3D打印文献,Direct laser writing,Principles and materials for scaffold 3D printing

Review ArticleDirect laser writing:Principles and materials for scaffold 3DprintingAlexandros Selimis a ,Vladimir Mironov b ,Maria Farsari a ,⇑a IESL-FORTH,N.Plastira 100,70013Heraklion,Crete,GreecebRenato Archer Information Technology Center CTI,Campinas,Brazila r t i c l e i n f o Article history:Received 23May 2014Received in revised form 21August 2014Accepted 5October 2014Available online 13October 2014Keywords:BiofabricationLaser-based 3D printing Photopolymersa b s t r a c tFor a great variety of research fields extending from photonics to tissue engineering applications,the requests for the construction of three-dimensional structures with high resolution grow more and more imperative.Towards this aim,the direct laser writing technique by multi-photon polymerization,due to its unique properties and characteristics,has proven to be an indispensable tool to high accuracy structuring and has been put on the map as an emerging technology for scaffold 3D printing.In the pres-ent review,the basic principles of multi-photon polymerization are presented,the experimental set-up requirements are described and the employed materials demands are thoroughly mentioned as well as the most representative examples of the recent developments in the field.Ó2014Elsevier B.V.All rights reserved.1.IntroductionDirect laser writing (DLW)by multi-photon polymerization (MPP)is a three-dimensional (3D)printing technology which allows the construction of readily assembled structures with sub-100nm resolution [1,2].It is based on the nonlinear photon absorption by photopolymers;the beam of an ultra-fast laser is tightly focused inside the volume of a transparent material,caus-ing it to absorb two or more photons and polymerize locally.Mov-ing the beam according to a path representing a Computer Aided Design/Computer Aided Manufacturing (CAD/CAM)model,one can fabricate a realistic micromodel of this design.DLW,along with classic stereolithography [3–5]and selective laser sintering [6,7],make up a versatile class of laser-based 3D printing techniques [8]which enable the fabrication of tailored structures directly from a computer data via a CAD/CAM pared to those two,DLW offers superior resolution and does not require the need of recoating or layer-by layer fabrication;on the negative side,it is much slower and requires expensive and specialized equipment.Pioneering work on single-photon DLW was performed in the early ‘90s [9,10].DLW by multi-photon polymerization was first demonstrated in 1997[11],and it was immediately embraced by the photonics community,as its capability for the fabrication of fully 3D,nanostructures was obvious [12,13].More recently it has found application in more diverse fields such as micro-optics[14,15],microfluidics [16],biomedical implants [17,18],and in 3D scaffolds for cell cultures and tissue engineering [19–21].Tissue engineering is defined as the discipline which applies the principles of engineering and life sciences toward the development of biological substitutes that restore,maintain,or improve tissue function or a whole organ [22].Creating an engineered tissue requires imitating the extracellular matrix (ECM);this entails find-ing a material suitable for the fabrication of a scaffold for a specific tissue engineering application,fabricating the scaffold,and seeding it with cells for cell culturing in vitro or in vivo [8,23,24].Micro-and nano-topography have been found to influence cell survival and proliferation [25];this is the main reason for which the use of DLW in scaffold fabrication has found explosive increase over the last few years.In this review,we describe direct laser writing and its applica-bility in cell culture and tissue engineering.We present the theory of multiphoton polymerization,and describe a typical experimen-tal set-up.We then describe the most widely used biodegradable and/or biocompatible materials for DLW.We finish by detailing recent developments in the field and the future prospects of the technology.2.Multiphoton absorptionThe physical phenomenon behind the operation of DLW is mul-tiphoton absorption.In this review,for simplicity reasons we shall concentrate on the theory of two-photon absorption;this can be easily extrapolated to three or more photons.Two-photon absorption (TPA)is defined as the simultaneous absorption of two photons of identical or different frequencies in/10.1016/j.mee.2014.10.0010167-9317/Ó2014Elsevier B.V.All rights reserved.⇑Corresponding author.E-mail address:mfarsari@iesl.forth.gr (M.Farsari).order to excite a molecule from one state(usually the ground state) to a higher state[26].The energy difference between these states is equal to the sum of the energies of the two photons(Fig.1).Two-photon absorption is a second-order process and as such,its strength depends on the square of the light intensity.It is several orders of magnitude weaker than linear absorption.TPA was theoretically predicted in1931by Maria Göppert-Mayer in her doctoral dissertation[27],however,it was not veri-fied experimentally until30years later by Werner Kaiser,when the invention of the laser permitted the generation of two-photon excitedfluorescence in a europium-doped crystal[28].The TPA cross section is measured in Göppert-Mayer(GM) units:1GM=1Â10À50cm4s moleculesÀ1photonÀ1.TPA can be measured