Materials Letters 237 (2019) 294–297

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Materials Letters
journal homepage: www.elsevier.com/locate/mlblue

Synthesis and characterization of a novel radiopaque dimethacrylate

zirconium containing pre-polymer for biomedical applications
C Vibha, P.P Lizymol
Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Thiruvananthapuram 695012, India

a r t i c l e i n f o a b s t r a c t

Article history: Novel radiopaque dimethacrylate zirconium containing pre-polymer [ZrR1] was synthesized using a sim-
Received 11 September 2018 ple single-pot modified sol gel method. The objective of the work is to check the feasibility of developing
Received in revised form 16 November 2018 photocured polymeric composite composites [ZrR1Q] using the novel zirconium containing pre-polymer
Accepted 17 November 2018
[ZrR1] and to investigate radiopacity and cytocompatibility of the composite. ZrR1Q exhibited cytocom-
Available online 17 November 2018
patibility, comparable radiopacity with tooth structure and low polymerization shrinkage.
Ó 2018 Elsevier B.V. All rights reserved.
Keywords:
Radiopaque pre-polymer
Modified sol gel method
Cytocompatibility
Polymerization shrinkage

1. Introduction trimethylbenzoyl) phosphine oxide (TPO), 2-hydroxy-4-

Potential mechanical and advantageous biocompatible proper-
ties of Zirconium (Zr) compounds increases its demand in biomed-
ical applications as dental implants and ceramic crowns in
dentistry [1,2], total knee [3] and hip replacement, and ossicular
chain reconstruction [4]. Habitually Zr has been integrated in filler
part of the bio-composites as zirconia (ZrO2) to induce radiopacity,
superior mechanical properties and good chemical and biological
compatibilities [5]. A radiopaque polymer offers the advantage of
being traceable via X-ray imaging. Radiopacity has been integrated
in polymeric materials by incorporating metal powders/salts/ coat-
ing halogen moieties by blending with polymers. Incongruity of
these additives results in degradation leading to systemic toxicity
and deterioration in mechanical properties of polymer [6]. The ver-
satility of zirconia in biomedical applications gives us an insight to
develop a novel zirconium containing radiopaque resin with poly-
merizable multifunctional groups. The objective of the present
work is to synthesize radiopaque resin and prepare photocured
composites out of it. Radiopacity and cytocompatibility of the com-
posite material was evaluated.
2. Materials and methods
2.1. Materials
3- trimethoxy silyl propyl methacrylate, triethylene glycol
dimethacrylate (TEGDMA), zirconyl chloride, diphenyl (2, 4, 6
E-mail address: lizymol@sctimst.ac.in (P.P Lizymol)
methoxybenzophenone, 4-(dimethylamino) phenethyl alcohol,
4-methoxy phenol, phenyl salicylate and 2, 6 di-tert-butyl -4-
methyl phenol were purchased from Sigma-Aldrich, US. Diethyl
ether (LR grade), Hydrochloric acid (Analytical grade) used in the
study were purchased from S.D. Fine Chemicals, Mumbai, India.
2.2. Methods
2.2.1. Synthesis and characterization of radiopaque pre-polymer
Radiopaque pre-polymer containing alkoxideof zirconium with
polymerizable dimethacrylate groups (ZrR1) was synthesized via
modified sol-gel technique. ZrR1 was characterized using Refrac-
tive index (RI), Fourier Transform infrared spectroscopy (FT-IR)
and Fourier Transform Raman spectroscopy (FT-Raman).
2.2.2. Refractive index
Refractive index of the resin was measured using an Abbey
refractometer (ATAGO 3T, Japan).
2.2.3. Fourier Transform infrared spectroscopy
FT-IR spectrum was recorded at room temperature by applying
a thin film on NaCl cell with a FT-IR spectrometer (Jasco FT-IR
6300, Japan). The scanned wave number range was 4000–
600 cm 1, at 4 cm 1 of spectral resolution.
2.2.4. Fourier Transform Raman spectroscopy
FT-Raman spectrum was recorded using a FT-Raman spectrom-
eter (Model Bruker RFS 100/s) with Nd:YAG (1064 nm) laser and a

