Vinyl esters: Low cytotoxicity monomers for the fabrication of biocompatible 3D scaffolds by...

Vinyl Esters: Low Cytotoxicity Monomers for theFabrication of Biocompatible 3D Scaffolds by LithographyBased Additive Manufacturing

CHRISTIAN HELLER,1,2 MARTIN SCHWENTENWEIN,2 GUENTER RUSSMUELLER,3 FRANZ VARGA,4

JUERGEN STAMPFL,1 ROBERT LISKA2

1Institute of Materials Science and Technology, Vienna University of Technology, Favoritenstraße 9-11,1040 Vienna, Austria

2Division of Macromolecular Chemistry, Institute of Applied Synthetic Chemistry, Vienna University of Technology,Getreidemarkt 9/163/MC, 1060 Vienna, Austria

3Department of Cranio- Maxillofacial and Oral Surgery, Medical University of Vienna, Wahringer Gurtel 18-20,1090 Vienna, Austria

4Ludwig Boltzmann Institute of Osteology, Vienna Hanusch-Hospital, Heinrich-Collin-Str. 30, 1140 Vienna, Austria

Received 7 September 2009; accepted 11 September 2009DOI: 10.1002/pola.23734Published online 5 November 2009 in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: Lithography based additive manufacturing technologies (AMT) like ster-eolithography or digital light processing have become appealing methods for the fab-rication of 3D cellular scaffolds for tissue engineering and regenerative medicine. Tocircumvent the use of (meth)acrylate-based photopolymers, that suffer from skin irri-tation and sometimes cytotoxicity, new monomers based on vinyl esters were pre-pared. In vitro cytotoxicity studies with osteoblast-like cells proofed that monomersbased on vinyl esters are significantly less cytotoxic than (meth)acrylates. Photoreac-tivity was followed by photo-differential scanning calorimetry and the mechanicalproperties of the photocured materials were screened by nanoindentation. Conversionrates and indentation moduli between those of acrylate and methacrylate referencescould be observed. Furthermore, osteoblast-like cells were successfully seeded ontopolymer specimens. Finally, we were able to print a 3D test structure out of a vinylester-based formulation by l-SLA with a layer thickness of 50 lm. For in vivo testingof vinyl esters these 3D scaffolds were implanted into surgical defects of the distalfemoral bone of adult New Zealand white rabbits. The obtained histological resultsapproved the excellent biocompatibility of vinyl esters. VVC 2009 Wiley Periodicals, Inc.

J Polym Sci Part A: Polym Chem 47: 6941–6954, 2009

Keywords: additive manufacturing technology; biocompatibility; biomaterials;in vivo testing; microstereolithography; photopolymerization; vinyl esters

INTRODUCTION

Additive Manufacturing Technology (AMT) tech-niques like stereolithography (SLA),1 3D print-ing,2 selective laser sintering,3 and fused deposi-tion modeling4 enable the fabrication of structures

Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 47, 6941–6954 (2009)VVC 2009 Wiley Periodicals, Inc.

Correspondence to: R. Liska (E-mail: [email protected])

6941

with defined geometry and pore structure andhave become very appealing methods for the pro-duction of three-dimensional scaffolds for severaltissue engineering and regenerative medicineapplications in the recent past.5,6 SLA has gainedincreasing interest especially due to the high fea-ture resolution and good surface quality comparedto other AMT techniques.7 It is based on a layer-by-layer curing of a light-sensitive resin via pho-topolymerization. Radicals or cations are beingformed upon excitation of a photoinitiator (PI)by an UV-Laser inducing polymerization andtherefore, a phase transition of the resin fromliquid to solid occurs. Such materials curingthrough radical polymerization are most com-monly based on acrylate or methacrylate chem-istry. Suitable biocompatible spacers for thesetype of reactive groups are based on carbohy-drates,8 poly(ethylene glycol),9,10 polyesters,11

peptides,12,13 and chitosan.14 It has to be notedthat the main disadvantage of methacrylates istheir limited reactivity due to the sterical hin-drance and inductive stabilization of the formedradical of the additional methyl group. Acrylatesare considerably more reactive but also show asignificant tendency toward Michael additionside reactions with amino groups of proteins orDNA giving hydrolytically noncleavable aliphaticadducts. This frequently results in some signifi-cant skin irritation or sometimes toxic effectsmaking them very controversial for biomedicalapplications.