by several techniques;the most common being two-photon excitedfluorescence and nonlinear transmission(z-scan) [29].As TPA is a second-order nonlinear optical process,and there-fore is most efficient at very high intensities,pulsed lasers are usu-ally employed in its measurement[26]ser sourceUntil recently,the laser source typically would be a Ti:Sapphire femtosecond oscillator operating at around800nm.However,as second-harmonicfibre lasers have become more reliable and affordable,they have taken over;they would typically operate at 780nm.DLW laser sources usually have a pulse length of less than 200fs and a repetition rate of50–80MHz.The energy required for the polymerization process depends on the material,the photoini-tiator and the focusing,but is usually in the order of a few nanojo-ules per pulse.There are also examples of an amplified ultrafast YAG(yttrium aluminium garnet)laser operating at a green wavelength(second harmonic)being used.In this case,the pulse length is sub-picosec-ond,and the laser repetition rate lies in the kHz range.The use of this type of light source is not very common in DLW,most likely due to its high cost and the availability of alternative,low cost sources.3.2.Motion systemIn order to‘write’the3D structure into the photopolymer using the focused laser beam as the tip of a pen,one needs to move either the beam focus,or the sample.The former can be done with galva-nometric scanners,while the latter with high-resolution xyz stages.When using galvo scanners,the photopolymerized structure is generated in a layer-by-layer format.The CAD/CAMfile is‘sliced’and each slice is exported as an.stlfile(STereoLithography).After the‘writing’of each slice is completed,the sample moves on the z-axis using a high-resolution translation.In addition to the z-axis movement stage,xy-axes movement stages are also required,to allow large-scale movement.When using xyz stages for structure writing,there is no need to slice the design,as the stages can move in all directions.It is pos-sible to use piezoelectric stages,which have nm-scale accuracy. These stages can travel small distances-a few hundred microme-ters at best,at tens of microns per second speed.In this case,it is also useful for these stages to be positioned on large-scale move-ment stages(such as linear or step-motor stages).Using galvo scanners has the advantage of high scanning speeds (up to m/s)and ease to fabricated curves.However,as the beam moves,it can be distorted as the objective,even if it is a-planar one,does not provide a completelyflatfield-of-view;for this rea-son,galvo scanners are not used for applications where high accu-racy is needed(such as photonics applications).On the other hand, they are very commonly used in bio-applications,where nanome-ter accuracy is not essential,and where scales are large,so high building speed is desired.In practice,most systems have both galvanometric scanners and high-accuracy xyz stages,and switch between them depending on the application requirements.Both systems have high reproducibility.3.3.Focusing opticsThe high intensities required for multi-photon absorption and high structuring resolution necessitate a tightly focused laser beam and,thus,a microscope objective has to be used;when the numer-ical aperture(N.A.)of the objective is higher than1,immersion oil is used for index matching.Galvo scanners have to be adapted to accommodate microscope objectives,as usually they are designed to take lenses with long focal lengths.One disadvantage of using microscope objectives is the short working distance(typical values:170l m–1mm),which limits the height of the fabricated structures(typical values: 30l m–1mm).To address this issue,‘‘Dip-in’’Lithography[30]Diagram of energy transitions in two-photonFig.2.A typical experimental set-up consisting of a light source,beam and samplemovement components,beam control and focusing optics,and a vision system.Engineering132(2015)83–89and the similar widened objective working two-photon polymeri-zation(WOW-2PP)[31]were developed.In this case,the micro-scope objective is immersed into the liquid photopolymer,so the working distance becomes an irrelevant factor.While this approach somehow solves the problem,it only works with liquids, while a lot of DLW materials are gels.3.4.Beam intensity controlBeam on–off control can be achieved by using a fast mechanical shutter or an acousto-optic modulator,while beam intensity control can be achieved using neutral densityfilters,a variable attenuator,or a combination of a polarizer and a waveplate.3.5.Control softwareThe optical and mechanical components described above need to be controlled centrally and synchronized.This can be performed using the commercially available software3DPoli,or by integrating laser scanning software,such as Scanner Application Modules-light (SAMlight),into homemade software.It should be noted that nowadays there are several companies providing DLW machines,using various laser sources and scanning configurations[32–35].The experimental procedure for fabricating a3D structure by DLW is shown in Fig.3.(I)The laser beam is tightly focused into the volume of the material.(II)Either the focused beam or the sam-ple move following a computer-generated pattern.(III)After the laser writing of the structure,the sample is immersed into an appropriate developer.