two-photon absorption (TPA) forexcitation of photoinitiator molecules shown in Figure 10. Toachieve TPA, a femtosecond laser is utilized.65,66 A virtual stateis created where two-photon of same or different frequency areabsorbed by the photoinitiator (Figure 10). This enables longerwavelength to possess a similar electronic excitation like a singlephoton which requires much higher energy.65,66 This non-linearity of TPA not only excites the photoinitiator moleculeswith less energy but material cross-linking can occur within theimmediate vicinity of the focal volume,67 leading to significantlyhigher resolutions than other direct write techniques (up to 100nm).67

Ultrashort pulsed lasers such as femtosecond lasers aregenerally used for multiphoton processes, as they are based on

Figure 9. (a) Laser material interactions between different types oflaser. (b) Holes drilled in 100 mm thick steel foils by ablation usingdifferent laser pulses. Left is with a fs laser and the right is with ananosecond (ns) laser. Reproduced with permission from ref 64.Copyright 2014 Nature Publishing Group.



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second or third order nonlinear optical phenomena which aremuch more efficient at higher intensities. TPP provides severaladvantages over conventional processes especially in thefabrication of small medical devices. There is a wide range ofnear-infrared transparent materials such as GelMA and PEG-DA which are biocompatible and suitable for tissue engineer-ing.68 The cost should be reduced as no cleanroom facilities

and specialized equipment besides the laser are required69 ascompared to other fabrication techniques such as deep reactiveion etching and lithography.70 This flexibility in the future maylead to fabrication of a medical device within close proximity ofan operating room or another clinical site.

3.2.4. Higher Degree of Freedom By Combining LaserLithography and Ablative Laser Machining. By tuning

Figure 10. (a) Mechanism of simultaneous excitation by TPA. (b, c) Schematic representation of two methods for increasing the TPA probability inwhich density of photon is increased by (b) spatial compression using high numerical aperture (NA) objectives, (c) temporal compression usingultrafast lasers. Reproduced with permission from ref 71. Copyright 2013 Intech Open.

Figure 11. (a) Scheme presenting the fabrication process of 3D ship-in-a-bottle biochip by hybrid fs laser microprocessing. It proceeds with fs laserscanning followed by 1st annealing, HF etching, 2nd annealing, polymer coating, TPP, and development. (b−e) Images of 3D Y-shapedmicrochannel (b) laser scanning and the 1st annealing, (c) HF etching, (d) 2nd annealing, (e) 3D polymer microstructure by TPP. (f) SEM imagesdemonstrating 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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different wavelengths provided by the laser, subtractivefabrication, i.e., ablation or selective etching, and additivefabrication, i.e., TPP, can be achieved together with just onelaser set up. Sugioka and team were able to combine bothmethods thus creating a hybrid femtosecond laser micro-fabrication to achieve true 3D glass/polymer compositebiochips with multiscale features and high performance72

(Figure 11).Biochips or lab on the chip is a “miniaturized laboratory”

where multiple biological reactions can be detected, separated,and analyzed within one chip in a highly efficient and sensitiveway. Additional advantages include low material consumption,low cost, and compactness.For these different features to befabricated on the biochip, researchers have used a variety ofmethods such as photolithography,73 soft lithography,74,75 andnanoimprint lithography,76 although additional postprocessingmay be required as well.When comparing the hybrid technique to the methods

mention above, three advantages can be observed. (1) True 3Dmicrofluidic structures can be fabricated without complicationssuch as stacking and bonding/sealing with other substrates. (2)Maskless and free-form technology allows for a morebiomimetic environment for cell culture.77−79 (3) Additionalpolymer microstructures that provide additional functionalitycan be integrated after the chip had been made.To fabricate the biochip using the hybrid technique, two

main steps are involved. For creating the 3D hollowmicrochannels, TPA was achieved using a femtosecond (fs)laser on the photosensitive Foturan glass. Surface smoothness isimproved by thermal annealing. Next, 3D polymer micro-structures were fabricated by TPP for chip functionalization.This hybrid method allows the user to create biochips withdifferent functionalities with better speed and greaterflexibility.80

3.3. Materials for laser Direct Writing and Multi-photon Polymerization. To create internal 3D structures,materials must not be able to have any linear absorption at thewavelength of the femtosecond laser. The use of femtosecondlaser have advantages over another laser such as (1) thenonlinear nature of the absorption confines any inducedchanges to the focal volume and (2) the absorption process isindependent of the material, enabling optical devices to befabricated in compound substrates of different materials. Thereare two main materials used for 3D fabrication of scaffolds: (1)glass and (2) photopolymers.

Glass. Glass is one of the preferred choices for makingmicrofluidics for biological application. It is chosen because ofits low background fluorescence, high strength, and thermo-conductivity.81 The excellent inertness of glass makes itbiocompatible with low nonspecific absorption. However, it isnot gas permeable making less feasible for long-term cellstudies. Traditional glass microfluidic chips are created byetching into the glass by wet or dry methods with the formationof fluidic features such as valves and pump require bonding orhybrid layer attachment to enclose.81 With the use of afemtosecond laser, whole microfluidics chips can be fabricated,even with micro components such as micropumps andmicrovalves.82 Table 2 shows the summary of materials andlaser used.

Photopolymer. Photopolymers are polymer materials whichphysical or chemical properties undergo a change whenexposed directly or indirectly with light.91 It comprises acomposite material containing at least two basic components:(1) a polymerizable material possessing polymerizable func-tional groups to form the polymeric structure backbone, and(2) a photoinitiator to provide the active species after absorbingthe laser energy for the polymerization.27,91 To date, a largecombination of polymeric materials and photoinitiatorcombinations have been used biological applications (Figure3). Most materials are in the form of negative photoresists suchas acrylate materials, hybrid materials, and hydrogels.92 In thefollowing sections, we will discuss briefly on the photoinitiatorsand hydrogels for bioapplications.

Photoinitiators. There are two main classes of a photo-initiator based on the nature of active species they generate: (1)radical photoinitiators and (2) cationic photoinitiators. Radicalphotoinitiators generate free radicals which initiates thepolymerization of acrylates or vinyl ethers while cationicinitiators produce cations, which are used for the polymer-ization of epoxides or vinyl ethers.27 Nowadays, efforts arebeing made to synthesize fast and efficient photoinitiatorsspecifically for multiphoton applications.93 Moreover, there arelots of efforts to synthesize biocompatible photoinitiators,explicitly for bioapplications. Classic dyes such as Bengal Rose,Eosin, and Nile Red and biomolecules such as Flavinmononucleotide are some of the more common naturalbiocompatible photoinitiators available.27

Hydrogels. Hydrogels are materials capable of retainingwater in it 3D polymeric network. Most common hydrogelspossess only 0.5−20 wt % of dry polymer mass with the

Table 2. Summary of Materials and Lasers Used for Glass

type of glass laser source part of the chips/resolution application ref

photostructurableglass (Foturan)

ytterbium-doped femtosecond fiber lasers with a pulse of 150 fs,wavelength of 775 nm wavelength, and repetition rate of 1 kHz

microchannels up to 150 μm

“nanoaquariums” for the studies ofmotility of bacteria (Euglenagracilis)

