Optimized Photoinitiator for Fast Two-Photon Absorption ... · DOI: 10.1002/adem.201600686...

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DOI: 10.1002/adem.201600686
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Optimized Photoinitiator for Fast Two-Photon AbsorptionPolymerization of Polyester-Macromers for TissueEngineering**

By Leander Poocza, Michael Gottschaldt, Eric Markweg, Nicole Hauptmann,Gerhard Hildebrand, David Pretzel, Matthias Hartlieb, Christian Reichardt, Joachim Kubel,Ulrich S. Schubert, Olaf Mollenhauer, Benjamin Dietzek and Klaus Liefeith*

The efficiency of two-photon polymerization (TPP) techniques depends on the photoinitiator (PI)systems. Objective of this study was to enhance the performance of a cyclopentanone-based PI byintroducing polar side chains in order to increase the solubility of the PI in the investigatedmacromonomers urethanedimethacrylate (UDMA) and lactide-caprolactone-methacrylate (LCM).The conditions for TPPwere investigated in writing power/speed arrays. To confirm the high reactivityof the new PI a maximum speed experiment at a concentration of 2% was performed and a TPPstructure in the dimensions of several centimeters could be written, which to our knowledge has not yetbeen reported elsewhere.

1. Introduction

Biocompatible polymers are applied in several biomedicalapplications, such as scaffolds for tissue engineering inparticular for the design of bioactive carriers for drug deliveryand targeted therapeutics as well as for bioactive coatings, orelectrospun fibres.[1–3] Over the last decades strategies focusedon the preparation of multifunctional monomers that containtwo or more polymerizable groups to form cross-linkedscaffolds of definedmicrostructures[4,5] by two-photon absorp-tion (TPA) initiated two-photon polymerization (TPP). The useof TPP produced scaffolds in biomedical applications hassignificantly increased in recent years[6] and the so produced

[*] Prof. Dr. K. Liefeith, L. Poocza, Dr. N. HauptmannDr. G. HildebrandInstitute for Bioprocessing and Analytical MeasurementTechniques e.V., Rosenhof, 37308 Heilbad Heiligenstadt,GermanyE-mail: [email protected]. PooczaUniversity of Valladolid, Paseo de Bel�en 19, 47011 Valladolid,SpainDr. M. Gottschaldt, Dr. D. Pretzel, Dr. M. HartliebProf. Dr. U. S. SchubertLaboratory of Organic and Macromolecular Chemistry, FriedrichSchillerUniversity Jena,Humboldtstrasse 10, 07745 Jena,Germany

Jena Center for Soft Matter, Friedrich Schiller University Jena,Philosophenweg 7, 07735 Jena, Germany

DOI: 10.1002/adem.201600686 © 2017 WILEY-VCH Verlag GmbH &ADVANCED ENGINEERING MATERIALS 2017,

materials have the potential to replace state-of-the-art implantsand allografts.[5,7] It is common knowledge, that in vivo nearlyall tissue cells reside ina3Dextracellularmatrix (ECM). For thatreason, the establishment of 3D scaffolds to reproduce thecomplex and dynamic environment of tissues and organs isdirectly associated with the development and adaptation of avariety of fabrication processes tomeet the requirements of cellgrowth, organization, and differentiation. Having in mind, theenormous importanceofhierarchical structures, forexample, ofbiological load carriers like bones, tendons, or ligaments,techniques like TPP gain valuable ground, which allow the

E. Markweg, O. MollenhauerTETRA Society for Sensoric, Robotics and Automation mbH,Am Wald 4, 98693 Ilmenau, GermanyC. Reichardt, J. Kubel, Dr. B. DietzekInstitute of Physical Chemistry and Abbe Center of Photonics,Friedrich Schiller University Jena, Albert- Einstein-Strasse 9,07743 Jena, Germany

Prof. Dr. B. DietzekLeibniz Institute of Photonic Technology, Albert-Einstein-Straße 9, 07743 Jena, Germany

[**] This work was supported by the European Commission (grantnumber: 263363), and the Federal Ministry for Educationand Research (FKZ: 03WKCB01C). (Supporting informationis available online from Wiley Online Library or from theauthor).

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L. Poocza et al./Optimized Photoinitiator for Fast Two-Photon Polymerization

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formationandfabricationofanartificial 3DECM(scaffold) over
several orders of magnitude (nano to centimeter range).The effect of TPA, described in 1931 by Göppert-Mayer

makes use of wavelength in the near-infrared (NIR) that canlead to an excited state by the simultaneous absorption of twophotons. The probability of TPA, which quadraticallydepends on the laser intensity, is highest in the focus of thelaser beam. This non-linear effect together with the highpenetration depth of the NIR light, can be used for thespatially defined resolution of excitation in various applica-tions.[8,9] So far, two-photon polymerized structures are usedas prototypes or in in vitro experiments.[6,10] The advantage ofTPP in contrast to other structuring techniques, like lithogra-phy or 3D printing, is the possibility to create any 3Dstructureswith a defined nano and/ormicro resolution. Since,this technique is known to be rather slow to be commerciallycompetitive, the optimization of the compounds like PI andmacromonomers is essential for better radical translation andincreased efficiency.[11]