Hence, there is a strong requirement for otherpolymerizable groups that can be photopolymer-ized and structured with stereolithography. Vinylacetate is a radically polymerizable monomershowing low toxicity.15 Poly(vinyl acetate) is usedin the food industry being FDA approved for theuse as chewing gums and cheese rinds.16 Further,poly(vinyl esters) give poly(vinyl alcohol) as theirdegradation product upon hydrolysis, a water-soluble and biocompatible polymer that has beenused for long-term implants, including bioartifi-cial pancreas, cartilage, nonadhesive film, andesophagus or scleral buckling material.17 How-ever, vinyl acetate is a very volatile monomer andtherefore, only higher molecular weight vinylesters are eligible for l-SLA. In literature, thereare some examples for the application of poly(vi-nyl esters) in the field of medical polymers.Yoshioka et al. use thermally polymerized vinyllaurate copolymers with N-vinyl pyrrolidone asdrug delivery agents18 and Miura et al. describethe thermal polymerization of enzymatic gluco-

conjugates with vinyl esters as possible polymersfor biomaterials.19

Compared to acrylates and methacrylates, onlya few vinyl esters are commercially available. Thereason might be that the common synthetic routeto vinyl esters using mercury(II)acetate as a cata-lyst is very sensitive towards functional groupslimiting the monomer design.20 Alternative meth-ods use Pd(II) salts as catalyst21,22 or seleniumbased precursors.23 Unfortunately, these proce-dures suffer from being very expensive or requir-ing a multistep synthesis, respectively. This mightalso be the reason for the fact that only a few pub-lications deal with the photopolymerizationbehavior of vinyl esters. Hoyle et al. describe thephotopolymerization behavior of divinyl fuma-rate24 and mono- and multivinyl ester monomerscontaining thioether linkages.25 According toL. M. Mironovich divinyl tri(ethylene glycol)bis(o-phthalate) could be photocured into thin poly(vi-nyl ester) films in presence of transition metalb-diketonates.26 Yet there is still no literature forvinyl ester-based photopolymers shaped by AMTor biomedical applications thereof.

Therefore, the focus of this present work is abasic investigation of different vinyl ester-basedmonomers (Fig. 1), MonoVinyl esters: hexanoicacid vinyl ester (6MV), decanoic acid vinyl ester(10MV), DiVinyl esters: succinic acid divinyl ester(2DV), adipinic acid divinyl ester (4DV), subericacid divinyl ester (6DV), sebacic acid divinyl ester(8DV), and TriVinyl ester: trimer fatty acid triv-inyl ester (FTV)) concerning their suitability forthe production of biomaterials by the means of l-SLA and comparing them to commercially avail-able (meth)acrylate references [MonoAcrylate:lauryl acrylate (12MA), DiAcrylate: 1,4-butane-diol diacrylate (4DA), MonoMethacrylate: laurylmethacrylate (12MM), DiMethacrylate: 1,4-buta-nediol dimethacrylate (4DM)] and thermoplasticpolyester references [poly(lactic acid) (PLA) andpoly(e-caprolactone) (PCL)]. These and severalother vinyl esters were recently claimed in a pat-ent for biomedical applications.27

The monomers were tested with respect totheir cytotoxicity in cell culture tests with osteo-blast-like cells. As photopolymerization is a pre-requisite for a successful structuring, photoreac-tivity of the monomers were conducted by meansof photo-differential scanning calorimetry (DSC).The mechanical properties of the resulting poly-mers were screened by nanoindentation to obtainan indentation modulus and hardness. Forselected materials, the Young’s modulus and

6942 HELLER ET AL.

Journal of Polymer Science: Part A: Polymer ChemistryDOI 10.1002/pola

flexural strength were determined by 3-point-bending tests. Finally, a suitable formulation wasselected for the 3D-fabrication of a cellular struc-ture by SLA.

EXPERIMENTAL

Materials and Characterization

Reagents and monomers and PCL were pur-chased from Sigma-Aldrich, Fluka and TCIEurope and were used without further purifica-tion. Trimer fatty acid (Unidyme 60) was pur-chased from Arizona Chemicals. PLA sampleswere obtained from BioSorbTM FX plates (Linva-tec Biomaterials). The solvents were dried andpurified by standard laboratory methods.28 1HNMR and 13C NMRspectra were measured with aBRUKERAC200 FT NMR-spectrometer. Thechemical shift is displayed in ppm (s ¼ singulett,d ¼ duplet, t ¼ triplett, m ¼ multiplett). Deutero-chloroform (CDCl3) with a grade of deuteration ofat least 99.5% was used as solvent. ATR-FTIRmeasurements were carried out on a Biorad FTS135 spectrophotometer with a Golden Gate MkIIdiamond ATR equipment (L.O.T.).

GC-MS runs were performed on a Thermo Sci-entific GC-MS DSQ II using a BGB 5 column (L ¼30 m, d ¼ 0.32 mm, 1.0 lm film, achiral) with thefollowing temperature method (injection volume:1 lL): 2 min at 80 �C, 20 �C/min until 280 �C,2 min at 280 �C. Viscosity measurements of themonomers were performed on a Physica MCR 300with a plate-plate arrangement. Tests were car-ried out at 25 �C at a shear rate of 100 s�1.

General Procedure for the Synthesis of theVinyl Esters

Mercury(II)acetate (3 mol %) and 500 ppm of hy-droquinone as inhibitor were added to a suspen-sion of the appropriate acid in a large excess ofvinyl acetate (2 mL/mmol acid group). After stir-ring the mixture for 20 min under argon atmos-phere, 0.1 mol % p-toluene sulfonic acid wasadded and the reaction mixture was refluxed for4–24 h. After cooling the resulting solution wasdiluted with ethyl acetate and extracted with 2 NNaOH. The organic layer was dried over sodiumsulfate and concentrated. The crude product waspurified by flash chromatography on silica gel (pe-troleum ether/ethyl acetate).