83

photostructurableglass (Foturan)

ytterbium-doped femtosecond fiber lasers with a pulse of 360 fs,wavelength of 1045 nm, and repetition rate of 200 kHz

microchannels up to 100 μm “Nanoaquariums” for the studies ofmotility of bacteria (Phormidium)

84

fused silica substrate ytterbium-doped femtosecond fiber lasers with a pulse duration of460 fs, wavelength of 1047 nm, and repetition rate of 500 kHz

microchannels up to 100 μm mammalian cell sorting 85

Foturan glass laser with a pulse duration of 150 fs and wavelength of 775 nm microplate acting as a microvalvein a microreactor up to 1 mm

control the flow direction of fluids inthe microreactor

86

porous glass andfused silica

laser with a pulse duration of 340 fs, wavelength of 1045 nm, andrepetition rate of 200 kHz

micropump, micromixer,nanograting up to 40 nm

control the flow direction andmixing of fluids in the fluidic chip

87

silica glass Ti: sapphire laser with 20-Hz laser pulses and wavelength of 800 nm optical rotator of different shapesup to 12 μm

acting either as a pump or mixer 88

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

3D multilayer microfluidic chipsup to 150 μm

increasing the flexibility andcomplexity of chip

89

silica glass Ti: sapphire laser with a the pulse duration of ∼100 fs, wavelengthof 800 nm, and a repetition rate of 250 kHz

nanofluidic channels up to 50 nmperiodic nanograting

DNA analysis, e.g., stretching ofDNA molecule

90



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Table 3. Summary of Photopolymers with the Specific Photoinitator and Laser Wavelength for Biological Applications

materialnatural orsynthetic

photo-initiatorlaser source cells application ref

bovine serum albumin (BSA) 1.5 × 10-4 M, or1% w/w fibrinogen natural Rose Bengal (1 × 10−4 M)

titanium: sapphire fem-tosecond laser (100fs, 76 MHz, 700−1000 nm)

mouse-lympho-cytic leukemiacells (L1210cell), fibroblast,neurons

drug delivery, de-vices of cellsorting, cell en-capsulation, andtissue engineer-ing 94, 95

trimethylol-propane triacrylate (TMPTA) synthetic

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

titanium: sapphire fem-tosecond laser (100fs, 76 MHz, 700−1000 nm)

L1210 cell, fibro-blast neurons

drug delivery, de-vices of cellsorting, cell en-capsulation, andtissue engineer-ing 94, 95

collagen (type I, II, IV) naturalmodified benzophenonedimer (BPD)

titanium: sapphire fem-tosecond laser withpulse energies at the500 pJ/pulse

primary humandermal fibro-blasts tissue scaffold 96

collagen type I natural Rose Bengal amine derivative

titanium: sapphire fem-tosecond laser (100fs, 76 MHz, 780−750nm)

drug delivery ortissue scaffold 97

BSA natural

methylene blue (1.2−3 mM)or flavin adenine dinucleo-tide (FAD; 5 mM)

titanium: sapphire laser(76kHz, 730−740nm) E. coli cell studies 98, 99

BSA natural

flavin adenine dinucleotidedisodium salt hydrate(1-4mM)

laser (790 m), enteringthe microscope were30-60 mW.

neuroblastoma-glioma cells(NG108-15) tissue scaffold 100

PEG-DA (MW 742) synthetic 2 wt% (Irgacure 369, Ciba)

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

ovine endothelialcells (ECs) tissue scaffold 101

2-hydroxyethyl methacrylate (HEMA) and PEG-DA synthetic

2,2-dimethoxy-2-phenyl ace-tophenone (Irgacure 651)/2-photon sensitive chromo-phore (AF240). P127 act asa surfactant

titanium: sapphire laseroperating mode-locked (~80 MHz,~200 fs) at a near-infrared wavelengthof 780 nm was usedfor fabrication tissue engineering 102

hyaluronic acid-glycidyl methacrylate (HAGM)or HA−PEG-DA natural Irgacure 2959

ytterbium femtosecondlaser pulses (250 fs,21 MHz, 520 nm)

human dermalHFF-1 fibro-blasts andhuman osteo-blast-like cellline MG-63 tissue engineering 103

gelatin-methacrylamide (GELMA) natural Irgacure 2959

femtosecond laseremitting at around515 nm

primary adipose-derived stemcell (ASC)/porcine mesen-chymal stemcells

tissue engineeringscaffold

104,105

vinyl ester derivative of gelatin hydrolysate natural

WSPI ,4- bis(4-(N,N-bis(6-(N,N,N-trimethylammo-nium)hexyl)amino)-styryl)-2,5-dimethoxybenzene tet-raiodide

Ti: sapphire laser sys-tem (80fs, 75 MHz,800 nm)

human osteosar-coma cell lineMG63 cell studies 106

PEG-DA synthetic G2CK, E2CK, P2CK

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

MG63 osteosar-coma cells andoutgrowth en-dothelial cells(OEC) tissue engineering 107

GELMA natural G2Ck and P2CK

Ti: sapphire femtosec-ond laser (80fs, 75MHz, 800 nm)

MG63 osteosar-coma cells tissue engineering 108

PEG-DA (302MW and 742mw) synthetic

Michler’s ketone (4,4′-bis(di-ethylamino)benzophe-none) / irgacure 2959 and369

Ti: sapphire femtosec-ond laser pulses(120 fs, 80 MHz,780 nm)

L929 mouse fi-broblasts

cell studies andtissue engineer-ing 109

PEG-DA, 700 Da synthetic WISP

pulsed, laser beam of aTi: sapphire laser(100fs, 73 MHz,810 nm)

Caenorhabditiselegans scaffold 110

combination of acrylamide (AAm), N,N′-meth-ylenebis(acrylamide)(MBAAm), acryla-mide (AAm), N,N′-methylenebis(acrylamide)(MBAAm) synthetic

Benzil and 2-benyl-2-(dime-thylamino)-4′-morpholino-butyrophenone

Ti: Sapphire laser (80 fs780 nm, 82 MHz)

biomedical appli-cations (micro-actuators andmicro manipula-tors) 111

PEG-DA, 700 Da synthetic

2,7-bis(2-(4-pentaneoxy-phe-nyl)-vinyl)anthraquinone(N) with a C2vsymmetrical

Ti: sapphire femtosec-ond laser beam (120fs, 80 MHz, 780 nm) scaffold 112



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remaining part consisting of water which provides a highresemblance to the extracellular matrix and providing goodbiocompatibility with living organisms. In addition, the soft andelastic properties minimize irritation and damage to surround-ing tissues. For the preparation of hydrogels, well-designedpolymeric chemical structure and architecture is essential.Depending on their origin, the polymeric compositions areclassified into natural polymer hydrogels (e.g., collagen, dextran,fibrin, chitosan) synthetic polymer hydrogels (e.g., poly-(ethylene glycol) derivatives) and their combination (e.g.,collagen-acrylate, alginate-acrylate). Depending on the bio-logical applications each type of hydrogels has its advantagesand disadvantages. For example, natural hydrogels are moresuited for supporting cellular activates while being easilydegraded with water-soluble enzymes; however, they are moresusceptible to pathogens, causing an immune response, batchvariation, and low mechanical property. On the other hand,synthetic hydrogels can be precisely controlled and adjustablefor a wide range of applications with better mechanical strengthbut low biodegradability (degradation via hydrolysis, solubiliza-

tion, and mechanical erosion), inherent bioactive propertiesand possibly toxic substances released are some of itsdisadvantages.

