photopolymerization of biocompatible phosphorus-containing vinyl esters and vinyl carbamates

Photopolymerization of Biocompatible Phosphorus-Containing Vinyl

Esters and Vinyl Carbamates

CLAUDIA DWORAK,1 THOMAS KOCH,2 FRANZ VARGA,3 ROBERT LISKA1

1Institute of Applied Synthetic Chemistry, Divison of Macromolecular Chemistry, Vienna University of Technology,

Getreidemarkt 9/163, 1060 Vienna, Austria

2Institute of Materials Science and Technology, Vienna University of Technology, Favoritenstrasse 9-11, 1040 Vienna, Austria

3Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK, AUVA Trauma Centre Meidling,

4th Medical Department, Hanusch Hospital, Heinrich-Collin-Strasse 30, 1140 Vienna, Austria

Received 22 January 2010; accepted 14 April 2010

DOI: 10.1002/pola.24072

Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: Phosphorus-containing vinyl esters and vinyl carba-

mates were synthesized as new biocompatible and degradable

photopolymers. Reactivity of the monomers with one, two, and

three polymerizable double bondswas evaluated by photo-differ-

ential scanning calorimetry. With respect to their potential appli-

cation in the biomedical field, studies on cytotoxicity, mechanical

stability, and hydrolytic erosion behavior of the poly(vinyl alco-

hol)-based derivatives were performed.VC 2010Wiley Periodicals,

Inc. J PolymSci Part A: PolymChem 48: 2916–2924, 2010

KEYWORDS: biocompatibility; photo-differential scanning calo-

rimetry; photopolymerization; radical polymerization

INTRODUCTION Photoinduced polymerization is still one ofthe most prospective growing industrial fields due to itsadvantages in technological applications, such as coatings,printing inks, etc. The method satisfies the needs for fastcuring processes, reduction of volatile compounds, and eco-nomical fabrication.1 With respect to these features, photo-polymerization also finds more attraction in the huge area ofbiomedical applications. Current research deals with thepreparation of superior biodegradable polymers as implantsin the mammalian body. The requirements for such materialsare high: low toxicity of the polymer and its products of de-gradation as well as easy fabrication and handling. Besidesthe need for mechanical strength, these materials shouldalso show high affinity for cells, as for example, osteoblasts,which array onto the implants and activate the formation ofnew human tissue. Finally, the implants should be able to de-grade under physiological conditions on a defined timescale.2 Especially, photopolymerized hydrogels show severaladvantages in the use as tissue engineering scaffolds due totheir high water content and tissue-like elastic properties,offering proper conditions for cell promotion and tissueregrowth.3

Scaffolds based on classical polyacrylates proved to be harm-ful due to residual functional groups, monomer and erosionproducts, which have an adverse effect on the cells alreadyadhered to the scaffold’s surface and inhibit further cell ad-hesion by Michael addition to amino or thiol containinggroups in proteins or DNA.2,4 Additionally, radical polymer-

ization of acrylates forms a high molecular polymer back-bone (e.g., 70,000 g mol�1) that cannot be transportedwithin the human body after degradation. In contrast to that,a significant higher number of chain transfer reactions yieldsa low-molecular polymer backbone (e.g., 3000 g mol�1) inthe case of vinyl esters.5 Recently, our working group pub-lished studies2 on the use of polyvinyl carbonates and poly-vinyl carbamates as biocompatible polymers with respect tobone replacement material. Those polymers are based onnontoxic poly(vinyl alcohol), PVA, which is left after hydro-lytic degradation. PVA is a water soluble and biocompatiblepolymer that has been used for long-term implants, includ-ing bioartificial pancreas, cartilage, and esophagus or scleralbuckling material.6

Inorganic phosphates already attracted much attention ascomponents in numerous biomaterials: For example, theinfluence of hydroxyapatite modified tissue engineering scaf-folds has been investigated in various studies.7,8 Phosphorus-containing polymers also gained increasing interest duringthe recent years due to their excellent properties, offering awide field of applications. They are used as flame retardantcoatings,9–11 corrosion protection,12 and biocompatible mate-rials.13,14 Also the use of polyphosphoesters as biodegrad-able hydrogels has already been presented in literature.15–17

Therefore, the purpose of our studies was the developmentof biocompatible and hence biodegradable phosphorus-con-taining polymers with a polyvinyl alcohol-based backbone.Herein, we describe the synthesis and characterization of

Correspondence to: C. Dworak (E-mail: [email protected])

Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 48, 2916–2924 (2010) VC 2010 Wiley Periodicals, Inc.

2916 INTERSCIENCE.WILEY.COM/JOURNAL/JPOLA

different types of phosphorus containing vinyl esters andvinyl carbamates (Fig. 1). So far only the mono- and trivinylesters of phosphoric acid have already been subjected toradical polymerization by Gefter and Kabachnik,18 but noinvestigations were performed on their use as biodegradablepolymers.

We present the results of radical polymerization experimentsby photo-Differential Scanning Calorimetry (Photo-DSC), fol-lowed by the calculation of the double bond conversion(DBC) and the theoretical heat of polymerization, DH0,P,using ATR IR spectroscopy and peak fit analysis.