2DV. Yield: 37%; 1H NMR (200 MHz, CDCl3, d,ppm): 7.25 (2H, dd, J ¼ 13.89 Hz, J ¼ 6.26 Hz,H2C¼¼CHA); 4.89 (2H, dd, J ¼ 13.89 Hz, J ¼ 1.76Hz, AHC¼¼CHH); 4.58 (2H, dd, J ¼ 6.26 Hz, J ¼1.76 Hz, AHC¼¼CHH); 2.73 (4H, s, ACH2A); 13CNMR (50 MHz, CDCl3, d, ppm): 169.2 (C¼¼O),141.0 (HC¼¼CH2), 98.0 (HC¼¼CH2), 28.5 (2 �ACH2A); IR (ATR, cm�1): 1750, 1618, 1361, 1131;GCMS (m/z): 170.94, 127.00, 98.94, 71.07, 55.07.

6DV. Yield: 44%; 1H NMR (200 MHz, CDCl3, d,ppm): 7.26 (2H, dd, J ¼13.84 Hz, J ¼ 6.21 Hz,H2C¼¼CHA); 4.85 (2H, dd, i ¼ 13.84 Hz, J ¼ 1.47Hz, AHC¼¼CHH); 4.54 (2H, dd, J ¼ 6.21 Hz, J ¼1.47 Hz, AHC¼¼CHH); 2.37 (4H, t, ACOACH2A);1.65 (4H, q5, ACOACH2ACH2A); 1.48–1.25 (4H,m, ACH2A); 13C NMR (50 MHz, CDCl3, d, ppm):170.6 (C¼¼O), 141.1 (HC¼¼CH2), 97.4 (HC¼¼CH2),33.7 (2 � ACH2ACO), 28.6 (2 � ACH2A), 24.3(2 � ACH2A); IR (ATR, cm�1): 2939, 2865, 1754,1647, 1136; GC-MS (m/z): 209.02, 207.04, 191.10,79.09.

Figure 1. Vinyl ester monomers and (meth)acrylate references.

VINYL ESTER-BASED 3D SCAFFOLDS 6943


8DV. Yield: 47%; 1H NMR (200 MHz, CDCl3, d,ppm): 7.25 (2H, dd, J ¼ 14.07 Hz, J ¼ 6.25 Hz,H2C¼¼CHA); 4.84 (2H, dd, J ¼ 14.07 Hz, J ¼1.56Hz, AHC¼¼CHH); 4.52 (2H, dd, J ¼ 6.25 Hz, J¼ 1.56 Hz,AHC¼¼CHH); 2.35 (4H, t,ACOACH2A);1.62 (4H, q5, ACOACH2ACH2A); 1.29 (8H, m,ACH2A); 13C NMR (50 MHz, CDCl3, d, ppm): 170.7(C¼¼O), 141.0 (HC¼¼CH2), 97.4 (HC¼¼CH2), 33.9 (2� ACH2ACO), 29.0 (2 � ACH2A), 28.9 (2 �ACH2A), 24.5 (2 �ACH2A); IR (ATR, cm�1): 2933,2862, 1754, 1648, 1137; GC-MS (m/z): 211.02,166.98, 121.06, 93.06, 81.09, 55.07.

FTV. Yield: 75%; 1H NMR (200 MHz, CDCl3, d,ppm): 7.30 (3H, dd, J ¼ 6.26 Hz, J ¼ 14.09 Hz,H2C¼¼CHAOACO); 4.88 (3H, dd, J ¼ 1.47 Hz,J ¼ 13.99 Hz, AHC¼¼CHH); 4.55 (3H, dd, J ¼1.57 Hz, J ¼ 6.26 Hz, AHC¼¼CHH); 2.39 (6H, t,ACH2ACOOA); 1.56–0.86 (99H, bm, alkyl-pro-tons); 13C NMR (50 MHz, CDCl3, d, ppm): 170.84(C¼¼O)), 141.19 (HC¼¼CH2), 97.38 (HC¼¼CH2),33.9, 31.9, 29.7, 29.4, 29.2, 29.0, 28.0, 26.7, 24.6,22.7, 20.6, 14.1 (alkyl-carbons); Mn (by NMR)�975 g/mol.