There are major challenges toward commercialization oftwo-photon based rapid prototyping due to the longprocessing time of comparatively small structures.[12] Up tonow, structures produced by TPP are limited in theZ-dimension to a few millimeters.[13–15] This is caused bythe restricted working distance of the objective and therefractive index of the photoresist, that limits controlledpenetration of the photoresist to 2–3mm.[14] Technicalinnovations begin to overcome the problems of the reducedZ-size of structures, but toward larger structures productiontimes increase by approximately three orders of magnitude.The major determining factors of processing time are speed ofthe laser beam and initiator efficiency. Nowadays, laser basedrapid prototyping facilities allow the application of extremehigh velocities with a sufficient precision/resolution of thefabricated structures. This is realized by using a combinationof optical and mechanical positioning systems. The opticalsystem, like a supporting galvano-scanner, is responsible forthe movement of the laser beam. Mechanical positioningsystems realize a simultaneous movement of the sample by aprecise high-speed control of a XYZ-stage, which carries thework piece or the precursor-system that has to be struc-tured.[16] With this, state-of-the-art laser facilities enable forwriting speeds of up to 1ms�1 at big distances. By utilizinggalvano-scanners writing speeds of more than 100m s�1 canbe accomplished theoretically.

To further accelerate the writing process, the introduc-tion of a faster chemistry is necessary. The conversion oflaser light energy into the chemical photoresists must beoptimized. One possibility is to increase the concentrationof the used photoinitiator (PI) to match the faster devicevelocities. But, this can cause unknown changes in thematerial characteristics due to the enhanced concentrationof incorporated PI. Asides impairing the material proper-ties, also biocompatibility issues arise with higher amountsof PI, which cannot be per se be classified as biologicallyharmless.[17,18] When it comes to the application of the

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generated constructs in biomedical applications, initiatormigration issues have been considered. Most of the researchhas been done in the investigation of photocured polymersfor food packing, where migration related harmful sideproducts are of refs.[19,20] In principle, the migration canoccur by set-off, by diffusion, and by gas-phase transport.Norrish type I and type II migration, especially occur onaromatic compounds, where relatively stable radicals canbe formed. For example, one route for acetophenone basedPIs, like Irgacure 369 (IC369), is to generate their ownscission products (type I), which are in this case smallaromatic radicals that can react to potentially harmful sideproducts.[21] One route to avoid the self-scission and to limitdiffusion, are bigger initiator molecules (macroinitiators),or cyclic ketones (cyclopentanone based PIs). Nevertheless,these compounds are still able to initiate migration in aNorrish type II mechanism by electron transfer.[20] There-fore, from a chemical point of view, for a selected polymersystem the photoinitiator has to be optimized. In general,the ideal PI possesses a large two-photon cross-sectioncombined with low fluorescence quantum yield. Thiswould lead to less radiative deactivation and a higherpopulation of the active state for initiatingthe polymerization.[11] Several donor-p-donor and donor-p-acceptor-p-donor systems,[11,22,23] similar as variousacetophenons[24–26] have been proven to be efficient PIsfor TPP. But, it has also been shown that a suitable“interaction” of the used PI with the macromonomers (e.g.,in terms of solubility of the PI in the macromonomer) is justas important as a high GM-value.[27]

In keeping with the maxim “like dissolves like” thehypothesis of our approach was, that the integration ofadditional ester groups into a known cyclopentanone basedchromophore structure with a high two-photon cross sectionshould result in an enhanced activity of the PI in thepolymerization of polyesters, which are a key component inrecently developed bio-degradable polymers.[28–32] After thesuccessful establishment of an effective synthesis route of thenew PI (BA740), its potential for TPP was investigated interms ofwriteability and process velocities. In comparison to awidely used commercial system of IC369 and UDMA.[33,34]

The study further includes other PIs (Figure 1), tested in bothprecursor systems to explore the determinants of writeabilityin the LCM system.[24,25,35]

2. Experimental Section

2.1. MaterialsN,N-Dihydroxyethylaniline, acetic acid anhydride, sodium

sulfate, phosphoroxy-chloride, cyclopentanone, cyanuricchloride, d,l-Lactide, e-caprolactone, diethylenglycol (DEG),stannous 2-ethylhexanoat, triethylamin, methacryloyl chlorid(MACl, 1846), hydroquinone monomethyl ether (MEHQ),urethanedimethacrylate (UDMA), and solvents of reagentgradewere purchased fromSigma-AldrichGermany. Irgacure907 (IC907) was purchased from TCI Europe, Irgacure 369

lag GmbH & Co. KGaA, Weinheim DOI: 10.1002/adem.201600686ADVANCED ENGINEERING MATERIALS 2017,

Fig. 1. Structures of the precursor systems and the tested PIs.