To gain insight into the biological compatibility of com-pounds resulting from unpolymerized monomers or productsfrom the erosion process, the influence of the monomers oncell multiplication, viability, and the expression of alkalinephosphatase (ALP) activity of osteoblasts were investigated.Mechanical stability of some crosslinked polymers was eval-uated by nanoindentation. Erosion behavior of selectedcrosslinked polymers was monitored by weight loss of thesamples under alkaline and acidic conditions.

EXPERIMENTAL

MaterialsTriethylphosphite, ethyl diphenyl phosphinite, phosphorusoxychloride, and n-butyllithium were purchased from Sigma-Aldrich. Diethyl chlorophosphate and chlorovinyl formatewere purchased from Fluka. Reference monomers such aslauryl acrylate (LA) and lauryl methacrylate (LMA) werepurchased from Sigma-Aldrich. Divinyladipate (DVA) and n-decanoic acid vinyl ester (DVE) were obtained from TCI. Tri-methylolpropane triacrylate (TMPTA) was provided by Cog-nis and trimethylolpropane trimethacrylate (TMPTMA) waspurchased from ABCR. 2-Hydroxy-2-methyl-1-phenyl-1-prop-anone (Darocur 1173), phenyl bis(2,4,6-trimethyl benzoyl)phosphine oxide (Irgacure 819), and hexane-1,6-diol diacry-late (HDDA) were received as a gift from Ciba SC and IvoclarVivadent, respectively. All reagents were used without fur-ther purification. The solvents were dried and purified bystandard laboratory methods. Column chromatography was

performed on VWR silica gel 60 (0.040–0.063 mm).2-Hydroxyethyl vinyl carbamate19 and ethyl dichlorophos-phate20,21 were prepared according to procedures stated inliterature.

Characterization1H and 13C NMR spectra were recorded on a Bruker AC-200FT-NMR spectrometer with CDCl3 as solvent. 31P NMR spec-tra were measured on a Bruker Avance 250 MHz NMR spec-trometer. Thin layer chromatography analysis was performedon silica gel 60 F254 aluminum sheets from Merck. Gas chro-matography/mass spectrometry was performed on a Hew-lett–Packard 5890/5970 B system using a fused silica capil-lary column (SPB-5, 60 m � 0.25 mm). MS spectra wererecorded using EI ionization (70 eV) and a quadrupole ana-lyzer. ATR FTIR measurements were carried out on a BioradFTS 135 spectrophotometer with a Golden Gate MkII dia-mond ATR equipment (L.O.T.). GPCs were measured on aWaters GPC system, which consists of a Waters 515 HPLCpump with an in-line Degasser AF Waters 717plus autosam-pler, a Waters HSPgel column (PS Stds 370–177,000 gmol�1) and a Waters 2410 RI detector. Photo-DSC was con-ducted with a Netzsch DSC 204 F1 Phoenix equipped withan autosampler. The compounds were irradiated with filteredUV-light (280–500 nm) by a double light guide (EXFO Omni-cure 2001) attached to the top of the DSC unit. The defaultlight intensity at the tip of the light guide was 3000 mWcm�2. All measurements were carried out in an isocraticmode at 25 �C under a nitrogen atmosphere. To permit anoxygen free irradiation of the samples a nitrogen purge(�20 mL/min) was used for at least 5 min before themeasurements.

Nanoindentation experiments were conducted on a Nano-indenter XP from MTS Systems. The polymer disks (5 � 1mm2) were prepared by UV-curing of a formulation contain-ing selected monomers and 2 wt % of a photoinitiator (Irga-cure 819) with a broadband UV-lamp. The samples wereimmersed in ethanol for 3 days to extract residual monomer,equilibrated 3 days in deionized water and dried in vacuo at40 �C for 8 h. The sample disks were fixed on an aluminumcylinder with a two-component glue. To obtain a smooth sur-face of the disks, the samples were barsed and polished withsandpaper of different grain size. For the indentation experi-ments, a diamant pyramide according to Berkovich wasused. The penetration depth was 2 lm with a penetrationvelocity of 0.1 lm s�1. The holding time at maximum loadwas 30 s, after that the sample disk was unloaded. From theunloading curve the indentation hardness and modulus werecalculated according to Oliver and Pharr.22

The samples for the erosion experiments were prepared inthe same manner as for nanoindentation testing. After dryingin vacuo, the polymer disks were incubated in 2 mL of NaOH(pH 12.0) or HCl (pH 1.0) solution at 37 �C and 65 �C,respectively. At different time points, the solution wasremoved and the disks were thoroughly rinsed with waterand dried in vacuo at 40 �C for 8 h. The dry weight was

FIGURE 1 Structures of the investigated phosphorus contain-

ing monomers.

ARTICLE

P-BASED BIOCOMPATIBLE PHOTOPOLYMERS, DWORAK ET AL. 2917

measured on a Sartorius ME235P balance. The experimentswere conducted in triplicate.

SynthesisDiethyl Phosphonovinyl Formate (E1)Triethyl phosphite (3.13 g, 19 mmol) was added dropwise at0 �C over a period of 15 min to the chlorovinyl formate (2.0g, 19 mmol). After the addition was finished, the reactionmixture was stirred at room temperature for additional 2 h.To remove the evolving ethyl chloride, the solution washeated gently to 40 �C for 30 min. The slightly yellow crudeproduct was purified by vacuum distillation to yield 3.3 g(83%) of a colorless liquid. Yield: 83%. Bp: 125–128 �C (8mbar).