4. APPLICATIONS IN CELL BIOLOGYFemtosecond-laser-based 3D printing of biomaterials and cellsscaffolds can be potentially used for various biomedicalapplications, from drug delivery and diagnostic testing to invitro tissue engineering and regeneration.

4.1. 3D cell cultures. Klein et al. have fabricated polymercomposite microstructures of with distinct protein-bindingproperties as shown in Figure 12. Ormocomp was being used asa biocompatible photoresist for the protein-binding cubes inthe scaffolds.119 Protein-repelling frameworks were generatedby using photoresist composed of PEG-DA and 3% (w/w)Irgacure 369 as a photoinitiator, while PETTA was used ascross-linker to enhance the mechanical properties of these 3Dscaffolds. Various Concentration of PETTA (0, 4.8, 9.1, 33.3,100% (w/w)) in PEG-DA were prepared and 2D patterns werefabricated by DLW. Moreover, Ormocomp squares (40 μm ×

Table 3. continued

materialnatural orsynthetic

photo-initiatorlaser source cells application ref

structure and 2-hydroxy-propyl-β-cyclodextrin

matrix metalloproteinase-sensitive peptide(GGPQGIWGQGK, abbreviated PQ) into thebackbone of PEG-DA derivative Synthetic

2,2-dimethoxy-2-phenyl- ace-tophenone (DMAP)

laser tuned to 720 nmwith a scan speed of25 μs/pixel and anintensity of 60 mW/μm2 was then used toexcite

human umbilicalvein endothelialcells (HUVECs,

scaffold/cell stud-ies 113

AKRE (an acrylate based custom made photo-polymer AKRE37 consisting of tris (2-hydroxyethyl) isocyanurate triacrylate and 4,4’-bis(di-methyl amino) benzophenone), ORMOSIL(hybrid organic−inorganic SZ2080 material)ORMOCER and biodegradable PEG-DA (MW258)

syntheticand natu-ral

Ti: sapphire laser withaverage output powerof 500 mW (80fs, 80MHz, 800 nm)

primary stem cellculture derivedfrom adult rab-bit muscle scaffold 114

PEG-DA, MW 700 synthetic1% (w/v) of the photoinitia-tor Irgacure 819

800 nm Ti: sapphirefemtosecond laser tissue scaffold 115

PEG-DA MW 700 syntheticlithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)

Ti: sapphire femtosec-ond laser (80MHz,800nm) with a max-imum power of 350mW.

anchorage-de-pendent embry-onic fibroblast10T1/2 cell line cell studies 116

fibrinogen, fibronectin, lectin Con A, BSA natural Rose Bengal

Ti: sapphire laser(100fs, 76 MHz, and800 nm)

Scaffolds andECMs for tissueengineering. 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 areapproximately 5 mW,or about 100 pJ perpulse

H9 embryonicstem cell-de-rived humanmesenchymalstem cells(MSCs) scaffolds 118

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 color for demonstration)were attached to the PEG-DA beams with precision. (c) Ormocomp cube (red color) in high magnification. Reproduced with permission from ref119. Copyright 2011 John Wiley and Sons.



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40 μm) were embedded in a PEG-DA with increasing PETTAconcentrations.PEG-DA-4.8% PETTA pillars having ∼7 μm diameter and

∼23 μm height interconnected by 1 μm diameter beams having10 μm length (Figure 12 a) were fabricated by DLW.Subsequently, the scaffolds were developed by isopropylalcohol (IPA) and casted with the ormocomp (secondphotoresist). Ormocomp cubes having the dimensions of 2.5μm were adhered in the middle of PEG-DA beams bypolymerization; after the development of first microstructures(Figure 12 b, c). Spatially controlled 3D microstructurescontaining protein binding ormocomp cubes and proteinrepellent PEG-DA scaffold has been fabricated by DLW. Aspredicted upon the cultivation of fibronectin, ECM proteinpreferentially binds to the ormocomp part, followed by theculture of fibroblasts on the scaffolds. Interestingly Klein et al.have found that cells were adhered to ormocomp or connectedthrough the several cubes and studied via confocal microscopy(Figure 13 a). 3D growth pattern of cells within theinterconnected can be observed in Figure 13b, c. Immunostain-ing by actin has demonstrated the periphery of the adheredcells while cell contact sites were majorly and the ormocompcubes not on PEG-DA part was revealed by paxillin staining(Figure 13d−f). It was revealed that the cells cultured in thesescaffolds specifically form cell adhesion sites in the ECMfunctionalized sections. This research has shown the futurepossibilities for systematic studies of spatial ligand distributionsand stiffness of the scaffold on the cell behavior in 3Denvironments.4.2. Micro Molding of Biological Scaffolds. Koroleva et

al. has generated TPP scaffolds and replicated it by using micromolding.120 These microstructures were fabricated via TPP ona glass substrate and a negative mold of PDMS was prepared bypolymerizing it at 100 °C for 1 h. PDMS was separated byusing the piezo stage and the mold was covered byphotosensitive PLA polymer. The desired biocompatible PLAmicrostructures were polymerized by the UV light andseparated by using piezo stage. The well-defined hexagonal

hollow cylinders having height, wall thickness and diameter of300, 20, and 100 μm were obtained. The photocured materialcan be easily recovered from the PDMS mold and can be usedfor subsequent replicated batches of the PLA. Figure 14 showsthe SEM image of the TPP scaffold and replicated scaffolds.

Koroleva et al. seeded primary Schwann cells on themicroreplicated 3D scaffolds for 7 days and observed it bySEM.120 It was observed that the Schwann cells adhered to thePLA 3D microstructures in spindle-like and flat cellmorphologies. The spindlelike cells adhered on the 3Dstructures (Figure 15a), whereas flat morphology cells boundedbetween the cells and the walls of the PLA scaffold (Figure15b), indicating cell growth.

4.3. Cell-Matrix Invasion. Cell-matrix invasion is animportant part of the pathological process which includesleukocyte extravasation and cancer cell metastasis. In recentyears cell matrix invasion was studied by the migration of cellswithin the polymer scaffolds which plays the role of ECM. Inorder to study the cell matrix invasion, Greiner et al. havefabricated the well-designed polymer cell scaffolds DLW asshown in Figure 16. 3D microstructures of biocompatiblepolymer PETTA with different mesh sizes (2,5 and 10 μm)

Figure 13. Confocal images showing the cell growth in 3D scaffolds. Primary chicken fibroblasts were cultivated in the scaffolds and immunostainedfor fibronectin (red), f-actin (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 celladhering 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.

Figure 14. SEM images of the microstructures fabricated by (left) TPPand (right) microreplication. Reproduced with permission from ref120. Copyright 2013 IOP publishing.