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(IC369) from Ciba AG and, 2, 5-bis[[4-(N,N dimethylamino)phenyl]methylene]cyclopentanone (BDEA, from AlindaChemical Ltd.).

2.1.1. Synthesis of BA740, N,N-Bis-(2-(Acetyloxy)-Ethyl)Aniline (1)[36]

The colorless solution of 54.33 g (0.299mol)N,N-dihydrox-yethylaniline, together with 120mL (1.2mol) acetic acidanhydride was stirred at 60 to 65 �C for 2 h. After cooling,the mixture was poured into ice water and the aqueous layerwas extracted with chloroform. Then, the organic phase waswashed until neutrality and dried over sodium sulfate. Afterevaporation of the solvent, the product was obtained ascolorless oil. Elemental anal. calc. for C14H19NO4 (26 531 gmol�1) [%]: C 63.38, H 7.22, N 5.28; found [%]: C 63.61, H 7.36,N 5.41. The product could be used for the next step withoutany further purification.

2.1.2. 4-[N,N-Bis[2-(Acetyloxy)Ethyl]Amino]-Benzaldehyd(2)[37]

Twenty-seven gram (0.102mol)N,N-bis-(2-acetoxyethyl)aniline (1) dissolved in 22.7 g (0.31mol) DMF were cooleddown to 0 �C. Afterward 15.7 g (0.102mol) phosphoroxy-chloride were added slowly and a yellowish precipitate wasformed. The temperature was raised slowly to 20 �C and,finally, the mixture was heated to 70 �C and stirred for 3 h.After cooling to room temperature, the mixture was pouredslowly into ice water. The aqueous phase was neutralizedby addition of 5N NaOH. The formed yellow precipitatewas collected and washed with water. Recrystallizationfrom cyclohexane yields 17.9 g of the product (60%, yellowcrystals, mp¼ 62 �C). Elemental anal. calc. for C15H19NO5

DOI: 10.1002/adem.201600686 © 2017 WILEY-VCH Verlag GmbH & CADVANCED ENGINEERING MATERIALS 2017,

(293.32 gmol�1) [%]: C 61.42, H 6.53, N 4.78; found [%]: C61.87, H 6.52, N 4.90.

2.1.3. 2,5-Bis[4-[N,N-Bis-[2-(Acetyloxy)Ethyl]Phenyl]-Methylene]-(2E,5E)-Cyclopentanone (BA740)

4-[N,N-Bis[2-(acetyloxy)ethyl]-amino]-benzaldehyd (2) (4 g,13.6mmol), cyclo:pentanone (576mg, 6.8mmol) and cyanuricchloride (252mg, 1.37mmol) together with one drop of waterwere added into a flask. The initially dark green mixture washeated to 75 �C under vigorous stirring for 1 h. After cooling toroom temperature, the raw product was dissolved in chloro-form, washed with water and dried over sodium sulfate.Purificationof theproductwasrealizedbytwodifferentcolumnchromatographies (silica gel 60, 1: eluent tert-butyl-methylether, Rf¼ 0.21; 2: ethyl acetate, Rf¼ 0.67). The product isisolated as dark red oil with a yield of 35% (1.5 g). Elementalanal. calc. for C35H42O9N2 (634.73gmol�1) [%]: C 66.23; H 6.67;N 4.41; found: C 66.35; H 6.78; N 4.31. 1H NMR (300MHz,CDCl3, d in ppm): 2.05 (s, 12 H, CH3-acetyl), 3.05 (s, 4 H, CH2

cyclopentanone), 3.67 (t, 8 H, N-CH2), 4.26 (t, 8 H, O-CH2), 6.80(d, 4 H, CH arom.), 7.51 (m, 6 H, ¼CH, CH arom.); 13C NMR(CDCl3,d inppm):20.9 (CH3acetyl), 26.6 (CH2cyclopentanone),49.6 (N-CH2), 61.2 (O-CH2), 111.9, 132.8 (CHarom.), 125.1, 133.2(Cquart. arom.), 134.0. 147.9 (C¼C), 170.9 (COO�), 196.1 (C¼O).ESI MS (m/z): 657.27 [MþNa]þ.

2.2. Photophysical CharacterizationTo investigate the TPA cross section of the photoinitiators,

the Z-Scan technique[38,39] was applied (Figure 2). Theexcitation beam (λ¼ 805 nm) was generated by a CoherentLibra Ti:Sapphire Amplifier (Seed; Vitesse, Pump: Evolution),the laser delivered a pulse train of 74 fs (FWHM) pulses at a

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pulse-to-pulse repetition rate of 1 kHz. A small portion of the
light was split off by a beam splitter and its intensity wascontinuously monitored by a photodiode for intensityreference. The main part of the beam was focused into thesample (1mm cuvette), which could be moved axially alongthe optical axis of the beam by a computer controlled stepmotor. As a function of the sample position with respect to thefocus of the laser beam (Figure 2), the transmitted intensity ofthe laser beam was monitored by a second slow photodiode.Suchmeasurementswere performed for each photoinitiator atfour different input powers. Each experiment was repeatedthree times.