Photo-Differential Scanning CalorimetryExperiments

Photo-differential scanning calorimetry (Photo-DSC) was conducted with a modified ShimadzuDSC50 equipped with an aluminum cylinder.29

Photoreactivity of the monomers was tested byweighing �3 mg of the sample into an aluminumDSC pan, which was subsequently placed in theDSC chamber. The samples were purged with aN2 flow (�50 mL/min) for 5 min and irradiatedwith filtered UV-light (320–500 nm) from an ExfoOmniCureTM series2000 with a light intensity of1000 mW/cm2 at the exit of the light guide (8.8mW/cm2 at the surface of the sample). The meas-urements were carried out with 0.5 wt % (for thelow molecular weight monomers) and 5 wt % (forthe FTV containing formulations) of bis(2,4,6-tri-methylbenzoyl)-phenylphosphine oxide (IrgacureV

R

819, CibaVR

) as PI at ambient temperature undernitrogen atmosphere. The samples were exposedto the light for at least 10 min, and the heat flowwas recorded as a function of time. The doublebond conversion (DBC) and rate of polymerization(Rp) were determined as described previously.30

For vinyl esters the theoretical heat ofpolymerization (DH0,P) was set to 87.8 kJ/mol, foracrylates 80 kJ/mol and 60 kJ/mol formethacrylates.31

Biocompatibility/Cytotoxicity Experiments

Cell Culture

MC3T3-E1 cells (donated by Dr. Kumegawa, Mei-kai University, Department of Oral Anatomy,Sakado, Saitama, 35002 Japan) were cultured inalpha MEM (Biochrom, Austria) supplementedwith 4.5 g/L glucose, 5% fetal calf serum (FCS,Biochrom, Austria), and 10 lg/mL gentamycin.The cells were kept in humidified air under 5%CO2 at 37 �C. They were subcultured twice aweek using 0.001% pronase E and 0.02% EDTA inphosphate-buffered saline (PBS).

Cell Viability, Cell Multiplication, and AlkalinePhosphatase Activity

For the experiments, the cells were seeded at adensity of 20,000 cm2 in 24-well culture platesand cultured over night. On the next day, the me-dium was changed and the cells were treated withdecreasing concentrations of the monomers (10, 5,2.5, 1.25, 0.63, 0.31 and 0.16 mM) for 5 days andcompared to untreated cells. Thereafter, cell via-bility was addressed by incubation of the cultureswith a colorimetric growth indicator based on thedetection of cellular metabolic activity (EZ4U,Biomedica, Austria). Furthermore, amount ofDNA of the cultures were measured by fluores-cence with Hoechst 33258 dye as a surrogate ofthe cell number. Alkaline phosphatase activitywas measured in the same cultures by means ofp-nitrophenyl-phosphate and normalized to theDNA-amount.32 All experiments were performedas triplicate.

To address whether cells grow on the material,polymer-coated coverslips were placed into a mul-tiwell plate, fixed with agarose and sterilized for30 min in a distance of 14 cm with a 15 W UVlamp (Sylvana, Austria). MC3T3-E1 cells wereseeded at a density of 50,000/cm2 onto the cover-slips and cultured for three days. After the culturetime the metabolic activity of living cells wasdetermined by an EZ4U test (Biomedica, Austria)via photometric measurements (405 vs. 490 nm).

Preparation of Polymer Coatings on Coverslips

Unmodified circular glass platelets (Menzel-Glaser, diameter: 12 mm; thickness: 0.19–0.23mm) were treated with acetone, with 1 M NaOHsolution for 3 h under reflux and 1 M HCl solutionovernight under reflux. The platelets werewashed with distilled water and dried for 10 min

6944 HELLER ET AL.


at 105 �C. To introduce methacrylate groups onthe glass surface, the platelets were refluxed for15 min in a solution of 0.95 mL of 3-(trimethoxysi-lyl)propyl methacrylate and 1 mL of water in50 mL of 2-propanol. After washing with 2-propan-ol the modified platelets were dried at 100 �C for10 min.33

One drop of a monomer formulation containing1 wt % of IrgacureV

R

819 was pipetted onto aPTFE sheet (NowofolV

R

, Novoflon ET-6235) andthe modified glass platelet was carefully placed onthe sheet. The samples were irradiated for 10 minwith UV light (mercury lamp broadband irradia-tion). Afterwards, the glass platelets with thepolymer films were extracted for 24 h with EtOHand distilled water, respectively.

Mechanical Testing by Nanoindentation

Nanoindentation experiments were carried out ona Nanoindenter XP, MTS Systems. Cylindricalspecimens with 5 mm in diameter and a thicknessof 1 mm were prepared in silicone moulds using0.5 wt % of IrgacureV

R

819 (5 wt % in cases of theFTV containing formulations) followed by photo-curing for 5–30 min using a broadband UV lamp.Samples were then glued onto an aluminum cylin-der with an epoxy-based adhesive and the surfacewas grinded and polished.

The specimens were indented with a velocity of20 nm/s until an indentation depth of 2 lm wasreached. From the recorded load versus displace-ment data indentation hardness (HIT) and inden-tation modulus (EIT) can be extracted.34,35

HIT was calculated starting from the maximumforce Fmax by applying eqs (1) and (2):

HIT ¼ Fmax

24:5 � hc2

(1)

hc ¼ hmax � e � ðhmax � hrÞ (2)

where Fmax is the maximum force in N, hmax isthe penetration depth at maximum force in m, hris the intersection of the tangent of the unloadingcurve at maximum load with the x-axis in m and ean indenter constant.