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were fabricated. Additionally, fibronectin was coated to enhancethe cell adhesion and matrix invasion.121

It is well-known fact that cells change their morphology whilemigrating through the pores of ECM during embryogenesis,wound healing, or metastasis. Greiner et al. have studied theeffect of nucleus stiffness on the scaffold invasion. MEF WTand MEF lmna KO cells were cultured on the scaffolds with 10μm pores for 24 h and were fixed and labeled with DRAQ5 tovisualize the nucleus of the cells. It was interesting to observethat both cells have different invasive behavior (Figure 17A, B).55% of the MEF WT cells were able to enter in the scaffold,whereas 80% of MEF lmna KO cells were partial or fullyembedded in the scaffold (Figure 17C). MEF lmna Ko cellshave a large percentage of cell matrix invasion as compared toMEF WT cells. The nucleus of MEF lmna KO cells was smallerthan the MEF WT cells, which deforms its morphology duringcell migration. Moreover, additional AFM and invasion studieshave also confirmed the link between nucleus stiffness andscaffold population.Thus, 3D printed scaffolds with predefined pore sizes can be

used for cell migration and invasion studies with different ECMmolecules. Moreover, 3D printed scaffolds can be potentiallyused for screening and determining cancer stages based on thenuclear mechanics and matrix invasion.

4.4. Targeted Cell Delivery. Kim et al. have demonstratedmultifunctional microrobots for targeted cell delivery using 3Dlaser lithography.122 Ni and Ti layers were coated fabricatedmicrorobots 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 cultureon the fabricated microstructures and were affixed usingparaformaldehyde solution. SEM imaging was performed after96 h. Figure 18 showed SEM and confocal microscopy imagesof the microrobots. Interestingly filopodia formation wasobserved during cell migration, as shown in Figure 18b,which was the indication of the cell interaction withmicrorobots. There were no signs of cytotoxicity andbiocompatibility analysis was confirmed by the cell adherence,migration, and proliferation of the structures. The movement ofmicrorobots was demonstrated by the application of theexternal magnetic field, which can be used for in vivo for thecell transportation, gene delivery, and drug delivery applica-tions.

5. CONCLUSION AND FUTURE PROSPECTSThis Review showed the recent trends of cell biologyapplications enabled by ultrashort femtosecond laser pulses,

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

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

Figure 17. Fibroblast invasion into 3D scaffolds on glass. (A, B) Topand side view 3D reconstructions of wild-type mouse embryonicfibroblasts (MEF WT) and lmna knockout MEFs(MEF lmna KO) on3D scaffolds with a mesh size of 10 μm. The cell nuclei of MEF WTand MEF lmna KO either localized on top of the 3D scaffold, or theywere partially or fully embedded within the 3D structure (nuclei 1/4red, F-actin 1/4 green, 3D scaffold 1/4 white). (C) Significantly morecell nuclei were partially or fully embedded in the KO cell line than forWT cells (chi-square test; *, p < 0.05; n > 72 cells from 5 independentexperiments). Reproduced with permission from ref 121. Copyright2014 Elsevier.



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followed by the latest lithography studies and systemdevelopments. DLW and multiphoton polymerization havegained a lot of attention because of a higher degree of freedomfor generating free-form 3D structures precisely by using visibleand NIR wavelength. As introduced, in the recent studies, therehas been much more focus on the lithography techniques basedon ultrashort pulse lasers rather than using traditional UV- ormask-based lithography for biological applications. In addition,femtosecond laser based 3D printing can be used for cell-to-cellinteraction studies in the confined microenvironment, cellproperty measurements, bio MEMs, and microfluidics in thenear future. DLW of multiple cells infemtosecond-laser-based3D lithography has emerged as a powerful tool and will expandits own leadership in the field of tissue engineering and cellbiology.

■ AUTHOR INFORMATION

Corresponding Authors*E-Mail: [email protected].*E-mail: [email protected]

ORCIDYoung-Jin Kim: 0000-0002-4271-5771Yong-Jin Yoon: 0000-0002-3885-4947Author Contributions†C.M.B.H. and A.M. have equal contribution.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This study was financially supported by Singapore NationalResearch Foundation (NRF-NRFF2015-02), Singapore Minis-try of Education (MOE) under its Tier 1 Grant (RG85/15,RG35/12, and RGC4/13), and the Singapore Centre for 3DPrinting. It was also supported by a research collaborationagreement by Panasonic Factory Solutions Asia Pacific(PFSAP) and Singapore Centre for 3D Printing (RCA-15/027).

■ REFERENCES(1) Drury, J. L.; Mooney, D. J. Hydrogels for tissue engineering:scaffold design variables and applications. Biomaterials 2003, 24 (24),4337−4351.(2) Langer, R.; Vacanti, J. Tissue engineering. Science 1993, 260(5110), 920−926.(3) Workman, J. K.; Myrick, C. W.; Meyers, R. L.; Bratton, S. L.;Nakagawa, T. A. Pediatric organ donation and transplantation.Pediatrics 2013, 131 (6), e1723−e1730.(4) Overby, K. J.; Weinstein, M. S.; Fiester, A. Addressing consentissues in donation after circulatory determination of death. AmericanJournal of Bioethics 2015, 15 (8), 3−9.(5) Danie Kingsley, J.; Ranjan, S.; Dasgupta, N.; Saha, P.Nanotechnology for tissue engineering: Need, techniques andapplications. J. Pharm. Res. 2013, 7 (2), 200−204.(6) Lim, J. Y.; Donahue, H. J. Cell sensing and response to micro-andnanostructured surfaces produced by chemical and topographicpatterning. Tissue Eng. 2007, 13 (8), 1879−1891.(7) Jeon, H.; Simon, C. G.; Kim, G. A mini-review: cell response tomicroscale, nanoscale, and hierarchical patterning of surface structure.J. Biomed. Mater. Res., Part B 2014, 102 (7), 1580−1594.(8) Greiner, A. M.; Richter, B.; Bastmeyer, M. Micro-engineered 3Dscaffolds for cell culture studies. Macromol. Biosci. 2012, 12 (10),1301−1314.(9) Zhang, A. P.; Qu, X.; Soman, P.; Hribar, K. C.; Lee, J. W.; Chen,S.; He, S. Rapid fabrication of complex 3D extracellular microenviron-ments by dynamic optical projection stereolithography. Adv. Mater.2012, 24 (31), 4266−4270.(10) Huang, T. Q.; Qu, X.; Liu, J.; Chen, S. 3D printing ofbiomimetic microstructures for cancer cell migration. Biomed.Microdevices 2014, 16 (1), 127−132.(11) Zhu, W.; O’Brien, C.; O’Brien, J. R.; Zhang, L. G. 3D nano/microfabrication techniques and nanobiomaterials for neural tissueregeneration. Nanomedicine 2014, 9 (6), 859−875.(12) Cukierman, E.; Pankov, R.; Stevens, D. R.; Yamada, K. M.Taking Cell-Matrix Adhesions to the Third Dimension. Science 2001,294 (5547), 1708−1712.(13) Yang, S.; Leong, K.-F.; Du, Z.; Chua, C.-K. The design ofscaffolds for use in tissue engineering. Part II. Rapid prototypingtechniques. Tissue Eng. 2002, 8 (1), 1−11.(14) Mishra, A.; Aswal, V. K.; Maiti, P. Nanostructure toMicrostructure Self-Assembly of Aliphatic Polyurethanes: The Effecton Mechanical Properties. J. Phys. Chem. B 2010, 114 (16), 5292−5300.(15) Mishra, A.; Purkayastha, B. P. D.; Roy, J. K.; Aswal, V. K.; Maiti,P. Tunable Properties of Self-Assembled Polyurethane Using Two-Dimensional Nanoparticles: Potential Nano-biohybrid. Macromolecules2010, 43 (23), 9928−9936.(16) Mishra, A.; Maiti, P. Morphology of polyurethanes at variouslength scale: The influence of chain structure. J. Appl. Polym. Sci. 2011,120 (6), 3546−3555.(17) Mishra, A.; Das Purkayastha, B. P.; Roy, J. K.; Aswal, V. K.;Maiti, P. Nanoparticle Controlled Self-Assembly in Varying ChainExtended Polyurethanes as Potential Nanobiomaterials. J. Phys. Chem.C 2012, 116 (3), 2260−2270.(18) Mishra, A.; Singh, S. K.; Dash, D.; Aswal, V. K.; Maiti, B.; Misra,M.; Maiti, P. Self-assembled aliphatic chain extended polyurethanenanobiohybrids: Emerging hemocompatible biomaterials for sustaineddrug delivery. Acta Biomater. 2014, 10 (5), 2133−2146.(19) Mishra, A.; Seethamraju, K.; Delaney, J.; Willoughby, P.; Faust,R. Long-term in vitro hydrolytic stability of thermoplastic polyur-ethanes. J. Biomed. Mater. Res., Part A 2015, 103 (12), 3798−3806.(20) Gupta, K. K.; Kundan, A.; Mishra, P. K.; Srivastava, P.; Mohanty,S.; Singh, N. K.; Mishra, A.; Maiti, P. Polycaprolactone compositeswith TiO2 for potential nanobiomaterials: tunable properties usingdifferent phases. Phys. Chem. Chem. Phys. 2012, 14 (37), 12844−12853.(21) Ho, C. M. B.; Mishra, A.; Lin, P. T. P.; Ng, S. H.; Yeong, W. Y.;Kim, Y.-J.; Yoon, Y.-J. 3D Printed Polycaprolactone Carbon Nanotube