Synthesis of the statistical macromonomer of lactic acid(LA) and e-caprolactone (CL) with methacrylate end groupswas adapted from Davis et al.[35] In brief, d,l-lactide (23.63 g,163.95mmol), e-caprolactone (3.83 g, 33.55mmol), and DEG(2.00 g, 18.85mmol) were added to a 500mL Schlenk roundbottom flask. Then stannous 2-ethylhexanoat (300mL,0.33mmol) was added and the mixture reacted overnight at130 �C. After cooling to RT and flushing with argon,methylene chloride (100mL) was added and the solutionwas cooled to 0 �C. Then TEA (4.71 g, 46.54mmol), and MACl(4.33 g, 41.42mmol) dissolved in methylene chloride (75mL)were added dropwise over a period of 2 h. The flask waswarmed to RTand stirred overnight. The solution was filteredand the methylene chloride evaporated under reducedpressure. The resulting viscous liquid was dissolved in ethylacetate (50mL), filtered again and precipitated into n-hexane(500mL) under stirring. The precipitate was dried anddissolved again in methylene chloride, washed with aqueoushydrochloric acid (3%, 2� 100mL), saturated aqueoussodium bicarbonate solution and saturated aqueous sodiumchloride solution (200mL), and dried overmagnesium sulfate.Mono methyl ether of hydroquinone (MEHQ; 300ppm) wasadded for stabilization. The process results in a yield of 70% ofa colorless liquid, with a molecular mass of 1 800 gmol�1

(calculated from 1H-NMR). (LA:CL 18:2). 1HNMR (300MHz,CDCl3, d in ppm): 1.2–1.6 (m, CH3- LA, �CH2- CL), 1.8 (s,CH3-), 2.3 (m, methylene protons adjacent to carbonyl carbonin CL), 3.6 (m, DEG ether protons), 4.0–4.2 (m, CH2-O), 5.0 (m,LA backbone), 5.5 (s, CH2¼C), 6.2 (s, CH2¼C).

2.3. TPP Structuring of UDMA and LCMFor processing, the precursors LCMandUDMA, they, were

mixed with the initiators IC369, IC907, BDEA, and BA740 (0.2and 2.0wt.%) dissolved in acetone (0.2 gmL�1) and stirred

Fig. 2. Scheme of the used Z-Scan technique.


overnight at 60 �C to ensure a thorough mixing and toevaporate traces of acetone, which impair the writing results.

For the first writeability trials, line experiments wereperformed. We did not obtain structuring with the followingprecursor systems: LCM with IC369 (0.2wt%), IC907 (0.2 and2.0wt%). BDEAwas not soluble in both precursors. Therefore,these precursor/PI mixtures were excluded from furtherinvestigations. The quantification of the obtained structureswas performed by SEM after development in acetone.

The TPP facility (M3DL, LZH Hannover, Germany)equipped with a femtosecond laser source, with 800 nmwavelength and 3W cw (continuous wave) power, delivers140 fs pulses at 80MHz repetition rate (Ti:sapphire; VISION II,Coherent, USA). The beam is focused by an x63 objective lenswithNA 0.75 (LD-Plan-NEOFLUAR, Zeiss, Germany) into thephotoresist. Structures were produced by moving the samplewith a three axis nanopositioning stage according to thecomputer model supported by a galvano-scanner (Aerotech,USA), as already mentioned. Power levels noted weremeasured in the focal point immediately behind the objective.

2.4. Line ExperimentA frame of 1 200� 100mmwaswritten first to fix the lines in

place. Line length was set to 100mm and line to line distancewas 100mm. All lines were written in triplicates. The powerremained constant at 125mWand speed was varied from 0.25to 10.00mms�1. Structures were then developed in acetone,and dried at ambient temperature. The line thickness wasmeasured from SEM images.

2.5. StructuringThe test structure was a Schwarz P unit cell volume of

250� 250� 250mm3. Power was varied from 50 to 180mWand speed from 0.50 to 5.00mms�1. Prior to the writing of theSchwarz P arrays, a base plate (2.5� 2.5mm2) was polymer-ized to fix the test structures at a defined position (Hatch andSlice of 2mm).

Experiments for maximal speed estimation were writtenwith a galvano-scanner unit (Aerotech) at maximum power(400mW) with a 20� objective 0.5 NA (EC-EPIPLAN-NEOFLUAR; Zeiss, Germany) and with 5.00mms�1 axisvelocity, as well as 50–250mms�1 scanner velocity. Theresulting structure consisted of 32 Schwarz P unit cells. It is ofvalue to note, that the application of an industrial TPPproduction unit (MBZ � TPP) enabled the fabrication ofcomparatively large scaffolds. TheMBZ� TPP is a productionunit equipped with a fiber laser and based on a special planarpositioning system.[40] A maximum speed of 500mms�1 canbe reached using this system. The area, which is accessible forwriting can be expanded to 200� 200� 100mm3. All compo-nents are products of TETRA (PPS 2020D, TETRA, Germany;Linear drive N2-EM-LPS007-50D, TETRA, Germany). Thelaser power was varied from 30 to 120mW. TheMBZ� TPP isequipped with a femtosecond laser source (TOPTICAPhotonics AG, Germany) with a wavelength of 780 nm anda continuous material supplying system.