EIT was calculated starting from the slope ofthe unloading curve’s tangent at the maximumload as shown in eqs (3) and (4):

EIT ¼ 1� ðmsÞ2

1Er� 1�ðmiÞ2

Ei

(3)

Er ¼ffiffiffip

p� S

2 �ffiffiffiffiffiffiAp

p (4)

With ms being the Poisson’s ratio of the sample(ms ¼ 0.35), mi the Poisson’s ratio of the indentertip (for diamond 0.07), Er the reduced modulus ofthe indentation contact in MPa, Ei modulus of theindenter tip in MPa (1140GPa for diamond), S thecontact strength in N/m (defined as the resistanceof two particles against their mutual displace-ment) and Ap the projected area in m2. At leastseven measure points were taken for eachsample.

Mechanical Testing by 3-Point Bending

Beams with dimensions of 50 � 5 � 2 mm3 wereprepared using PTFE moulds. 5 wt % of IrgacureV

R

819 were dissolved in the monomer and theresulting formulations were photocured for 20min using an UV–vis lamp. The specimens weremeasured on a MTS Synergie 2000 with a spandistance of 20 mm and at a strain rate of 0.5 mm/min, resulting in a curve of stress as a function ofstrain. The Young’s modulus (E) was calculatedfrom the slope of linear region of the curve.Results were obtained in triplicate.

Stereolithography

The fabrication of the 3D-parts was done using al-SLA system developed by the Laser ZentrumHannover (LZH), as described previously.36,37 Theutilized laser (Electronics 355 nm Quasi-CWLaser System XCYTE) was a neodymium dopedyttrium aluminum garnet (Y3Al5O12) laser(Nd:YAG) with a frequency multiplied wavelengthof 355 nm (tripled), equipped with an acousto-optic modulator (AOM, Isomet AO ModulatorRFA9 � 0–110 Series) and a scanner (SCANLAB-hurrySCAN 14). IrgacureV

R

819 was used as PIand 2,20-dihydroxy-4,40-dimethoxybenzophenone(HMB) as an UVabsorber.

With the parameters optimized by penetrationtests,36 3D structures were printed onto an alumi-num plate. The CAD model of the target structurewas sliced into layers of 50 lm; the hatching andthe line reduction were adjusted in order to obtainoptimum results. The noncured resin within thepores of the structure was rinsed with 2-propanolby using an ultrasonic bath before a post curingstep under the UV lamp was performed. The



resulting structure was examined by means ofscanning electron microscopy (SEM).

In Vivo Testing

As an established animal model in biomaterialresearch, adult female New Zealand white rabbitswere used for in vivo testing of polymers based onvinyl esters.38 Scaffolds of 5 � 5 � 8 mm3 contain-ing a 3D-microstructure were fabricated (Fig. 2).

All surgical procedures were performed afterapproval by the local ethics committee and undergeneral anesthesia. After layer-wise preparationto the rabbit’s distal femoral bone, a drilling holeof 5-mm diameter and 10-mm depth was placed(Fig. 3). Then one 3D-scaffold was press-fittedinto the surgical defect and the wound closedlayer-wise.

A total number of 6 animals was tested byimplanting a 3D-scaffold and observation periodsof 4 and 8 weeks were selected to allow sufficientinteraction between biomaterial and surroundingbone. At the end of observation the animals were

sacrificed, and bone samples containing theimplanted 3D-scaffolds were harvested. Histologi-cal samples were prepared for nondecalcified his-tology utilizing a modification of the Donath tech-nique,39 ground down to a thickness of 5–10 lmand stained with 1% thionine for transmissionlight microscopic evaluation.

RESULTS AND DISCUSSION

Synthesis

For the synthesis of vinyl esters, vinyl acetate isused in most cases as a vinyl group donatingagent in the presence of mercury(II)acetate20 or aPd(II)21,22 salt catalysts in literature. However,Pd(II) salts are rather expensive and the methodusing Hg(II) salts is intolerable to nucleophilicgroups due to the deactivation of the intermedi-ately formed Hgþ salt. Another synthetic route toobtain vinyl esters is a multistep reaction usingphenylselenium ethanol.23 For this basic study, al-iphatic vinyl esters without additional functional

Figure 2. Macroscopic (left) and SEM-detail view (right) of the used 3D-scaffolds.[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3. Cadaver specimen (left) and operative situs (right) of an implanted3D-scaffolds into the distal femoral bone using press-fit fixation. [Color figure can beviewed in the online issue, which is available at www.interscience.wiley.com.]

6946 HELLER ET AL.


groups were considered. Therefore, the methodusing a Hg(II) salt and p-toluene sulfonic acid asa catalyst was chosen (Scheme 1) because of itsstraight forward synthesis with comparativelygood yields. To circumvent residual traces of toxicHg(II) salts within the purified monomer, themore expensive and more time-consuming syn-thetic routes (Pd, Se) could be favored for futureinvestigations.