https://doi.org/10.1016/j.matlet.2018.11.098
0167-577X/Ó 2018 Elsevier B.V. All rights reserved.
 C Vibha, P.P Lizymol / Materials Letters 237 (2019) 294–297
liquid nitrogen cooled Ge-diode detector. The laser power used
was150 mw, and 50 scans were used for each material.
2.3. Preparation of photocured composites
Photocured composite [ZrR1Q] was prepared from ZrR1 by the
previously patented procedure [7]. The filler used for the prepara-
tion of the paste consists of silanated quartz and fumed silica. The
control Bis GMA based composite (BGMA), one among popular
dimethacrylate resin based composite used in biomedical applica-
tions, was prepared as per the reported procedure [8].
2.4. Evaluation of photocured composites
2.4.1. Linear polymerization shrinkage
Polymerisation shrinkage of ZrR1Q was performed as per the
reported procedure [9] and compared with control BGMA.
2.4.2. Radiopacity evaluation
For qualitative radiopacity evaluation, scout images of ZrR1Q,
BGMA and natural tooth was recorded using Micro CT (m-CT Scanco
40, Switzerland) and compared. Extracted human premolar tooth
received from Government Dental College, Thiruvanathapuram,
Kerala, by the indications of the orthodontic specialist as per Insti-
tutional Ethics Committee’s (IEC) approval order IEC/C/02/GDC7
21-03-2015, was used for the radiopacity evaluation. For quantita-
tive radiopacity evaluation, two sets of photocured samples were
radiographed with aluminum stepwedge (aluminum alloy EN
1050 containing 99.5% of Al) which was used as a reference recom-
mended by ISO 4049 [10]. The stepwedge had six steps, with thick-
ness of 0.5, 1, 1.5, 2, 2.5, and 3 mm and the mean value was taken
for gray scale evaluation. The radiographic images were acquired
with a storage phosphor plate system (3  4 cm Digora Imaging
Plate and Digora FMX scanner; Soredex Corporation, Tuusula, Fin-
land) and a digital X-ray machine (Planmeca Prostyle Intra, Plan-
meca Oy, Helsinki, Finland) operating at 70 kV and 8 mA with a
total filtration equivalent of 2.0 mm of Al. The exposure time was
0.20 s and focus-to-object distance was 40 cm. The graph was plot-
ted with grayscale intensity against thickness of aluminium wedge
using ImageJ software and the radiopacity of the sample was eval-
uated. Mean gray scale intensities of the samples were obtained
from ImageJ software.
Fig. 1. (a) FTIR spectrum of ZrR1 and (b) FT-Raman spectrum of ZrR1.
295
2.4.3. In vitro cytotoxicity and MTT assay
In vitro cytotoxicity test using direct contact method was per-
formed as per ISO 10993-5 [11] by using L929 mouse fibroblast
cells. Ultra high molecular weight poly ethylene was used as neg-
ative control and Stabilised PVC disc as positive control. Test sam-
ples (6 numbers) were steam sterilized at 121 °C for 20 min. Test
samples, negative controls and positive controls in triplicate were
placed on the cells. The cell monolayer was examined microscopi-
cally after incubation for 24–26 h at 37 ± 1 °C. MTT assay was per-
formed at the end of the test procedure for measuring the
metabolic activity of cells to reduce yellow coloured tetrazolium
salt 3-(4, 5-Dimethyl thiazol-2-yl)-2, 5- diphenyltetrazolium bro-
mide to purple colour. The colour developed was quantified by
UV spectrophotometeric method. Absorbance at 570 nm was
measured.
2.4.4. In vitro cell adhesion study
An in vitro cell adhesion study was performed using L929
mouse fibroblast cell lines. The cells were sub cultured and seeded
on test materials (6 numbers) along with control glass cover slip at
a density of 1x104 cells/cm2 and incubated for 48 h at 37 ± 1 °C
under humidified atmosphere containing 5% CO2. The cell seeded
test material and glass cover slips were fixed in 4% paraformalde-
hyde for 48 h. The samples were rinsed thrice with 0.1 M phos-
phate buffered saline followed by permeabilisation with 0.1%
Triton X-100 in PBS for 1 min. The samples were rinsed 3 times
with PBS and treated with Rhodamine Phallodin (1:100) for
15 min. The samples and controls were observed under fluores-
cence microscope Leica N2.1 filter cubeDMI6000, (ExBp515-560)
EmiLP590.
2.5. Statistical analysis
The data obtained for evaluation and comparison of polymerisa-
tion shrinkage of ZrR1Q against Control BGMA in the present study
is subjected to statistical analysis. The population is normal and
the data presented a normal distribution so we performed Stu-
dent’s t-test along with ANOVA.
3. Results and discussions
FT-IR spectrum of ZrR1 [Fig. 1(a)] exhibited typical silane peaks
as reported earlier [12]. An intense peak for –OH group around
296 C Vibha, P.P Lizymol / Materials Letters 237 (2019) 294–297