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2.6. SEM ImagingThe morphology of TPP structures was investigated by

scanning electron microscopy (SEM) using a Zeiss EVO LS 10.All TPP-structured samples were prepared using an internalstandard protocol (propanol series, critical CO2-drying, Au-sputtering) to avoid typical artifacts. All SEM investigationswere performed under high vacuum conditions withaccelerating voltages around 15 keV, and a working distanceof 10–12mm. Specific imaging settings like magnification areshown in the data zone of the corresponding SEM images.

Fig. 3. Z-Scan measurement of BA740 in chloroform at an input power of 67GWcm�2.Symbols represent experimental data, while solid line refers to Gaussian fit.

3. Results and Discussion

3.1. SynthesisIn order to obtain the PI functionalized with ester groups

in the periphery, N,N–dihydroxyethylanilin was acetylatedto give the diester 1, which was formulated with DMF/POCl3 to 4-[N,N-bis[2-(acetyloxy):ethyl]amino]-benzalde-hyd (2). The synthesis of BA740 was finally achieved byaldolcondensation of cyclopentanone with 2 in a procedureadopted from literature[41] using cyanuric chloride as thecatalyst (Scheme 1).

3.2. Photophysical CharacterizationThe result of a Z-Scan measurement is shown in Figure 3.

The minima characterize the TPA of the sample in the beamfocus. A Gaussian fit was used to analyze the data andto determine the transmission T at the minimum. WithEquation 1, the TPA cross section s was calculated:

s ¼ 1� Tð ÞEPh

T l I0 NA c2ð1Þ

where EPh is the photon energy, l is the sample path length, I0is the input power, NA is the Avogadro constant, and c is thesample concentration.

The Z-Scan experiments of the reference sample Irgacure369 and Irgacure 907 showed no discernible TPA signal, whilefor the bis(benzylidene)cyclopentanone, derivatives a signal

Scheme 1. Schematic representation of the synthesis of the new photoinitiator.


based on TPAwas observed for all input intensities. Due to thefact, that increasing the laser power results in higher ordernon-linear effects like thermal lensing,[39,42] it is conceivablethat the measurement of the two-photon absorption cross-section was adversely impacted. The results of the Z-Scanexperiment with the lowest input energy, corresponding to 67GW cm�2, were used to determine the TPA cross section[43]

(Table 1).In agreement with literature, the TPA cross section for

BDEAwas determined to 315 GM.[44,45] BA740 revealed a TPAcross section of 177 GM. This fact illustrates, that the twophoton absorption cross section is not the only relevantparameter in this context and that other factors, for example,the quantum yield of radical formation and/or the initiatorrate, have also to be taken into account.[46,47] A first indicationfor the efficiency of BA740 in forming radicals was obtainedfrom considering the emission quantum yields of thecompounds investigated, in this study (Table 1).

Under the assumption that the non-radiative decay of thephotoexcited state is dominated by radical formation, anincreased emission quantum yield points to a reduced


Table 1. Calculated TPA cross section s and standard deviation ss of the Z-Scanexperiment with an input power I0 of 67GWcm�2 and emission quantum yield Fe

of the bis(benzylidene)cyclopentanone derivates

PI s in GM Ss in GM Fe in %

IC369/IC907 [40] <10 – –

BA740 177 3.2 6.4BDEA 315 5.7 12.5


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propensity of radical formation. For the compounds dissolvedin chloroform, a two times higher emission quantum yield ofBDEA (12.5%) compared to BA740 (6.4%) was observed. Thisindicates that for BA740, the probability to form radicals isincreased indicating a higher efficiency as photoinitiatorsystem for two-photon polymerization processes.

3.2.1. TPP ExperimentsThe polymer precursor LCMwas synthesized according to

Davis et al.[35] and Felfel et al.[48] As PIs for structuringexperiments BA740 was chosen and compared to commer-cially available IC369 and IC907. BDEA was insoluble inthe polymer precursors used for this work even at lowconcentrations and could not be further investigated in termsof writeability. All experiments were performed using a broadpower spectrum (50–200mW measured behind the objectivelens) and a broad speed range (0.50–5.00mms�1), in order tobe able to generate comparable data (structures) for the mostefficient system (BA740/LCM) and the less efficient system(IC907/UDMA).