The divinyl esters were prepared from the cor-responding acids, succinic acid, suberic acid, andsebacic acid, respectively, in an excess of vinyl ac-etate, giving the monomers in 37–47% yield. Sincethose low molecular weight vinyl esters tend tohave a large shrinkage during photopolymeriza-tion, a high molecular weight vinyl ester derivateof trimer fatty acid was prepared under similarconditions with a yield of 75%.

Photo-DSC Experiments

Photo-DSC is a excellent technique to evaluatethe photoreactivity of monomers and therefore,the suitability for the l-SLA process. The photo-polymerization of the new vinyl ester monomersand their corresponding (meth)acrylate referenceswas monitored by the means of DSC to investi-gate the polymerization kinetics and the conver-

sion of the double bonds as a function of exposuretime. The samples were exposed under nitrogenatmosphere to filtered UV–vis light (320–500 nm)with an intensity of 8.8 mW cm�2 at the surface ofthe samples (corresponding to 1000 mW cm�2 atthe tip of the light guide) and tmax, RP and DBCwere calculated from the heat flow versus timegraph. Generally, for these experiments 0.5 wt %of IrgacureV

R

819 were used as PI.Figure 4 shows that among monovinyl mono-

mers photoreactivity, expressed by a low tmax anda high rate of polymerization, decreased in orderof acrylate 12MA [ vinyl ester 10MV � vinylester 6MV [ methacrylate 12MM. Values forDBC increase in the same order. The photoreac-tivity of the divinyl monomers having the samespacer length between the polymerizable groupsincreased in the same order as their monovinylrepresentatives; acrylate 4DA [ vinyl ester4DV[methacrylate 4DM. Surprisingly, the vinylester derivative 4DV showed similar values forDBC compared to the acrylate reference 4DA giv-ing conversions of 79% and 78%, respectively. ADBC in that range is a typical value for highlycrosslinked photopolymers due to limited diffu-sion and mobility at higher conversions. Further,the influence of the spacer length was investi-gated showing a lower rate of polymerization with

Figure 4. Results for tmax, Rp, and DBC of mono- and divinyl esters.

Scheme 1. Synthesis of vinyl esters.



increasing spacer length in order 4DV [ 6DV [8DV, which is consistent with a higher doublebond concentration. In case of the derivative 2DVthe positive effect of the short spacer length onthe photoreactivity seems to be overcompensatedby an enhanced limitation of diffusion at higherconversions due to high crosslinking, overallresulting in a lower value for RP and DBC (72%)compared to 4DV (79%).

To decrease the shrinkage of parts built byl-SLA the high molecular weight FTV (Mn � 975g/mol) should be used as comonomer. Mixtures ofFTV and 4DV (Fig. 5) showed a gradual decreaseof tmax and increase of DBC with higher contentsof the low molecular weight crosslinker, asexpected. Therefore, the mixture with the highestcrosslinker content 4DV:FTV 3:1 has shown to bethe best suitable for l-SLA being only slightly lessreactive than pure 4DV.

Viscosimetry

The viscosity of the monomer formulation is animportant property for the l-SLA process. Lowviscosity materials allow the coating of reasonablythin layers and faster production of the parts byavoiding high settling times. The viscosity of themono- and divinyl esters and their (meth)acrylatereferences is listed in Table 1.

Monovinyl esters as well as divinyl esters showlower viscosity than their respective (meth)acry-late references resulting in a faster settling pro-cess of vinyl ester-based resins. As argued in thesection Synthesis, mixtures containing the highmolecular weight FTV have to be evaluated toavoid high shrinkage by decreasing crosslinkingdensity in the photopolymer (Table 2).

The viscosity of pure FTV is relatively highcompared to the commercial stereolithography

resin Renshape SL7570 (g ¼ 210 mPa s).37,40 Byadding the low molecular weight 4DV, the viscos-ity can be decreased dramatically while crosslinkdensity and photoreactivity (see section Photo-DSC) are increased.

Biocompatibility/Cytotoxicity

Influence of the Monomers on Osteoblast-LikeCells Activity

Potentially, photopolymers are known to releaseresidual monomers, photoinitiators and similarproducts resulting from decomposition processesinto the environment. For this reason it is of sig-nificant interest how these chemicals influencebone cell proliferation and differentiation. Weaddressed the parameters by measuring cell via-bility, cell multiplication and ALP-activity thatindicates proceeding osteoblastic differentiation ofthe cells (Table 3). To compare the toxicity of themonomers we incubated MC3T3-E1 cells withincreasing concentrations of the monomers andapproximated the concentration where the half ofthe cells survived. This concentration was denotedas LC50. The ALP-activity was measured at amonomer concentration of 10 mM and compared tocontrols except for 6MV where 1.25 mM was used.

Compared to the methacrylate (4DM) andespecially to acrylate (4DA) reference, the vinyl

Figure 5. Results for tmax, Rp, and DBC of4DV:FTV mixtures.