(IV)The freestanding structure is revealed.4.Materials for laser polymerization4.1.IntroductionIn general,a material suitable for structuring with DLW includes at least two components:(i)a monomer,or a mixture of monomers/oligomers,which will provide thefinal polymer and (ii)a photoinitiator,which will absorb the laser light and provide the active species that will cause the polymerization.Several monomer/oligomer and photoinitiator combinations have been used for this purpose.These are mostly negative photoresists such as hydrogels,acrylate materials[36,37],the epoxy-based photore-sist SU-8[13],and hybrid materials[38,39].Recently,redox and Diels–Alder photopolymerization have also been reported [40,41].In the following sections,we will discuss briefly these photoinitiators and materials.4.2.PhotoinitiatorsDuring polymerization a monomer is converted into polymer and this transformation can be induced by light.In classic photoli-thography,a photoinitiator absorbs the light and produces an active species which causes the photopolymerization.In two-pho-ton polymerization,however,things are more complicated,and these extra requirements must be fulfilled[36,42]:Both the photoinitiator and the monomer/oligomer are trans-parent at the laser wavelength used,so that the laser beam can be focused inside the volume of the material without being absorbed at the surface.The monomer/oligomer needs to be transparent at the two-photon absorption wavelength(k/2);if it is not,then the photo-polymer is likely to be burnt or ablated.The photoinitiator needs not only to absorb at the two-photon wavelength,but also to have a high two-photon cross-section,a high radical quantum yield and highly-active radical speciesgenerated;typically,if any two of these three are large enough, the initiator will normally be efficient for two-photon polymerization.In classic lithography,there are two types of photoinitiators: radical photoinitiators and cationic photoinitiators.All the materi-als developed to date for DLW employ radical photoinitiators; these,upon light irradiation,generate free radicals,which initiate a polymerization process of acrylates or vinyl ethers.Cationic ini-tiators are photo-acid generators that produce cations upon light irradiation and are used for the polymerization of epoxides or vinyl ethers[43–45];to the best of our knowledge,there are no materi-als to date,specifically developed for DLW employing cationic polymerization,however,it is employed in the most commonly used photolithography resist,SU8.An effective two-photon polymerization(2PP)photoinitiator has a high quantum yield in the generation of the active moieties, high thermal stability and stability in darkness and is highly solu-ble in the polymerization medium[46].The most commonly used free radical photoinitiator is benzophenone and its derivatives [47,48].Nowadays,there is a lot of concentrated effort to synthesize fast and efficient photoinitiators specifically for multi-photon applica-tions[49].In addition,there is a lot of work toward biocompatible photoinitiators,specifically for bio-applications.To this end,classic 3.DLW experimental procedure(I)beam focusing(II)laser writing(III)development(IV)completed structure.dyes such as Bengal Rose,Eosin,Nile Red,biomolecules such as fla-vin mononucleotide,but also novel,synthetic photoinitiators have been used [37,50–55]anic photopolymersThe first materials used for two-photon polymerization were acrylate photopolymers [56].These materials have several properties useful for DLW,such as low cost and easy availability,transparency at the visible and near infrared (NIR),rapid polymer-ization with low shrinkage and,after polymerization,mechanical and chemical stability.In bio-applications,acrylate photopolymers have been used mostly for the fabrication of components for cell migration studies.In a pioneering work,Tayalia et ed a simple triacrylate photopolymer to develop a three-dimensional model of the extra-cellular matrix,which provided precise and independent control of architectural parameters for controlled cell adhesion and migration studies [57].More recently,using an acrylate photopoly-mer,3D porous structures were fabricated inside the closed micro-channel of a commercially available polymer chip for analysis of directed cell migration (Fig.4)[58].In all cases to date,however,the acrylate was not suggested as a biomaterial for medical implants or scaffolds.Two-photon polymerization of SU-8was first demonstrated by Witzgall et al.[59].Since then,SU-8has been used by a large num-ber of the research teams involved in DLW [60–64].There are few examples,however,of SU-8used in DLW for tissue engineering and microfluidic applications,with most of the latter being with one photon polymerization [65].4.5.Photosensitive hybrid materialsOver the last few years,a lot of DLW materials research has focused on the development of photosensitive hybrid composites [39].Especially silicate-only based photopolymers have proved to be a very popular choice,as they are commercially available and they combine properties of silicate glasses such as hardness,chem-ical and thermal stability,and optical transparency with the laser processing at low temperatures of organic polymers;properties impossible to be achieved with just inorganic or polymeric materi-als [66–68].The most widely used and studied silicate material is the photopolymer ORMOCER Ò(and its different