As a first approach, a line array of 12 precursor/PImixtureswas generated including all three PIs at two concentrations(0.2 and 2.0wt%) with two different polymer precursors,commercial UDMA, and the biodegradable polyester LCM.Secondly, the conditions were varied in a writing power/speed array to determine the structure stability relationship.The good performance of the LCM mixture containing 2%BA740 allowed for a maximum speed experiment. Withoptimized parameters, we were able to write a TPP structurein the dimensions of several centimeters, which to the best ofour knowledge has not yet been reported elsewhere.

3.2.2. Line ExperimentsTo investigate the writeability of the precursor materials,

lines of different writing speeds were written into a singlesupport structure (Figure 4d) to keep the building time short.The acetophenone based PIs showed less efficiency in TPP.Concentrations higher than 1% were needed to structurethe precursors. IC907 reacted under the given conditionsonlynwith UDMA2%, and the resulting lines were comparablethin (Figure 4). Moreover, thickness fluctuations ranging from7.4 to 3.8mmwere observed. The lines appear not straight, butslightly curved atwriting speeds higher than 0.25mms�1, andsignificant mechanical instabilities were seen above a writingspeed of 2.5mms�1.


With IC369 polymerization of LCM and UDMA wasonly possible at high PI concentrations of 2wt%. The linethicknesses in UDMA/IC3692% and LCM/IC3692% werecomparable. UDMA/IC3692% lines had a stronger tendencyto form curvature at high writing speed. It should bementioned that the performance of IC369 was significantlybetter in comparison with IC907.

Polymerization with BA740 was more effective, lines couldbe obtained in both precursors even at low concentrations. Theline thickness in UDMA/BA7400.2% ranged from 12.6–5.2mm,which is comparable with the data obtained with UDMA/IC3692%, despite the achieved tenfold dilution. A furtherimprovement of the polymerization efficiency, with thickerlines was observed, when raising the concentration (UDMA/BA7402%). However, this improvement was seen primarilybelow 1.0mms�1. As expected, the activity of LCM/BA7400.2%

was already significantly higher and considerable thicker lineswere generated. In detail, at velocities lower than 2.5mms�1,line thickness ranged from 17.0–22.7mm, whereas withhigher velocities, the thicknesses approximated to UDMA/BA7402%. LCM/BA7402% showed dramatic increase ofpolymerization with a maximum diameter of 45mm at0.25mms�1, which was more than twice the diameter ofUDMA/BA7402%, and equals a fivefold larger polymerizedvolume. It can be concluded that the photoinitiator BA740 hasshown a significant superiority for achieving an efficientpolymerization process of biodegradable lactide-caprolactone-methacrylate (LCM) macromonomers. Only very slightmechanical instabilities were seen above writing speedsof 5.0mms�1.

3.3. StructuringFor the stability tests, structures based on Schwarz P unit

cells of 250mm (Figure 5d) were chosen.[49,50] These structuresconsist of triply periodic minimal surfaces (TPMS) and arepotential candidates for porous biomimetic scaffolds with ahomogenous stress and strain distribution.[47] Furthermore,these structures show a cell design that yields an optimizedmicrostructure for stiffness and fluid permeability.[51,52] Thismeans, that these structures show an optimal structurestability from a mechanical point of view and possibledeformations and instabilities observed in this study must becaused by an inadequate polymerization process during TPP.Hence, one can get an impression whether the degree ofpolymerization of the written structures is too low toguarantee the necessary structural stability, or too muchenergy is introduced into the system, so that the pores of thestructure are sealed by undefined bulk or auto-polymeriza-tion. For visualization, the written structures were colorranked from no/poor polymerization (white/blue) to thepoint where bulk polymerization, that is sealing the pores ofthe structure occurs (red), the results were, thus, displayed ina heat map (Figure 5).

The results of the writing process are summarized inFigure 5. There are two mayor trends, which are dominatingthe results. i) Enhanced efficiency of acetophenone based PIs


Fig. 4. TPP-Line experiments of the PIs in different polymer precursors. (a) in UDMA, (b) in LCM. (c) SEM micrographs of the lines written at different speed. (d) Scheme of theframe used to fix lines in place.


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(IC369 and IC907) was observed for UDMA compared to LCM(UDMA>LCM). For example, IC907 showed no reaction withLCM, IC369 only with low speed and high power (above100mW). ii) BA740 was much more reactive than theacetophenone based PIs and showed an inverted reactivitypattern (LCM>UDMA).

3.4. Effect of Different PIs on a Synthetic BiocompatibleUDMA

For IC907, as already indicated by the line experiments,only structures in UDMA were obtained, which were onlyachieved at high power (150–180mW) and very low velocities(< 1.0mms�1). The reactivity of IC369 in UDMA is slightlyhigher and stable structures could be achieved at alltested velocities by adjusting the power. Under the givenparameters, IC369 showed the whole range of results from toohigh energy levels leading to uncontrolled polymerizationreactions (120–180mW; <0.5mms�1) to subcritical energylevels without any polymerization (50mW; >1.0mms�1, and90mW;>5.0mms�1). A power-speed-relation can be defined,


where ideal structures can be achieved either by reducing thepower or raising the writing velocity.