Table 1. Dynamic Viscosity of the Monomers

Monomer g (mPa s)

6MV 0.510MV 1.012MA 4.312MM 4.32DV 2.64DV 2.76DV 3.38DV 4.64DA 5.24DM 4.4

Table 2. Dynamic Viscosity of the FTV ContainingFormulations

Monomer g (mPa s)

FTV 5604DV:FTV 1:3 784DV:FTV 1:1 214DV:FTV 3:1 7

6948 HELLER ET AL.


ester 4DA demonstrated significantly better toler-ance as demonstrated by both cell viability andcell multiplication; after treatment with the(meth)acrylates, most cells died and therefore,estimation of ALP-activity gave no clear results.The monovinyl esters 6MV and 10MV had eitherno significant influence or even increasedcell multiplication, respectively. Both chemicalsincreased ALP-activity. Short chain fatty acid dif-ferentially regulates proliferation and differentia-tion of cultured cells. Most studied are butyrateand the tumor drug valproate41; both regulateproliferation and differentiation by inhibiting his-tone deactylase activity, an important regulator ofgene expression and cell differentiation. Onemight speculate that the increase of ALP-activitycould be a result of a similar mechanism.

The divinyl esters of the dibasic acids showedan interesting behavior: the succinic acid divinylester (2DV) with two CH2 moieties demonstratednegative effects on both cell viability and cellmultiplication. With increasing number of CH2

moieties spacing both carboxylic groups, thegrowth inhibitory effect was attenuated, with8DV having no effect on these parameters any-more. Whether the increase of ALP-activityobserved with 2DV and 12MA is a result of theirgrowth inhibitory activity or these substancesindeed increased differentiation ob osteoblasticcells cannot be answered yet. In summary, com-pared to the (meth)acrylic components, the vinylesters clearly demonstrated low or no toxic effectson the osteoblasts, moreover, some vinyl esterspotentially increased cell multiplication and dif-ferentiation of such cells that could be beneficialfor bone cell behavior.

Cell Growth Behavior on Polymers

Coverslips were coated with the respective resin,UV cured, seeded with osteoblast-like MC3T3-E1cells und cultured for 4 days (Fig. 6). Thereafterthe cells were fixed and micrographs were taken.There was no noticeable difference in morphology

Table 3. Influence of the Monomers on Osteoblast-Like Cells

MonomerViability(LC50)

Cell Number(LC50)

ALP-Activity(% of Control)

6MV No influence No influence 127 (1.25 mM)10MV Increased Increased 135 (10 mM)12MA 2.0 mM 1.3 mM 300 (10 mM)12MM 3.1 mM 2.7 mM 6.1 (10mM)2DV 2 mM 1.4 mM 223 (10 mM)4DV 6.4 mM 1.0 mM 28.5 (10 mM)6DV 7.7 mM [10 mM 39.5 (10 mM)8DV [10 mM 10 mM 39.5 (10 mM)4DA \0.16 mM \0.16 mM n.d.4DM 1.3 mM 0.8 mM n.d.

Figure 6. MC3T3-E1 cells were grown on coverslips covered with 4DV (a), 4DA(b), and 4DM (c) for 4 days. Cells were fixed with 4% paraformaldehyde, magnifica-tion �40.



of the cells indicating no influence of the polymeron the differentiation of the cells.

Mechanical Properties

The mechanical properties were screened bynanoindentation, allowing a very fast and mate-rial-saving comparison of some basic mechanicalproperties of the investigated polymers. The in-dentation modulus and hardness (Fig. 7) weredetermined according to the procedure describedin the experimental section.

Comparing the polymer networks formed from4DV to its related acrylate reference 4DA, showssimilar results for the indentation modulus and ahigher value for the indentation hardness for thevinyl ester-based monomer. These findings of com-parable macroscopic properties coincide with thevalues for the double bond conversion in thePhoto-DSC section and indicate the suitability ofvinyl esters for photopolymerization-based AMTtechniques. The significant higher values for themodulus and hardness of the methacrylate refer-ence 4DM can be explained by the additionalmethyl group.

As expected, the polymers of the vinyl esterswith longer spacer 6DV and 8DV gave a continu-ing decrease of the modulus and hardness com-pared to 4DV since the longer spacer length leadsto a lower crosslink density of the polymers,which decreases the elastic modulus andhardness. The monomer with shorter spacer 2DVgave too brittle polymers to be measured. Themodulus of all photopolymers was between thethermoplastic reference substances PLA andPCL, respectively.

Homopolymers of the monovinyl ester mono-mers 6MV and 10MV remained liquids due to a

double bond conversion of just about 50% afterphotopolymerization making polymers thereofonly feasible by the use of high crosslinker con-tent. Figure 8 shows mixtures of 4DV with themonovinyl esters 6MV and 10MV and the highmolecular weight crosslinker FTV. As expected,in every case the modulus and hardness dropswith lower content of 4DV compared to the puredivinyl ester polymer, which allows the adjust-ment of a desired value for hardness or modulusby choosing the appropriate amount of comono-mer. Mixtures containing 6MV gave significantlyhigher moduli compared to the monovinyl esterwith a longer aliphatic chain 10MV, indicatingthat not only the content of comonomer plays akey role for the mechanical properties but also theside chain lengths.