Figure 18. (a) SEM image of a hexahedral microrobot after cell cultureand (b) high magnification SEM image showing filopodia.Confocalmicroscope images of the (c) hexahedral and (d) cylindricalmicrorobots after staining of the cells. Reproduced with permissionfrom ref. 122. Copyright 2014 John Wiley and Sons.



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Composite Scaffolds for Cardiac Tissue Engineering. Macromol. Biosci.2017, 17 (4), 1600250.(22) Teo, A. J. T.; Mishra, A.; Park, I.; Kim, Y.-J.; Park, W.-T.; Yoon,Y.-J. Polymeric Biomaterials for Medical Implants and Devices. ACSBiomater. Sci. Eng. 2016, 2 (4), 454−472.(23) Yang, S.; Leong, K.-F.; Du, Z.; Chua, C.-K. The design ofscaffolds for use in tissue engineering. Part I. Traditional factors. TissueEng. 2001, 7 (6), 679−689.(24) Gibbs, D. M.; Vaezi, M.; Yang, S.; Oreffo, R. O. Hope versushype: what can additive manufacturing realistically offer trauma andorthopedic surgery? Regener. Med. 2014, 9 (4), 535−549.(25) Too, M.; Leong, K.; Chua, C.; Du, Z.; Yang, S.; Cheah, C.; Ho,S. Investigation of 3D non-random porous structures by fuseddeposition modelling. International Journal of Advanced ManufacturingTechnology 2002, 19 (3), 217−223.(26) Vaezi, M.; Seitz, H.; Yang, S. A review on 3D micro-additivemanufacturing technologies. International Journal of AdvancedManufacturing Technology 2013, 67 (5−8), 1721−1754.(27) Selimis, A.; Mironov, V.; Farsari, M. Direct laser writing:Principles and materials for scaffold 3D printing. Microelectron. Eng.2015, 132, 83−89.(28) Lu, C.; Lipson, R. Interference lithography: a powerful tool forfabricating periodic structures. Laser & Photonics Reviews 2010, 4 (4),568−580.(29) Wolferen, H.; Abelmann, L. Laser Interference Lithography. InLithography: Principles, Processes, and Materials; Hennessy, T. C., Ed;Nova Science: Hauppauge, NY, 2011; 133−149.(30) Jeon, S.; Park, J.-U.; Cirelli, R.; Yang, S.; Heitzman, C. E.; Braun,P. V.; Kenis, P. J. A.; Rogers, J. A. Fabricating complex three-dimensional nanostructures with high-resolution conformable phasemasks. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (34), 12428−12433.(31) Jang, J. H.; Ullal, C. K.; Maldovan, M.; Gorishnyy, T.; Kooi, S.;Koh, C.; Thomas, E. L. 3D Micro- and Nanostructures via InterferenceLithography. Adv. Funct. Mater. 2007, 17 (16), 3027−3041.(32) Jang, J.-H.; Ullal, C. K.; Kooi, S. E.; Koh; Thomas, E. L. ShapeControl of Multivalent 3D Colloidal Particles via InterferenceLithography. Nano Lett. 2007, 7 (3), 647−651.(33) Harb, A.; Torres, F.; Ohlinger, K.; Lin, Y.; Lozano, K.; Xu, D.;Chen, K. P. Holographically formed three-dimensional Penrose-typephotonic quasicrystal through a lab-made single diffractive opticalelement. Opt. Express 2010, 18 (19), 20512−20517.(34) Ullal, C. K.; Maldovan, M.; Thomas, E. L.; Chen, G.; Han, Y.-J.;Yang, S. Photonic crystals through holographic lithography: Simplecubic, diamond-like, and gyroid-like structures. Appl. Phys. Lett. 2004,84 (26), 5434−5436.(35) Miklyaev, Y. V.; Meisel, D. C.; Blanco, A.; von Freymann, G.;Busch, K.; Koch, W.; Enkrich, C.; Deubel, M.; Wegener, M. Three-dimensional face-centered-cubic photonic crystal templates by laserholography: fabrication, optical characterization, and band-structurecalculations. Appl. Phys. Lett. 2003, 82 (8), 1284−1286.(36) Lei, M.; Yao, B.; Rupp, R. A. Structuring by multi-beaminterference using symmetric pyramids. Opt. Express 2006, 14 (12),5803−5811.(37) Chen, J.; Jiang, W.; Chen, X.; Wang, L.; Zhang, S.; Chen, R. T.Holographic three-dimensional polymeric photonic crystals operatingin the 1550nm window. Appl. Phys. Lett. 2007, 90 (9), 093102.(38) Born, M.; Wolf, E. Principles of Optics; Cambridge UniversityPress: New York, 1999; p 704.(39) Schulze, C.; Flamm, D.; Duparre, M.; Forbes, A. Beam-qualitymeasurements using a spatial light modulator. Opt. Lett. 2012, 37 (22),4687−4689.(40) Flamm, D.; Schulze, C.; Bruning, R.; Schmidt, O. A.; Kaiser, T.;Schroter, S.; Duparre, M. Fast M 2 measurement for fiber beams basedon modal analysis. Appl. Opt. 2012, 51 (7), 987−993.(41) Goodman, J. W. Introduction to Fourier Optics; Roberts &Company: Englewood, CO, 2005.(42) Arrizon, V.; Ruiz, U.; Carrada, R.; Gonzalez, L. A. Pixelatedphase computer holograms for the accurate encoding of scalar complexfields. J. Opt. Soc. Am. A 2007, 24 (11), 3500−3507.