1H NMR (250 MHz, CDCl3, d, ppm): 7.27 (dd, J1 ¼ 13.8 Hz, J2¼ 6.2 Hz, 1H), 5.06 (dd, J1 ¼ 13.7 Hz, J2 ¼ 1.7 Hz, 1H), 4.75(dd, J1 ¼ 6.1 Hz, J2 ¼ 1.8 Hz, 1H), 4.27 (q, J ¼ 7.0 Hz, 4H),1.13 (t, J ¼ 7.0 Hz, 6H); 13C NMR (50 MHz, CDCl3, d, ppm):161.2 (C¼¼O), 139.9 (CH¼¼CH2), 100.8 (CH¼¼CH2), 64.8(CH2CH3), 16.2 (CH2CH3);

31P NMR (100 MHz, CDCl3, d,ppm): �4.2; IR (AT IR, thin film, cm�1): 1729 (C¼¼O), 1645(C¼¼C), 1273 (P¼¼O), 1011 (PAOAC); MS (m/z): calcd.208.15, found: 209.2 [M þ H]þ; ELEM. ANAL. Calcd. forC7H13O5P: C: 40.39, H: 6.30, found: C: 40.09, H: 6.14.

Diphenyl Vinyloxycarbonyl Phosphinoxide (P1)Ethyl diphenyl phosphinite (1.0 g, 4.3 mmol) was addeddropwise at 0 �C over a period of 15 min to the chlorovinylformate (0.69 g, 6.5 mmol). After the addition was finished,the reaction mixture was stirred at room temperature foradditional 3 h. To remove the evolving ethyl chloride, the so-lution was heated gently to 40 �C for 30 min. The crudeproduct was obtained as a white solid. After drying in highvacuum 1.1 g (99%) of pure product were obtained. Yield:99%.

1H NMR (250 MHz, CDCl3, d, ppm): 7.88 (m, J1 ¼ 6.3 Hz, J2¼ 0.7 Hz, ArH, 4H), 7.57 (m, ArH, 6H), 7.37 (dd, J1 ¼ 6.3 Hz,J2 ¼ 14.0 Hz, 1H), 5.14 (m, J1 ¼ 13.9 Hz, J2 ¼ 1.7 Hz, 1H),4.78 (m, J1 ¼ 6.1 Hz, J2 ¼ 2.0 Hz, 1H); 13C NMR (50 MHz,CDCl3, d, ppm): 169.6 (C¼¼O), 140.3 (CH¼¼CH2), 133.2 (ArC),131.9 (ArC), 129.2 (ArC), 128.9 (ArC), 127.1 (ArC ), 101.2(CH¼¼CH2);

31P NMR (100 MHz, CDCl3, d, ppm): 18.0; IR (ATIR, thin film, cm�1): 1720 (C¼¼O), 1645 (C¼¼C), 1647 (C¼¼C),1216 (P¼¼O), 1163 (PAOAC); MS (m/z): calcd. 272.24,found: 273.3 [M þ H]þ; ELEM. ANAL. Calcd. for C15H13O3P: C:66.18, H: 4.81, found: C: 66.55, H: 4.53.

General Procedure for the Preparation of the VinylEsters of Phosphoric Acid (V1, V2, V3)For preparation of the mono-, di-, and trivinyl ester, an equi-molar, twice or thrice molar amount of n-butyl lithium solu-tion (2.1 M in hexane) was added dropwise to 70 mL THFunder Ar-atmosphere at 0 �C. Then the solution was stirred30 min at 0 �C and 16 h at room temperature. This reactionmixture was added to the appropriate chlorophosphate at�76 �C. After the addition was finished, the mixture wasstirred 1 h at 0 �C, then 16 h at room temperature. Finally,the white precipitate was filtered off and the solvent was

removed by distillation under reduced pressure. The slightlyyellow crude products were purified by columnchromatography.

Diethyl Vinyl Phosphate (V1)Yield 60% of a colorless liquid. Rf ¼ 0.48 (PE:EE ¼ 4:1). 1HNMR (250 MHz, CDCl3, d, ppm): 6.56 (dd, J1 ¼ 12.6 Hz, J2 ¼6.3 Hz, 1H), 4.88 (dd, J1 ¼ 12.6 Hz, J2 ¼ 1.2 Hz, 1H), 4.55(dd, J1 ¼ 6.3 Hz, J2 ¼ 1.2 Hz, 1H), 4.15 (m, 4H), 1.33 (m,6H); 13C NMR (50 MHz, CDCl3, d, ppm): 142.0 (CH2¼¼CH),99.5 (CH2¼¼CH), 64.2 (CH2ACH3), 15.8 (CH2ACH3);

31P NMR(100 MHz, CDCl3, d, ppm): �3.5; IR (ATR IR, thin film,cm�1): 1645 (C¼¼C), 1271 (P¼¼O), 1012 (PAOAC); 820(C¼¼C); MS (m/z): calcd. 180.14, found: 181.3 [M þ H]þ.