It is in notable agreement with the described line experi-ments, that the polymerization initiated by BA740 was muchmoreefficient in comparisonwith IC369and IC907.This offers acertain potential to render the polymerization process techno-logicallymore effective,whenusingaPI concentrationof 2%or,alternatively, to reduce the PI concentration (0.2%) to bettermeet biocompatibility issues. Even with the lowest power andhighest speed traces of polymerized precursor were observed(50mW; 3.0–5.0mms�1). Looking onto the higher energies(180mW) an intensive bulk polymerization around thestructures occurred over the whole investigated speed-range(0.5–5.0mms�1). Further, parameter variation led finallyto optimal conditions at low power and low speed (50mW;0.5–1.0mms�1). When using concentrations of the PI of 0.2%,the resulting structures corresponded exactly to the defaultsettings of the software based control unit. So, a tenth of BA740concentration is needed for fabricating structures with a veryhigh quality. As a result, it can be stated that the fabrication of


Fig. 5. ResultsofTPPstructuringwithdifferentPIs. (a) IC907; (b) IC369,and(c)BA740. (d)CADsimulatedSchwarzPunit cell (side andplanview). (e):RepresentativeSEMmicrograph(plan view of Schwarz P unit cells with 250mm) of BA740 (0.2%) in LCM (highlighted in grey). The colored circles represent the obtained results in the heat maps (a–c). Dark Red: bulkpolymerization, loss of structure;Red: bulk polymerization is sealing the pores; Yellow:most precise 3DSchwarzP structure, bestTPPwriting conditions; Blue: polymerized structures arenot stable/load bearing; With x: not a single trace of polymerization, no TPP writing possible.


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structuresdefinedas optimumispossibleunder this conditionsand that optimal results were obtained for intermediate andhigh power values over the whole velocity range.

3.5. Effect Of Different Pis On Synthetic BiocompatiblePolyester

The designed bio-degradable polyester showed differentaffinity toward TPP with the different PIs. IC907 did not leadtoward TP induced polymer formation even at concentrationsof 2wt%. IC3692% led to optimal structures at high powers(150–180mW) and a low speed (< 1.0mms�1). By reducing thepower, structures became more unstable and below 100mW, it


was impossible to initiate a polymerization reaction. By raisingthe speed above 1.0mms�1, no polymer was formed. Thisreduced reactivity of the commercial PIs in polymerizing theLCM macromonomer could be overcome utilizing BA740.

Already at low concentrations of 0.2%, BA740 polymerformation was observed under all conditions with the novelPI, where the optimum parameter set was given at a higherspeed (3.0–5.0mms�1), when using laser power between120 and 180mW. Bulk polymerization was only observableabove 100mW combined with a writing speed slower than3.0mms�1 Evidently, it can be concluded that a concentrationof 2% of BA740 is far too high for the given parameter setting.


Fig 6. High speed experiments of LCM/BA7402% written with the galvano-scanner at 180mW. (a) STL-File of a Schwarz P scaffold with 32 unit cells. (b–d) SEMmicrographs of thewritten scaffolds with velocities of 50, 100 and 250mm s�1, respectively.


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On the basis of the before mentioned power-speed relation, ahigh speed experiment atmaximal powerwas conductedwiththis reactive precursor (LCM/BA7402%). This was necessaryfor the parameter optimization for writing a centimetersized TPP scaffold as displayed in Figure 7, with a total ofapproximately 150.000 unit cells. Here, the reduction ofwriting time per unit cell is crucial, since every second per unitcell equals to two days of writing (Table 2).

By means of the galvano-scanner, a writing speed of up to250mms�1 was realized to fabricate small scaffolds of2� 2� 1.5mm3 consisting of 500mm Schwarz P unit cells.Scaffolds were chosen instead of the single unit cell array toinvestigate the structural integrity.

Stable structuring was possible at writing speeds between50–100mms�1 (Figure 6), which is 20 times faster than theaxis acceleration in the previous experiments. At about250mms�1, which corresponds an increase of the writingvelocity by a factor of 50, the obtained structures are start tolook brittle and incomplete and the connection betweenadjacent unit cells seems to be inadequate, but still thestructures are visible after the development process.