(43) Flamm, D.; Naidoo, D.; Schulze, C.; Forbes, A.; Duparre, M.Mode analysis with a spatial light modulator as a correlation filter. Opt.Lett. 2012, 37 (13), 2478−2480.(44) Lutkenhaus, J.; George, D.; Moazzezi, M.; Philipose, U.; Lin, Y.Digitally tunable holographic lithography using a spatial lightmodulator as a programmable phase mask. Opt. Express 2013, 21(22), 26227−26235.(45) Bagal, A.; Chang, C.-H. Fabrication of subwavelength periodicnanostructures using liquid immersion Lloyd’s mirror interferencelithography. Opt. Lett. 2013, 38 (14), 2531−2534.(46) Campbell, M.; Sharp, D.; Harrison, M.; Denning, R.;Turberfield, A. Fabrication of photonic crystals for the visiblespectrum by holographic lithography. Nature 2000, 404 (6773), 53−56.(47) Gutierrez-Rivera, L. E.; Cescato, L. SU-8 submicrometric sievesrecorded by UV interference lithography. J. Micromech. Microeng. 2008,18 (11), 115003.(48) Hyuk Moon, J.; Yang, S. Creating Three-Dimensional PolymericMicrostructures by Multi-Beam Interference Lithography. J. Macromol.Sci., Polym. Rev. 2005, 45 (4), 351−373.(49) Lasagni, A. F.; Yuan, D.; Shao, P.; Das, S. PeriodicMicropatterning of Polyethylene Glycol Diacrylate Hydrogel byLaser Interference Lithography Using Nano-and Femtosecond PulsedLasers. Adv. Eng. Mater. 2009, 11 (3), B20−B24.(50) Park, S.-G.; Jeon, T. Y.; Yang, S.-M. Fabrication of Three-Dimensional Nanostructured Titania Materials by Prism HolographicLithography and the Sol−Gel Reaction. Langmuir 2013, 29 (31),9620−9625.(51) Boland, E. D.; Espy, P. G.; Bowlin, G. L. Tissue engineeringscaffolds. Encyclopedia of biomaterials and biomedical engineering 2004,1630−1638.(52) Ho, C. M. B.; Ng, S. H.; Yoon, Y.-J. A review on 3D printedbioimplants. International Journal of Precision Engineering andManufacturing 2015, 16 (5), 1035−1046.(53) Liu, W.; Li, Y.; Liu, J.; Niu, X.; Wang, Y.; Li, D. Application andperformance of 3D printing in nanobiomaterials. J. Nanomater. 2013,2013, 681050.(54) Ho, C. M. B.; Ng, S. H.; Li, K. H. H.; Yoon, Y.-J. 3D printedmicrofluidics for biological applications. Lab Chip 2015, 15 (18),3627−3637.(55) Gross, B. C.; Erkal, J. L.; Lockwood, S. Y.; Chen, C.; Spence, D.M. Evaluation of 3D Printing and Its Potential Impact onBiotechnology and the Chemical Sciences. Anal. Chem. 2014, 86(7), 3240−3253.(56) Gittard, S. D.; Narayan, R. J. Laser direct writing of micro-andnano-scale medical devices. Expert Rev. Med. Devices 2010, 7 (3), 343−356.(57) Xu, H.; Sun, H. Femtosecond laser 3D fabrication ofwhispering-gallery-mode microcavities. Sci. China: Phys., Mech. Astron.2015, 58 (11), 114202.(58) Zhang, W.; Han, L.-H.; Chen, S. Integrated Two-PhotonPolymerization With Nanoimprinting for Direct Digital Nano-manufacturing. Journal of Manufacturing Science and Engineering2010, 132 (3), 030907−030907−5.(59) Osellame, R.; Hoekstra, H. J. W. M.; Cerullo, G.; Pollnau, M.Femtosecond laser microstructuring: an enabling tool for optofluidiclab-on-chips. Laser & Photonics Reviews 2011, 5 (3), 442−463.(60) Srinivasan, R.; Sutcliffe, E.; Braren, B. Ablation and etching ofpolymethylmethacrylate by very short (160 fs) ultraviolet (308 nm)laser pulses. Appl. Phys. Lett. 1987, 51 (16), 1285−1287.(61) Kuper, S.; Stuke, M. Femtosecond UV excimer laser ablation.Appl. Phys. B: Photophys. Laser Chem. 1987, 44 (4), 199−204.(62) Gattass, R. R.; Mazur, E. Femtosecond laser micromachining intransparent materials. Nat. Photonics 2008, 2 (4), 219−225.(63) Korte, F.; Nolte, S.; Chichkov, B.; Bauer, T.; Kamlage, G.;Wagner, T.; Fallnich, C.; Welling, H. Far-field and near-field materialprocessing with. femtosecond laser pulses. Appl. Phys. A: Mater. Sci.Process. 1999, 69, S7−S11.