Ethyl Divinyl Phosphate (V2)Yield 36% of a yellow liquid. Rf ¼ 0.42 (PE:EE ¼ 3:1). 1HNMR (250 MHz, CDCl3, d, ppm): 6.57 (dd, J1 ¼ 6.6 Hz, J2 ¼1.4 Hz, 2H), 4.94 (dd, J1 ¼ 13.5 Hz, J2 ¼ 1.1 Hz, 2H), 4.62(m, 2H), 4.23 (m, 2H), 1.37 (m, 3H). 13C NMR (50 MHz,CDCl3, d, ppm): 141.9 (CH2¼¼CH), 100.8 (CH2¼¼CH), 65.4(CH2ACH3), 16.1 (CH2ACH3);

31P NMR (100 MHz, CDCl3, d,ppm): �7.7; IR (ATR IR, thin film, cm�1): 1644 (C¼¼C), 1279(P¼¼O), 1011 (PAOAC); 820 (C¼¼C); MS (m/z): calcd. 178.1,found: 179.2 [M þ H]þ; ELEM. ANAL. Calcd. for C6H11O4P: C:40.46, H: 6.22, found: C: 40.68, H: 6.11.

Trivinyl Phosphate (V3)18

Yield 26% of a colorless liquid. Rf ¼ 0.43 (PE:EE ¼ 5:1). 1HNMR (250 MHz, CDCl3, d, ppm): 6.56 (m, 3H), 4.97 (m, 3H),4.67 (m, 3H). 13C NMR (50 MHz, CDCl3, d, ppm): 141.3(CH2¼¼CH), 101.6 (CH2¼¼CH); 31P NMR (100 MHz, CDCl3, d,ppm): �12.2; IR (ATR IR, thin film, cm�1): 1643 (C¼¼C),1286 (P¼¼O), 1017 (PAOAC); MS (m/z): calcd. 176.11,found: 175.1 [M � H]�.

General Procedure for the Preparation of the Vinylcarbamates C1 and C22-Hydroxyethyl vinyl carbamate and an equimolar amount oftriethylamine were mixed together in dry THF. The reactionflask was cooled in an ice bath to 0 �C, then the correspond-ing chloroethyl phosphate was added dropwise in 4 mL ofdry THF over a period of 30 min. To prepare monovinyl car-bamate C1, an equimolar amount of monochloroethyl phos-phate was used. In case of C2, the molar ratio of 2-hydrox-yethyl vinyl carbamate and dichloroethyl phosphate was 2:1.After the addition was finished, the reaction mixture wasstirred at room temperature for 12 h. The white precipitate(triethylamine hydrochloride) was filtered off and the solventwas removed under reduced pressure. The slightly yellowoily residue was dissolved in 10 mL of distilled dichlorome-thane and washed with 5% sodium bicarbonate solution(3 � 10 mL). Then the organic layer was dried over sodiumsulfate and after filtration of the drying agent, the solventwas distilled off under reduced pressure. The crude productwas purified by column chromatography.

2-(Diethoxyphospholoyloxy) Ethyl Vinyl Carbamate (C1)Yield: 25% of a colorless oil. Rf: 0.28 (PE:EE ¼ 1:5). 1H NMR(250 MHz, CDCl3, d, ppm): 7.15 (dd, J1 ¼ 14.0 Hz, J2 ¼ 6.4,

JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA


1H), 5.69 (s, NAH, 1H), 4.71 (dd, J1 ¼ 14.0 Hz, J2 ¼ 1.3 Hz,1H), 4.40 (dd, J1 ¼ 6.3 Hz, J2 ¼ 1.2 Hz, 1H), 4.10 (m,CH2ACH3, PAOCH2, 6H), 4.48 (m, 2H), 1.32 (m, 6H). 13CNMR (50 MHz, CDCl3, d, ppm): 186.5 (C¼¼O), 141.9(CH2¼¼CH), 95.3 (CH2¼¼CH), 66.2 (PAOCH2ACH2), 64.1(CH2ACH3), 41.3 (HNACH2), 16.0 (CH2ACH3);

31P NMR (100MHz, CDCl3, d, ppm): 0.76; IR (ATR IR, thin film, cm�1):3277 (NAH), 1740 (C¼¼O), 1650 (C¼¼C), 1531 (CAN), 1248(P¼¼O), 1020 (PAOAC), 864 (C¼¼C); MS (m/z): calcd. 267.22,found: 224.2 [MAOCH¼¼CH2]

�; ELEM. ANAL. Calcd. forC9H18NO6P:C: 40.45, H: 6.74, N: 5.24, found: C: 40.19, H:6.61, N: 4.91.

Bis[2,20-(ethoxyphospholoyloxy)] Ethyl Vinyl Carbamate(C2)Yield: 43% of a slightly yellow, highly viscous oil. Rf: 0.23(PE:EE ¼ 1:5). 1H NMR (250 MHz, CDCl3, d, ppm): 7.16 (dd,J1 ¼ 14.3 Hz, J2 ¼ 6.3 Hz, 2H), 5.76 (s, NAH, 2H), 4.72 (dd,J1 ¼ 14.1 Hz, J2 ¼ 1.4 Hz, 2H), 4.42 (dd, J1 ¼ 6.3 Hz, J2 ¼1.6 Hz, 2H), 4.11 (m, CH2ACH3, POCH2ACH2, 6H), 3.47 (m,4H), 1.30 (m, 3H). 13C NMR (50 MHz, CDCl3, d, ppm): 186.5(C¼¼O), 141.8 (CH2¼¼CH), 95.5 (CH2¼¼CH), 66.5 (OCH2ACH2),64.5 (CH2ACH3), 41.3 (HNACH2), 16.1 (CH2ACH3);