3.5. Up-ScalingThe excellent performance of the LCM/BA740 system

possesses high potential to reduce the production time of 3Dscaffolds significantly. This seems to be quite importantespecially for the establishment of scaffolds with clinicallyrelevant dimensions in the centimeter range (Table 2).This advantage in production efficiency during photo

Table 2. Minimum processing times of scaffolds consisting of Schwarz P (500mm)3 unitwriting speed

UnitCells

UDMAIC3692%

UDMABA7400.2%

UDMABA7402%

(0.5mm)3 1 2.5min 2.5min 1.25min1mm3 8 20min 20min 10min[a]1 cm3 8000 13d 21 h 13d 21 h 6d 23 h

[a]extrapolated values from the processing time of a single unit cell.


polymerization of LCM polymers by TPP using BA740 canbe used in a different approach, where a novel device designthat enables structuring above the given Z-limit of 2mm wasapplied. In detail, this can be realized by moving the writtenstructure away from the focal plane, where the polymeriza-tion takes place and simultaneously filling the space above thealready written part of the structure with unreacted precursorsimilar than realized in a stereolithography process. Togetherwith the evaluated parameters of the high-speed experiment,it was possible to write a TPP structure as cylinder with aheight of 3.0 cm and a diameter of 2.5 cm with a definedmicrostructure. The production was done by writing theSchwarz P unit cells in a one by onemode. Consequently, afterone layer of Schwarz P cells was finished the next layer ofSchwarz P cells were written. The result of this procedure isshown in Figure 7.

4. Discussion

The functionalization of BDEA with polar side chainsenabled the solubility of the PI (BA740) in the investigatedmacromers. The present study was carried out to test thesuitability and efficiency of BA740 to initiate a light inducedpolymerization process, when using TPA structuringmethodsto fabricate scaffolds for bone tissue engineering based onbiodegradable LCM polyesters. We can conclude that it waspossible to create structures of the most reactive, as well as themost unreactive precursor/PI mixtures with the chosenvelocity and energy settings.

cells. Given are different precursor/initiator pairs on basis of the maximum possible

UDMAIC9072%

LCMIC3692%

LCMBA7400.2%

LCMBA7402%

12.5min 10min 2.5min 16 s100min 80min 20min 2min69d 11 h 55d 13 h 13d 21 h 1d 12 h


Fig. 7. (a) CAD model of the TPP industrial production unit (TETRA, Germany). (b): TPP written scaffold in cm dimensions (scale bar: 10mm) by the use of a LCM / BA740precursor system. (c): SEM micrograph (scale bar: 500mm) containing several Schwarz P unit cells.


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When comparing UDMA and LCM affinity of the chosenPIs, the efficiency of TPP polymer formation is contrary. Usingthe new developed BA740, more stable structures could befabricated with LCM in comparison to UDMA. The differenceis small at high speeds and small PI concentrations, buttremendous when reducing the speed and enhancing the PIconcentration (Figure 4). IC369 and IC907 show a betterperformance inUDMAand structures and lines can be createdunder conditions, where no polymerization of LCM wasobserved.

One reason for the different reactivity might be themolecular weight and the corresponding chain lengthbetween the crosslinking double bonds of the precursormaterials. UDMA with 470 gmol�1 is almost four timessmaller than the LCM precursor with a molecular weight of1 800 gmol�1. This deviation in double bond to double bonddistance or total double bond density, is certainly one factorthat influences the writeability of polymer precursors.

In dependence of the complexity and the size of the scaffoldstructure, the processing times can vary widely. Examples forprocessing times are rarely reported. An example given byStampfl et al. was 12min for a relatively small structure(300� 300� 80mm3 at 0.30mms�1),[12] which could beroughly compared with the volume of a single Schwarz Punit cell (Table 2), or as described by Koroleva et al.,[10] wherethe preparation of a 3mm3 scaffold (equals to 24 unit cells)took 50min. In UDMA, the processing time can be reduced bya factor of 2 when replacing IC369 with BA740. Alternatively,the writing speed can be increased by a factor of 2 at same PIconcentrations. This offers the opportunity to reduce theconcentration of the PI. The resulting process time for ascaffold in centimeter dimension is about one week. Theproduction efficiency of LCM scaffolds was increased by afactor of 50 due to higher sensitivity of BA740 in comparisonwith IC369, which indeed enables a competitive processingtime of 36 h. State of the art of writing big structures by TPP is

1600686 (10 of 12) http://www.aem-journal.com © 2017 WILEY-VCH Ve

the so-called dip-in-process. Using the novel device setup, wecould bypass the optical limitations of about 2mm focusdistance and to produce structures with centimeter dimen-sion, which to the best of our knowledge have not beenreported elsewhere.

5. Conclusions

The studies, in this article, represent a novel approach forthe generation of large scaffolds written by TPP for the use intissue engineering and regenerative medicine. In particular,we were able to modify a non-soluble cyclopentanone basedPI with a high two-photon cross section to obtain a PI solublein the LCMmacromonomer.Under the presented parameters,the BA740/LCM pair is clearly the most efficient systemleading to writingspeeds up to 250mms�1. With this system,we were able to write a structure in dimensions of 3.0 cm inheight and 2.5 cm in diameter by TPP, which give thepossibility, for example, to be investigated in animalstudies.[47,48] This opens the door for TPP to be applied infurther applications like CAD based personalized implantgeneration,when its high resolution can be combinedwith theproduction efficiency shown in this study. The potential ofBA740 to initiate a polymerization process of other photosen-sitive precursor systems is subject of further investigations.

Article first published online: xxxxManuscript Revised: December 5, 2016

Manuscript Received: September 30, 2016

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