To evaluate mixtures of 4DV with varying con-tents of the high molecular weight FTV to findthe most suitable formulation for l-SLA, 3-point-bending tests, giving the Young’s modulus E andthe flexural strength rB were conducted (Fig. 9).

Figure 8. Nanoindentation of monovinyl esters andFTV with 4DV.

Figure 9. 3-point-bending tests of 4DV:FTVmixtures.

Figure 7. Elastic modulus and hardness of lowmolecular weight crosslinkers determined by nanoin-dentation.

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Mixtures containing 3 parts of FTV and onepart of 4DV had an indentation modulus of just107 MPa and therefore, they were eliminatedfrom the 3-point-bending tests. The Young’s mod-ulus increases with higher content of the cross-linker 4DV but the flexural strength seems tohave a maximum for the 4DV:FTV 3:1 polymer.From optical observations pure 4DV seems tohave the highest shrinkage, which leads to easiercrack formation and lower toughness of theresulting polymer. Small amounts of FTV there-fore act as toughening additive. Correlating thesefindings with the results from the Photo-DSCexperiments, 4DV:FTV 3:1 seems to be the mostsuitable material for the shaping process byl-SLA.

Microstereolithography

The formulation of 4DV:FTV 3:1 was chosen asthe most suitable resin for l-SLA because of the

lowest viscosity and the highest photoreactivityand flexural strength among FTV containing mix-tures. Based on experiments in preliminary lightpenetration depth test,36 3 wt % of IrgacureV

R

819and 0.5 wt % of HMB were used as PI and UV-absorber, respectively. 3D cellular structures werebuilt with a laser power of 2mW, a building speedof 30mm/min and a layer thickness of 50lm. Aftercompletion of the structuring process the proto-type was rinsed with 2-propanol, followed by post-curing under the UV lamp. The CAD model andthe SEM images of the printed test structure aredisplayed in Figure 10.

In Vivo Testing

Three-dimensional-scaffolds made of the4DV:FTV 3:1 formulation were used for in vivostudies in New Zealand white rabbits. The heal-ing period was uneventful in all animals and his-tology showed that all 3D-scaffolds had beenimplanted correctly. No inflammatory round cells

Figure 10. CAD model (a) and SEM images (b–d) of the test structure.



were observed as an indicator for poor biocompati-bility.

Abundant formation of new bone around andinside the 3D-scaffolds was evident in all speci-mens (Figs. 11 and 12). Where bone was activelyformed, seams of osteoblasts and unmineralizedmatrix containing collagen fibrils were seen. In-cipient mineralization was indicated by calcifica-tion fronts.

SUMMARY

Lithography based AMT like stereolithographyhave gained increasing interest for biomedical

applications, but commonly used (meth)acrylate-based monomers have shown their drawbacksconsidering their potential irritation and some-times cytotoxicity in vivo. Therefore, differentvinyl esters were synthesized and have shownconsiderably lower cytotoxicity and better biocom-patibility in cell culture studies with osteoblastsas demonstrated by cell viability, cell multiplica-tion and ALP-activity than their (meth)acrylatereferences Furthermore, the polymers were ableto support cell growth of MC3T3-E1 osteoblasticcells.

Excellent photoreactivity between those ofacrylates and methacrylates was determined byPhoto-DSC experiments used as a benchmark tool

Figure 12. High power view of a specimen 8 weeks after implantation. Highamounts of newly formed bone inside the 3D-scaffold (left, �100 magnification) rep-resent the excellent osseointegration of the implant. Bone apposition especially alongthe serrated surface areas (right, �200 magnification) caused by the layered fabrica-tion process shows that this microstructure enhances the interaction between bioma-terial and living tissue.

Figure 11. Histological specimen showing a 3D-scaffold (left) 4 weeks after implan-tation (�10 magnification). Invasion of newly formed bone can be observed through-out the entire scaffold. A noticeable high bone-to-implant contact (right) underlinesthe good osteoconductive properties of the used formulation and proofs the biocom-patibility of the material.

6952 HELLER ET AL.


for the suitability for l-SLA. Nanoindentationexperiments were carried out as a screeningmethod for the modulus and the hardness andsupplemented with 3-point bending testing ofpure low molecular weight crosslinker 4DV andmixtures thereof with the high molecular weightFTV. Indentation und Young’s moduli comparableto (meth)acrylates and the thermoplastic and bio-degradable PLA and PCL references further indi-cate the suitability of vinyl ester based resins forbiomedical applications.

Finally, a test structure consisting of4DV:FTV 3:1, exhibiting the highest photoreac-tivity, best mechanical properties and lowest vis-cosity among FTV containing formulations,could be manufactured successfully throughshaping by l-SLA.

In vivo testing proofed the excellent biocompat-ibility of these materials and the microstructureobtained by AMT seems to enhance the interac-tion between biomaterial and living tissue clearly.

PIs from Ciba SC, and financial support by the AustrianScience Fund under contract no. P19387 and the sup-port of EXFO by providing an Omnicure 2000 unit arekindly acknowledged.

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