31P NMR(100 MHz, CDCl3, d, ppm): 0.69; IR (ATR IR, thin film, cm�1):3319 (NAH), 1721 (C¼¼O), 1652 (C¼¼C), 1528 (CAN), 1242(P¼¼O), 1022 (PAOAC), 865 (C[dsbond]C); MS (m/z): calcd.352.3, found: 355.3 [M þ 3H]þ; ELEM. ANAL. Calcd. forC12H21N2O8P: C: 40.91, H: 6.01, N: 7.95, found: C: 40.63, H:5.70, N: 7.90.

Cell Multiplication (DNA Amount) and AlkalinePhosphatase ActivityMC3T3-E1 osteoblast-like cells were seeded in culture dishesat a density of 20,000 cm�2 and grown in aMEM (a-mini-mum essential medium; Biochrom) containing 5% fetal calfserum (FCS; Biochrom), supplemented with 4.5 g L�1

L-glu-cose, 50 lg mL�1 ascorbic acid (Sigma) and 10 lg mL�1 gen-tamycin (Sigma). On the next day, the medium was changedand the cells were treated with increasing concentrations ofthe monomers (0.1, 0.05, 0.025, and 0.0125 M) for 5 daysand compared with untreated cells. Thereafter, cell viabilitywas addressed by incubation of the cultures with a colori-metric growth indicator based on the detection of cellularmetabolic activity (EZ4U, Biomedica, Austria). Furthermore,amount of DNA of the cultures were measured by fluores-

cence with Hoechst 33258 dye as a surrogate of the cellnumber. Alkaline phosphatase activity was measured in thesame cultures by means of p-nitrophenyl-phosphate and nor-malized to the DNA-amount.23

RESULTS AND DISCUSSION

SynthesisThe monovinyl esters E1 and P1 were synthesized accordingto the Michaelis-Arbuzov reaction.24 Hence, chlorovinyl for-mate was added to triethyl phosphate for compound E1 orto diphenyl ethyl phosphinite for compound P1, respectively(Scheme 1).

The Michaelis-Arbuzov reaction proceeds smoothly withoutneed for any solvent. Compound E1 was isolated as clear oil(83% yield) by vacuum distillation and compound P1 wasobtained as a white solid (99% yield) after removal of theresulting ethyl chloride and drying in high vacuum withoutfurther purification.

A convenient method to prepare esters of phosphoric acidsis to react phosphohalides, especially chlorides, with nucleo-philes, such as alcohols or primary amines, using triethyl-amine as HCl scavenger.25,26 Preparation of vinyl carbamatesC1 and C2 (Scheme 2) were performed by dropwise additionof the phosphorus compound to a THF solution of 2-hydrox-yethyl vinyl carbamate, which had been prepared from 2-amino ethanol and chlorovinyl formate following the proce-dure stated in literature.19 After purification by column chro-matography both products were obtained in 25–43% yield.

The syntheses of the mono-, di-, and trivinyl esters of phos-phoric acid were performed according to the methoddescribed by Johnston et al.27 By this simple two step reac-tion, the enolate of acetaldehyde is prepared by the cyclore-version of THF in the presence of n-butyllithium, followed bythe reaction with chlorophosphinates or chlorophosphonates(Scheme 3). This procedure proved to be more convenientthan other synthetic pathways described earlier in literature,involving the dehydrohalogenation of the corresponding 1-

SCHEME 1 Synthesis of the monovinyl esters E1 and P1.

SCHEME 2 Synthesis of the mono- and divinyl carbamates C1

and C2.

ARTICLE


chloroethyl phosphoric acid esters as presented by Gefterand Kabachnik.18

It has to be noted that classical lithiation reactions with n-butyllithium in THF as solvent are generally done at �78 �Cjust to avoid this cycloreversion. In our case, the addition ofn-butyllithium to THF occurred at 0 �C as the cycloreversionprocess only takes place at higher temperatures. Columnchromatography was used to purify the raw products insteadof vacuum distillation due to better product yield. Therefore,the vinyl esters V1, V2, and V3 were obtained in moderateyields from 26 to 60% after purification.

Cytotoxicity TestsCell viability and multiplication as well as ALP-activity ofosteoblasts are sensitive indicators of any substance for theircompatibility with the biological tasks of the cells. Especiallythe ALP-activity is an important indicator whether osteo-blasts proceed in their differentiation process or are blockedby a reagent.23 The osteoblast-like cells were treated withincreasing concentrations of the monomers for 5 days andcompared with the untreated cells. Although, some differen-ces between cell viability and cell number were found, bothparameters were comparable for all investigated monomers(Table 1). In this match, vinyl carbamate C1 and vinyl esterE1 had the least influence on cell multiplication. Except forcompounds C1 and E1, all monomers downregulated theALP-activity indicating some influence on the osteoblasticdifferentiation process. C1 had only marginal effect on ALP-activity (�13%), whereas E1 even increased (þ23%) the ac-tivity of the osteoblastic enzyme exhibiting a stimulatoryeffect on osteoblast differentiation. Compared with the refer-ence monomer LMA, our compounds showed better resultsby a factor 5–20.