3471 cm 1 confirmed the presence of free Si-OH groups in ZrR1
which will enhance the bioactivity of the resin [13]. The refractive
index of ZrR1 was found to be 1.4752.
Fig. 2. Comparison of linear polymerization shrinkage of ZrR1Q. with control
BGMA. *Indicates polymerization shrinkage of ZrR1Q. was significantly low
compared to control BGMA.
Si-O-CH3 stretching band of silica (1100 cm 1) of ZrR1 was very
strong in IR while extremely weak in Raman spectra [Fig. 1(b)].
Strong peaks at 1000–1200 cm 1 (O-Si-O asymmetric stretch)
and 700–800 cm 1 (Si-O-Si symmetric stretch) observed in FTIR
spectrum was absent in FT-Raman spectrum [12]. The other peaks
were 1717 cm 1 (C = O group), 1299 cm 1 (Si-O-Si asymmetric
stretch), 1639 cm 1 (C = CH2) and 2897–3000 cm 1 (C–H).
The polymerization shrinkage of the control material, BGMA
was reported to be 2.3–3% [14]. The shrinkage value obtained
for ZrR1Q was significantly low compared to BGMA
(p value < 0.00001), which is found to be ideal in biomedical
applications [Fig. 2]. The bond formed by the alkoxide is purely
electrostatic, satisfied the coordination number of the silane
precursor which will excellently crosslink with its valency and
providing overall stability to the system [9]. The presence
of polymerizable dimethacrylate monomers in the resin matrix
[ZrR1] alleviates the binding with the inorganic filler [9] by reduc-
ing the polymerization shrinkage of ZrR1Q.
Scout images of ZrR1Q were compared with BGMA and natural
tooth in Fig. 3a. ZrR1Q exhibited enhanced radiopacity compared
to BGMA while comparable radiopacity with tooth structure
[Fig. 3a]. The radiopacity was attributed by resin matrix to the

Fig. 3. Radiopacity measurement (a) Qualitatively by Micro-CT Scout images (i) ZrR1Q, (ii) Comparison of ZrR1Q with control BGMA and (iii) Comparison of ZrR1Q. with
natural tooth; (b) Quantitatively using Aluminium wedge (i) X-ray radiograph of aluminium wedge and samples and (ii) excel plot of grayscale intensity Vs thickness of
aluminium wedge.

Fig. 4. Cytocompatiblity study of ZrR10 (a) in vitro cytotoxicity of ZrR1Q, (b) MTT assay of ZrR1Q and (c) in vitro cell adhesion of ZrR1Q using L929 mouse fibroblast cells.
 C Vibha, P.P Lizymol / Materials Letters 237 (2019) 294–297
composite. ZrR1Q demonstrated radiopacity value of 2.7 (from
quantitative radiopacity measurement) [Fig. 3b] which is greater
than radiopacity of dentin and equal to dental enamel which
implies that ZrR1Q can be applied for both anterior and posterior
teeth restoration [15].
Fig. 4 showed the cytocompatibility of ZrR1Q using L929 mouse
fibroblast cell lines. The cells around ZrR1Q displayed spindle
shape morphology without cell detachment and lysis [Fig. 4a].
MTT assay quantified the possible toxicity of materials exerted
on cells [Fig. 4b]. The cell adhesion studies demonstrated that
L929 fibroblast cells adhered and spread well on ZrR1Q [Fig. 4c].
The non-cytotoxic nature along with good cell viability and cell
adhesion of the test sample proved that no monomer was leached
out from the sample [9].
4. Conclusion
A radiopaque composite based on novel radiopaque pre-
polymer containing alkoxide of zirconium was developed. The
new composite with low shrinkage and cytocompatibility will def-
initely be a potential candidate in biomedical field specifically for
orthopaedic, dental and bioactive coating applications.
Acknowledgement
Financial support from Kerala State Council for Science, Tech-
nology and Environment, Government of Kerala, India is gratefully
acknowledged. We thank, Director, Head and Dr. V. Kalliyana
Krishnan, BMT Wing, SCTIMST for extending the facilities.
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