2212


(64) Sugioka, K.; Cheng, Y. Ultrafast lasersreliable tools foradvanced materials processing. Light: Sci. Appl. 2014, 3 (4), e149.(65) Doraiswamy, A.; Jin, C.; Narayan, R.; Mageswaran, P.; Mente,P.; Modi, R.; Auyeung, R.; Chrisey, D.; Ovsianikov, A.; Chichkov, B.Two photon induced polymerization of organic−inorganic hybridbiomaterials for microstructured medical devices. Acta Biomater. 2006,2 (3), 267−275.(66) Narayan, R. Two Photon Polymerization: An Emerging Methodfor Rapid Prototyping of Ceramic―Polymer Hybrid Materials forMedical Applications. Am. Ceram. Soc. Bull. 2009, 88 (5), 20−25.(67) Serbin, J.; Egbert, A.; Ostendorf, A.; Chichkov, B.; Houbertz, R.;Domann, G.; Schulz, J.; Cronauer, C.; Frohlich, L.; Popall, M.Femtosecond laser-induced two-photon polymerization of inorganic−organic hybrid materials for applications in photonics. Opt. Lett. 2003,28 (5), 301−303.(68) Ovsianikov, A.; Passinger, S.; Houbertz, R.; Chichkov, B. N.,Three dimensional material processing with femtosecond lasers. InLaser Ablation and Its Applications; Springer: Berlin, 2007; pp 121−157.(69) Ovsianikov, A.; Chichkov, B.; Adunka, O.; Pillsbury, H.;Doraiswamy, A.; Narayan, R. Rapid prototyping of ossicularreplacement prostheses. Appl. Surf. Sci. 2007, 253 (15), 6603−6607.(70) Hemker, K.; Sharpe, W., Jr Microscale characterization ofmechanical properties. Annu. Rev. Mater. Res. 2007, 37, 93−126.(71) Spangenberg, A.; Hobeika, N.; Stehlin, F.; Malval, J.-P.; Wieder,F.; Prabhakaran, P.; Baldeck, P.; Soppera, O., Recent advances in two-photon stereolithography. In Updates in Advanced Lithography;InTech: Rijeka, Croatia, 2013.(72) Wu, D.; Wu, S.-Z.; Xu, J.; Niu, L.-G.; Midorikawa, K.; Sugioka,K. Hybrid femtosecond laser microfabrication to achieve true 3Dglass/polymer composite biochips with multiscale features and highperformance: the concept of ship-in-a-bottle biochip. Laser & PhotonicsReviews 2014, 8 (3), 458−467.(73) McCreedy, T. Fabrication techniques and materials commonlyused for the production of microreactors and micro total analyticalsystems. TrAC, Trends Anal. Chem. 2000, 19 (6), 396−401.(74) Qin, D.; Xia, Y.; Whitesides, G. M. Soft lithography for micro-and nanoscale patterning. Nat. Protoc. 2010, 5 (3), 491−502.(75) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G.M. Rapid Prototyping of Microfluidic Systems in Poly-(dimethylsiloxane). Anal. Chem. 1998, 70 (23), 4974−4984.(76) Guo, L. J.; Cheng, X.; Chou, C.-F. Fabrication of Size-Controllable Nanofluidic Channels by Nanoimprinting and ItsApplication for DNA Stretching. Nano Lett. 2004, 4 (1), 69−73.(77) Luo, Y.; Shoichet, M. S. A photolabile hydrogel for guided three-dimensional cell growth and migration. Nat. Mater. 2004, 3 (4), 249−253.(78) Albrecht, D. R.; Underhill, G. H.; Wassermann, T. B.; Sah, R. L.;Bhatia, S. N. Probing the role of multicellular organization in three-dimensional microenvironments. Nat. Methods 2006, 3 (5), 369−375.(79) Schindler, M.; Nur-E-Kamal, A.; Ahmed, I.; Kamal, J.; Liu, H.-Y.;Amor, N.; Ponery, A. S.; Crockett, D. P.; Grafe, T. H.; Chung, H. Y.;Weik, T.; Jones, E.; Meiners, S. Living in three dimensions. CellBiochem. Biophys. 2006, 45 (2), 215−227.(80) Cheng, Y.; Sugioka, K.; Midorikawa, K. Freestanding opticalfibers fabricated in a glass chip using femtosecond laser micro-machining for lab-on-a-chip application. Opt. Express 2005, 13 (18),7225−7232.(81) Nge, P. N.; Rogers, C. I.; Woolley, A. T. Advances inmicrofluidic materials, functions, integration, and applications. Chem.Rev. 2013, 113 (4), 2550−2583.(82) Sugioka, K.; Xu, J.; Wu, D.; Hanada, Y.; Wang, Z.; Cheng, Y.;Midorikawa, K. Femtosecond laser 3D micromachining: a powerfultool for the fabrication of microfluidic, optofluidic, and electrofluidicdevices based on glass. Lab Chip 2014, 14 (18), 3447−3458.(83) Hanada, Y.; Sugioka, K.; Kawano, H.; Ishikawa, I. S.; Miyawaki,A.; Midorikawa, K. Nano-aquarium for dynamic observation of livingcells fabricated by femtosecond laser direct writing of photo-structurable glass. Biomed. Microdevices 2008, 10 (3), 403−410.

(84) Hanada, Y.; Sugioka, K.; Shihira-Ishikawa, I.; Kawano, H.;Miyawaki, A.; Midorikawa, K. 3D microfluidic chips with integratedfunctional microelements fabricated by a femtosecond laser forstudying the gliding mechanism of cyanobacteria. Lab Chip 2011, 11(12), 2109−2115.(85) Choudhury, D.; Ramsay, W. T.; Kiss, R.; Willoughby, N. A.;Paterson, L.; Kar, A. K. A 3D mammalian cell separator biochip. LabChip 2012, 12 (5), 948−953.(86) Masuda, M.; Sugioka, K.; Cheng, Y.; Hongo, T.; Shihoyama, K.;Takai, H.; Miyamoto, I.; Midorikawa, K. Direct fabrication of freelymovable microplate inside photosensitive glass by femtosecond laserfor lab-on-chip application. Appl. Phys. A: Mater. Sci. Process. 2004, 78(7), 1029−1032.(87) Sugioka, K.; Cheng, Y. Fabrication of 3D microfluidic structuresinside glass by femtosecond laser micromachining. Appl. Phys. A:Mater. Sci. Process. 2014, 114 (1), 215−221.(88) Kiyama, S.; Tomita, T.; Matsuo, S.; Hashimoto, S. Laserfabrication and manipulation of an optical rotator embedded inside atransparent solid material. J. Laser Micro/Nanoeng. 2009, 4, 18−21.(89) Li, Y.; Qu, S. Water-assisted femtosecond laser ablation forfabricating three-dimensional microfluidic chips. Curr. Appl. Phys.2013, 13 (7), 1292−1295.(90) Liao, Y.; Cheng, Y.; Liu, C.; Song, J.; He, F.; Shen, Y.; Chen, D.;Xu, Z.; Fan, Z.; Wei, X.; et al. Direct laser writing of sub-50 nmnanofluidic channels buried in glass for three-dimensional micro-nanofluidic integration. Lab Chip 2013, 13 (8), 1626−1631.(91) Crivello, J. V.; Reichmanis, E. Photopolymer materials andprocesses for advanced technologies. Chem. Mater. 2014, 26 (1), 533−548.(92) Ciuciu, A. I.; Cywin ski, P. J. Two-photon polymerization ofhydrogels−versatile solutions to fabricate well-defined 3D structures.RSC Adv. 2014, 4 (85), 45504−45516.(93) Lu, W.-E.; Dong, X.-Z.; Chen, W.-Q.; Zhao, Z.-S.; Duan, X.-M.Novel photoinitiator with a radical quenching moiety for confiningradical diffusion in two-photon induced photopolymerization. J. Mater.Chem. 2011, 21 (15), 5650−5659.(94) Cunningham, L. P.; Veilleux, M. P.; Campagnola, P. J. Freeformmultiphoton excited microfabrication for biological applications usinga rapid prototyping CAD-based approach. Opt. Express 2006, 14 (19),8613−8621.(95) Pitts, J. D.; Campagnola, P. J.; Epling, G. A.; Goodman, S. L.Submicron multiphoton free-form fabrication of proteins andpolymers: studies of reaction efficiencies and applications in sustainedrelease. Macromolecules 2000, 33 (5), 1514−1523.(96) Basu, S.; Cunningham, L. P.; Pins, G. D.; Bush, K. A.; Taboada,R.; Howell, A. R.; Wang, J.; Campagnola, P. J. Multiphoton excitedfabrication of collagen matrixes cross-linked by a modifiedbenzophenone dimer: bioactivity and enzymatic degradation. Bio-macromolecules 2005, 6 (3), 1465−1474.(97) Pitts, J. D.; Howell, A. R.; Taboada, R.; Banerjee, I.; Wang, J.;Goodman, S. L.; Campagnola, P. J. New Photoactivators forMultiphoton Excited Three-dimensional Submicron Cross-linking ofProteins: Bovine Serum Albumin and Type 1 Collagen. Photochem.Photobiol. 2002, 76 (2), 135−144.(98) Kaehr, B.; Shear, J. B. Multiphoton fabrication of chemicallyresponsive protein hydrogels for microactuation. Proc. Natl. Acad. Sci.U. S. A. 2008, 105 (26), 8850−8854.(99) Kaehr, B.; Shear, J. B. Mask-directed multiphoton lithography. J.Am. Chem. Soc. 2007, 129 (7), 1904−1905.(100) Kaehr, B.; Allen, R.; Javier, D. J.; Currie, J.; Shear, J. B. Guidingneuronal development with in situ microfabrication. Proc. Natl. Acad.Sci. U. S. A. 2004, 101 (46), 16104−16108.(101) Ovsianikov, A.; Gruene, M.; Pflaum, M.; Koch, L.; Maiorana,F.; Wilhelmi, M.; Haverich, A.; Chichkov, B. Laser printing of cells into3D scaffolds. Biofabrication 2010, 2 (1), 014104.(102) Jhaveri, S. J.; McMullen, J. D.; Sijbesma, R.; Tan, L.-S.; Zipfel,W.; Ober, C. K. Direct three-dimensional microfabrication ofhydrogels via two-photon lithography in aqueous solution. Chem.Mater. 2009, 21 (10), 2003−2006.