Concerning the toxicity evaluation of the polymers used fornanoindentation and hydrolytic erosion studies the followingaspects have to be considered. Besides the residual mono-mer, which has been studied within this article, the photoini-tiator is the main extraction product. However, in a formerstudy, the photoinitiator (Irgacure 819) turned out to benontoxic to human osteoblast cells.28 For all experiments,these have been extracted before further investigation.

Photoreactivity of MonomersOne simple and accurate way to determine the performanceof a formulation is by photo-DSC. The reactivity can bederived from the time, which is needed to reach the maxi-

mum polymerization heat (tmax). The DBC and the rate ofpolymerization (RP,max, mol L�1 s�1) give additional informa-tion on the performance of a system. RP,max is calculatedfrom the height of the maximum (h; mW mg�1), and thedensity of the monomers (q; g L�1) using eq 1.

RPmax ¼ h� qDH0;P

(1)

To gain knowledge on the photoreactivity of a monomer it isalso essential to know the theoretical heat of polymerization(DH0,P). A well established method29 to determine this valueis to cure monomer formulations by photo-DSC that givesthe actual heat of polymerization (DHP) evolved under theseconditions. In combination with the DBC obtained from ATRIR analysis of this sample after curing by photo-DSC it ispossible to calculate the DH0,P using eq 2, where MM is themolecular weight of the monomer.

DH0;P ¼ DHP �MM

DBC(2)

To evaluate the reactivity of monomers and in terms of cal-culating their DH0,P, we used peak fitting (PeakFit V4.12, SSI)to determine the DBC of the polymers. This method has al-ready been established by Stansbury and coworkers30 forinvestigation of hydrogen bonding in methacrylate mono-mers and polymers. We have used this technique veryrecently31 for comparison of the fitted peak areas of theC¼¼C bond at 1660 cm�1 in the monomer and the polymerATR IR spectrum of acrylates, offering a more accurate waythan the simple calculation from the peak height or area ofunfitted IR data. In our investigations presented here wemonitored only the decrease of the C¼¼C stretching vibrationat 1640 cm�1, as the C¼¼C vibration at 810 cm�1 was uselessin this case due to strong overlapping with other bonds. TheP¼¼O bond at 1265 cm�1 served as internal reference in thephosphorus-containing monomers, whereas for the referenceacrylates, methacrylates, and vinyl esters the C¼¼O bond at1740 cm�1 was selected for this purpose. For the data fit,the ‘‘Residuals method’’ was chosen together with a ‘‘Loesssmoothing’’ algorithm. The fitting procedure was repeateduntil the square of the correlation coefficient, r2, exceeded0.99.

Figure 2 shows an example for the comparison of the ATRIR spectra of compound V1 as monomer and as polymer.

SCHEME 3 Reaction pathway for the synthesis of the mono-,

di-, and trivinyl ester of phosphoric acid V1–V3.

TABLE 1 Influence of the Monomers on Cell Multiplication,

Viability, and ALP-Activity on Osteoblasts

C1 C2 V2 V1 E1 P1 V3 LMA

Viability (mM)a 8 5 5 6 12 3 3 2

Cell number (mM)a 14 8 6 7 16 6 5 1

ALP-activity (%)b 87 44 75 76 123 38 60 6

a Concentration (mM) where the half-maximal value of viability or DNA-

content compared with control was found.b The ALP-activity is the maximum value that was found at the investi-

gated concentrations in % of the control. Controls: cells culture on a

standard culture dish.



By the photo-DSC experiments, we wanted to investigate theunknown photoreactivity of our phosphorus-containingmonomers. Measurements of compounds E1, V1–V3, andC1–C2 with 100 lmol g�1 Darocur 1173 as photoinitiatorwere carried out under nitrogen atmosphere. The mass ofthe samples was 9.0 mg 6 0.3 mg. As reference materialserved highly reactive acrylates and methacrylates. Less re-active vinyl esters were also used for comparison due totheir structural similarity to our compounds. Our phospho-rus-based monomers were compared with the reference ma-terial according to their number of polymerizable groups(mono-, di-, and tri-). From the data presented in Table 2,the most surprising result in the field of monvinyl monomerswas the very good performance of the vinyl ester E1 thatshowed an exothermic behavior almost comparable with thatof the acrylate, LA, combined with very high DBC (95%) andRP,max. Except of C1, the DBC of the monovinyl monomers,obtained by peak fit analysis of the ATR IR spectra, is ratherhigh (�90%), as expected. Considering the RP,max, all of ourmonovinyl compounds performed better than the methacry-late reference, LMA, with the vinyl carbamate C1 beingslightly better than the vinyl ester V1. Also tmax, the time toreach the maximum polymerization rate (RP,max) was in therange of the methacrylate. Generally, vinyl esters and vinylcarbamates showed higher values for the DH0,P than the ref-erence acrylates and methacrylates, which is also in good ac-cordance with experiments already stated in literature.32,33

The performance of the divinyl monomers V2 and C2 con-cerning RP,max was significantly poorer than for the referenceacrylate, HDDA, and the reference vinyl ester, DVA, althoughthe values for the DBC were comparable good. The high con-version of V2 might be explained by the size of the moleculethat allows migration even at higher conversions. Signifi-cantly faster curing of C2 can be assigned to the flexibility ofthe molecule and preorganization effects by hydrogen bond-ing.34 Interestingly, the DH0,P of C2 and V2 were in good ac-cordance with their monovinyl analogues C1 and V1.