formulations like ORMOCOMP Ò,ORMOCLEAR Òetc.),commercially available from Microresist Technologies,Germany [69],and it has been used for a variety of mostly photonic applications,but also as a scaffold for biomolecule immobilization and cell growth applications (Fig.5)[65,68].Other pure silicates have been used for this purpose as well [70].Materials which contain only silicon as an inorganic component do not allow,however,the optimization of the materials properties for specific applications.The versatile chemistry of hybrid compos-ites allows the co-polymerization of more than one metals and organic materials,enhancing the material mechanical stability and allowing its doping with desirable materials.There are a few examples of composite photosensitive hybrid materials used in DLW applications [38]:Ovsianikov et al.showed that under spe-cific fluence conditions,specific material combinations can be structured into complex 3D structures without shrinkage [71,72].Psycharakis et al.showed that Zirconium and Titanium doped sil-icates are biocompatible and can enhance cell growth [73].The same Zirconium material was employed by Raimondi to fabricate niches for cell growth [74],while different variants of Zirconium-Fig. 4.Acrylate 3D porous structures inside the closed microchannel of commercially available polymer chip for analysis of directed cell migration.Fig.5.(a)A 3D scaffold made of ORMOCER Ò[65]and (b)an ORMOCER Òstructure decorated with amyloid peptides [68].Engineering 132(2015)83–89that resembles the extra-cellular matrix[21,78].They can be broadly divided into two categories(i)synthetic hydrogels,and (ii)natural polymers and proteins.4.6.1.Synthetic hydrogelsThe most popular synthetic hydrogel not only in tissue engineering,but also in all kind of bio-applications,is Polyethylene glycol,commonly known as PEG[78].It is cheap,FDA(Food and Drug Administration)-approved and can exist in a lot of different formulations,which have been extensively used in one-photon and other2D and3D printing researches.Its use in DLW has been, however,somehow limited.First Malinauskas et al.made3D scaf-folds out of PEG using DLW[79],followed by other groups(Fig.7) [80–83].None of these groups,however,appeared to continue working with PEG,and its use in DLW-made scaffolds for tissue engineering has remained limited.4.6.2.Natural polymers and proteinsThe multiphoton polymerization of natural polymers and pro-teins has been pioneered by Campagnola and his co-workers, who worked with a variety of cross-linked proteins such as bovine serum albumin(BSA),fibrogen andfibronectin,and collagen [51,84–89].Seidlits et al.took it further,by crosslinking protein inside a hydrogel(hyaluronic acid)and using these scaffolds for neural cell guidance[90].The same team used a picosecond pulse duration green laser to structure pure BSA[91].BSA and also avidin using flavin mononucleotide were crosslinked by Turunen et al. (Fig.8a)[92],while Torgesen et al.photopolymerized BSA encap-sulating a live C-elegans worm(Fig.8b)[20].The same team mixed BSA with a modified gelatin,to make stable and well-defined DLW-made scaffolds[93].4.7.Chemically modified natural polymersSince natural polymers have not provided the resolution and mechanical integrity for fabrication of fully3D structures by DLW,a lot of researchers turned into modifying natural polymers. Claeyssens et al.synthesized and structured a novel,photostruc-turable composite based on polycaprolactone,[47]while the same researchers later moved to a polylactide-based polymer,which they used for the fabrication of3D structures for the alignment of neural cells(Fig.9a)[94]and study of porosity[95].Ovsianikov et al.worked on acrylating gelatin,while Kufelt et al.on Hyaluronic Acid(Fig.9b)[96].This avenue of research has provided the most promising results,as far as biodegradable materials are concerned.5.Summary and future directionsIn summary,the present review deals with the excitingfield of scaffold3D printing for tissue engineering in combination with the DLW technology.It is shown that DLW,in just a decade,has matured in a way that many laboratories apply this technique for diverse applications.Indeed,the great variety of applicable materi-als,ranging from synthetic polymers and hydrogels to natural bio-polymers and the well-defined structural features allow the realization of highly controlled test systems,where the physical, chemical and geometrical influences on cell behavior can be studied either separately or in combination.On the other hand, the deeper understanding of the crosslinking mechanisms andFig.6.(a)Mineralized3D scaffold and(b)a mineralized scaffold with osteoblastic cells[77].Fig.7.3D scaffolds made by DLW using PEG as a photopolymer[81].especially of the protein ones is still missing.Once these underly-ing mechanisms are revealed,the realization of highly controlled in vitro test systems can be achieved.However,the major issue which inhibits the wider spread of DLW technology concerns the low productivity.The current typical writing speed values are in the order of magnitude of mm/s which are insufficient for the real-ization of macroscopic scaffolds required for tissue engineering applications.Towards this improvement,an amalgam of process development and novel materials of high performance should show the right way and the 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