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(103) Kufelt, O.; El-Tamer, A.; Sehring, C.; Schlie-Wolter, S.;Chichkov, B. N. Hyaluronic acid based materials for scaffolding viatwo-photon polymerization. Biomacromolecules 2014, 15 (2), 650−659.(104) Ovsianikov, A.; Deiwick, A.; Van Vlierberghe, S.; Pflaum, M.;Wilhelmi, M.; Dubruel, P.; Chichkov, B. Laser fabrication of 3D gelatinscaffolds for the generation of bioartificial tissues. Materials 2011, 4(1), 288−299.(105) Ovsianikov, A.; Deiwick, A.; Van Vlierberghe, S.; Dubruel, P.;Moller, L.; Drager, G.; Chichkov, B. Laser fabrication of three-dimensional CAD scaffolds from photosensitive gelatin for applica-tions in tissue engineering. Biomacromolecules 2011, 12 (4), 851−858.(106) Qin, X. H.; Torgersen, J.; Saf, R.; Muhleder, S.; Pucher, N.;Ligon, S. C.; Holnthoner, W.; Redl, H.; Ovsianikov, A.; Stampfl, J.;Liska, R. Three-dimensional microfabrication of protein hydrogels viatwo-photon-excited thiol-vinyl ester photopolymerization. J. Polym.Sci., Part A: Polym. Chem. 2013, 51 (22), 4799−4810.(107) Li, Z.; Torgersen, J.; Ajami, A.; Muhleder, S.; Qin, X.;Husinsky, W.; Holnthoner, W.; Ovsianikov, A.; Stampfl, J.; Liska, R.Initiation efficiency and cytotoxicity of novel water-soluble two-photonphotoinitiators for direct 3D microfabrication of hydrogels. RSC Adv.2013, 3 (36), 15939−15946.(108) Ovsianikov, A.; Muhleder, S.; Torgersen, J.; Li, Z.; Qin, X.-H.;Van Vlierberghe, S.; Dubruel, P.; Holnthoner, W.; Redl, H.; Liska, R.;Stampfl, J. Laser photofabrication of cell-containing hydrogelconstructs. Langmuir 2014, 30 (13), 3787−3794.(109) Ovsianikov, A.; Malinauskas, M.; Schlie, S.; Chichkov, B.;Gittard, S.; Narayan, R.; Lobler, M.; Sternberg, K.; Schmitz, K.-P.;Haverich, A. Three-dimensional laser micro-and nano-structuring ofacrylated poly (ethylene glycol) materials and evaluation of theircytoxicity for tissue engineering applications. Acta Biomater. 2011, 7(3), 967−974.(110) Torgersen, J.; Ovsianikov, A.; Mironov, V.; Pucher, N.; Qin, X.;Li, Z.; Cicha, K.; Machacek, T.; Liska, R.; Jantsch, V.; Stampfl, J.Photo-sensitive hydrogels for three-dimensional laser microfabricationin the presence of whole organisms. J. Biomed. Opt. 2012, 17 (10),105008−105008.(111) Xiong, Z.; Zheng, M.-L.; Dong, X.-Z.; Chen, W.-Q.; Jin, F.;Zhao, Z.-S.; Duan, X.-M. Asymmetric microstructure of hydrogel: two-photon microfabrication and stimuli-responsive behavior. Soft Matter2011, 7 (21), 10353−10359.(112) Xing, J.; Liu, J.; Zhang, T.; Zhang, L.; Zheng, M.; Duan, X. Awater soluble initiator prepared through host−guest chemicalinteraction for microfabrication of 3D hydrogels via two-photonpolymerization. J. Mater. Chem. B 2014, 2 (27), 4318−4323.(113) Culver, J. C.; Hoffmann, J. C.; Poche, R. A.; Slater, J. H.; West,J. L.; Dickinson, M. E. Three-Dimensional Biomimetic Patterning inHydrogels to Guide Cellular Organization. Adv. Mater. 2012, 24 (17),2344−2348.(114) Malinauskas, M.; Danilevicius, P.; Baltriukiene, D.; Rutkauskas,M.; Zukauskas, A.; Kairyte, Z.; Bickauskaite, G.; Purlys, V.; Paipulas,D.; Bukelskiene, V.; Gadonas, R. 3D artificial polymeric scaffolds forstem cell growth fabricated by femtosecond laser. Lith. J. Phys. 2010,50 (1), 75.(115) Zhang, W.; Chen, S. Femtosecond laser nanofabrication ofhydrogel biomaterial. MRS Bull. 2011, 36 (12), 1028−1033.(116) Zhang, W.; Soman, P.; Meggs, K.; Qu, X.; Chen, S. Tuning thepoisson’s ratio of biomaterials for investigating cellular response. Adv.Funct. Mater. 2013, 23 (25), 3226−3232.(117) Basu, S.; Campagnola, P. J. Properties of crosslinked proteinmatrices for tissue engineering applications synthesized by multi-photon excitation. J. Biomed. Mater. Res. 2004, 71A (2), 359−368.(118) Su, P.-J.; Tran, Q. A.; Fong, J. J.; Eliceiri, K. W.; Ogle, B. M.;Campagnola, P. J. Mesenchymal stem cell interactions with 3D ECMmodules fabricated via multiphoton excited photochemistry. Bio-macromolecules 2012, 13 (9), 2917−2925.(119) Klein, F.; Richter, B.; Striebel, T.; Franz, C. M.; Freymann, G.v.; Wegener, M.; Bastmeyer, M. Two-component polymer scaffolds for

controlled three-dimensional cell culture. Adv. Mater. 2011, 23 (11),1341−1345.(120) Koroleva, A.; Gill, A.; Ortega, I.; Haycock, J.; Schlie, S.; Gittard,S.; Chichkov, B.; Claeyssens, F. Two-photon polymerization-generatedand micromolding-replicated 3D scaffolds for peripheral neural tissueengineering applications. Biofabrication 2012, 4 (2), 025005.(121) Greiner, A. M.; Jackel, M.; Scheiwe, A. C.; Stamow, D. R.;Autenrieth, T. J.; Lahann, J.; Franz, C. M.; Bastmeyer, M.Multifunctional polymer scaffolds with adjustable pore size andchemoattractant gradients for studying cell matrix invasion.Biomaterials 2014, 35 (2), 611−619.(122) Kim, S.; Qiu, F.; Kim, S.; Ghanbari, A.; Moon, C.; Zhang, L.;Nelson, B. J.; Choi, H. Fabrication and Characterization of MagneticMicrorobots for Three-Dimensional Cell Culture and TargetedTransportation. Adv. Mater. 2013, 25 (41), 5863−5868.



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