Similar to V2, the trivinyl ester V3 exhibited a high DBC(75%), but also an elevated tmax, (23 s) has to be acceptedcompared with the reference acrylate TMPTA and methacry-late TMPTMA, whereas RP,max was within scope of the meth-acrylate. Unfortunately, no proper trifunctional vinyl esterwas commercially available as reference compound. The

amazingly high DBC (75%), which is untypical for trifunc-tional monomers due to their extremely high crosslinkingability, might be again explained by the smallness of the mol-ecule which enables it to move through the polymer net-work, even if it becomes more and more rigid by crosslink-ing reactions. Similar as for the divinyl ester V2, thisbehavior might also explain the very broad exothermic peakin the photo-DSC. Again, the DH0,P for one vinyl double bondper mol (83.5 kJ mol�1) showed good consistency with themono- and the divinyl esters of phosphoric acid, V1 and V2.

The molecular weight of polymer samples made from themono vinyl esters E1 and V1 as well as from the mono vinylcarbamate C1 was determined by GPC analysis using poly-styrene standards. The Mn ranged from 37,100 Da (E1) to4900 Da (V1) and to 3800 Da (C2). These results were ingood accordance with molecular weights of polymers basedon vinyl esters from previous studies,5 which are muchlower than for common polyacrylates due to chain transferreactions. This can be explained by the high chain transferconstant, which is caused by the fact, that vinyl esters showa low monomer reactivity, whereas their radicals are highlyreactive.

Mechanical PropertiesNanoindentation experiments were conducted to study themechanical properties of selected polymers from monomerswith low cytotoxicity and/or high reactivity. By this methodparameters like Indentation modulus (IM), which is similarto Young’s modulus, and hardness (H) are easily accessible.The photopolymerization of the polymer disks (5 � 1 mm2)for the nanoindentation experiments was carried out in airwith a broadband UV-lamp. The kind of photoinitiator waschanged from Darocur 1173 to Irgacure 819, as the later hasexcellent photobleaching properties, thus being more suita-ble for broadband irradiation. Besides C2 and V3, nontoxic

FIGURE 2 Comparison of the ATR IR spectra for compound V1

as monomer and as polymer.

TABLE 2 Photo-DSC and ATR IR Data for Compounds E1,

V1–V3, C1–C2, and the Reference Monomers

Compound

DBC by

ATR-IR (%)

tmax (s) by

Photo-DSC

RP,max �103

(mol L�1 s�1)

DH0,P

(J mol�1)

E1 95 11.4 247 95,900

V1 90 19.5 32.8 83,500

C1 70 13.7 60.1 87,800

LA 91 4.0 359 77,600

LMA 86 15.8 18.6 66,900

DVE 88 7.8 85.7 81,600

V2 89 27.0 127 169,600

C2 59 7.3 68.2 173,700

HDDA 83 2.3 578 147,600

HDMA 62 4.9 231 120,500

DVA 90 12.4 299 168,100

V3 75 23.3 101 267,900

TMPTA 55 1.9 330 237,400

TMPTMA 41 3.5 102 206,300

ARTICLE


monovinyl ester E1 with variable amounts of V2 as cross-linker were chosen. As shown in Figure 3, the mechanicalproperties of nearly all crosslinked materials (except E1:V2¼ 9:1) superseeded the reference material poly(caprolac-tone), PCL, which is used as biodegradable polymer for vari-ous biomedical applications, for example, bone replacementor tissue engineering.35 Increasing amounts of V2 led topolymers, which were too brittle for preparation and testingby this method, therefore no data of mixtures > 50 wt % ofV2 are available. The polymer of C2 displayed better me-chanical properties than PCL, which were comparable withthe 3:1 mixture of E1:V2. Astonishingly, the polymer of V3exhibited even better mechanical properties than the semi-crystalline reference poly(lactic acid), PLA, which finds alsowide-spread use as biodegradable material. Moreover, the IMof the polymer of V3 already approached that of humanbone36 and also an extraordinary high hardness was foundfor this compound.

Of the polymer mixtures of E1 with various amounts of V2as crosslinker also swelling ability in PBS buffer (pH 7.4) af-ter 24 h was investigated. The mixture of E1:V2 ¼ 3:1 exhib-ited the highest swelling ration of 272%, compared with the1:1 composition with 179% and a sample containing onlyV2 which showed a swelling ability of only 26% after 24 h.

Erosion BehaviorFrom the degradation of polyphosphoesters it is known thatthe breakdown of such polymers by hydrolytic or enzymaticcleavage delivers finally phosphates and alcohol deriva-tives.37,38 Generally, it can be assumed that during the degra-dation nontoxic polyvinyl alcohol is released. Indeed, severalother phosphorus based degradation products could beexpected as it was shown in another study dealing with thehydrolytic degradation mechanisms of polyphosphoesters.39

PVA as a water-soluble, biocompatible degradation productis well known for many applications in the medical field.40,41

The phosphate containing side chains at the backbone of thePVA resulted in good mechanical stability and might favorbiocompatibility and biodegradability of the crosslinked poly-

mers V2 and V3. Erosion of polymers from C2 lead to modi-fied PVA with a 2-hydroxyethyl carbamate residue, which isalso used for contact lens materials.19

In accordance with literature42 and to gain a first estima-tion of the erosion behavior of our crosslinked polymers,hydrolysis experiments in NaOH solution with pH 12.0 at37 �C as well as in HCl solution with pH 1.0 at 37 �C and65 �C were performed, as the erosion under simulatedphysiological conditions (pH 7.4; 37 �C) is generally veryslow and can last up to years. Weight loss of the UV-curedpolymer disks (5 � 1 mm2) was measured by the gravi-metric method.40 The initial weight, (W0) of the sampleswas determined after drying for 8 h in vacuo. For measure-ment of the weight after defined times of mass loss (Wt),the solution was removed, the polymer disks were washedthoroughly with deionized water and dried for 8 h at 40�C in vacuo. The weight loss was calculated according toeq 3.

WL½%� ¼ W0 �Wt

W0� 100 (3)

The time until 50% of the weight of the PVA-based polymersof V2, V3, and C2 has been lost at pH 12.0 is shown in Fig-ure 4. After 24 days, full mass loss of all three compoundswas detected under alkaline conditions. Surprisingly, thepolymer of the trivinyl compound V3 decomposed faster

FIGURE 3 Indentation Modulus (IM) and hardness (H) values

for copolymers of E1 containing various amounts of V2 as

crosslinker to poly(caprolactone), PCL, poly(lactic acid), PLA,

and human trabecular (T12 vertebrae) bone.

FIGURE 4 Time (h) until 50% weight loss of the polymers from

compounds V2, C2, and V3 compared with PLA (NaOH solu-

tion, pH 12.0, 37 �C).

FIGURE 5 Time (h) until 50% weight loss of the polymers from

compounds V2, C2, and V3 (HCl solution, pH 1.0 at 37 �C and

65 �C).



than the very similar polymer of compound V2. As expected,vinyl carbamate C2 that has a much wider network by theethanolamine spacer groups, results in faster decompositionthan V2. Generally, all materials have similar erosion timesin the same order of magnitude than the reference materialPLA.

Also hydrolysis under acidic conditions was studied (HCl, pH1.0). The data are shown in Figure 5. As mass loss at lowertemperature (37 �C) was found to be quite slow, a secondexperiment was conducted at 65 �C. The reference materialPLA did not show any significant mass loss under acidic con-ditions over the monitored period of these experiments. Forthe highly crosslinked polymer of trifunctional compound V3acidic hydrolysis is comparatively slow and less temperaturesensitive. Less crosslinked polymer of C2 shows similar aciderosion as V3 at lower temperatures, but at higher tempera-ture it decomposes rather fast. Compared with V2, the massloss of the C2-polymer went more rapidly. This can easily beexplained by a lower crosslinking density of V2 (DBC 89%),allowing better access for water that is responsible for thehydrolytic degradation of the polymer.

The distinct hydrolysis behavior of compound V2 and V3might result on the one hand from the different networkdensity in the polymers, on the other hand also the type ofthe substituent (primary versus secondary alcohol) and dif-ferent mechanisms involved under alkaline and acidic condi-tions seem to be responsible. Generally, less crosslinked net-works as for V2 degrade faster than tightly crosslinkedstructures (V3). Surprisingly, under alkaline conditions con-trary behavior was observed. It has to be noted that not V3is comparatively too fast, but erosion of V2 is relatively slow.This might be explained by the fact that especially V2 forms5-membered rings during radical polymerization43 that arepresumably more stable toward a nucleophilic attack by thehydroxyl anion.

CONCLUSIONS

In this study, we presented the synthesis and photopolyme-rization behavior of phosphorus containing monomersbased on vinyl esters and vinyl carbamates with varyingnumber of polymerizable groups. All monomers, except P1,which was a solid, were polymerizable with a Type I pho-toinitiator, Darocur 1173, under irradiation with UV light.The diethyl phosphonovinyl ester E1 was the most reactivemonomer and together with vinyl ester V3 unusually highDBC was achieved. Cell viability and measurements on thedevelopment of ALP-activity of osteoblast-like cells MC3T3-E1 indicate that C1 did not significantly influence the dif-ferentiation of the preostoblastic cell line, whereas E1appeared to increase the differentiation process. Mechanicalproperties were determined by nanoindentation experi-ments of polymer disks. Almost all samples except E1 with10 wt % V2 as crosslinker were better than the referencePCL. Surprisingly, the highly crosslinked polymer of V3even displayed higher IM and H values than the second ref-erence, PLA. Furthermore, the IM of V3 already approachedthat of human bone. Erosion behavior of the polymers from

our di- and trivinyl monomers was slower under acidicconditions compared to the alkaline media, but still fasterthan for PLA. Concerning future application as biodegrad-able polymers or hydrogels, studies under physiologicalconditions are currently carried out and in vivo experi-ments will follow.

Financial support by the Austrian Science Fund FWF for thisproject, P19769-N14, is gratefully acknowledged.

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photopolymerization of biocompatible phosphorus-containing vinyl esters and vinyl carbamates

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