Template for Electronic Submission to ACS JournalsZirconium Amine
Tris(phenolate): A More Effective Initiator for Biomedical
Lactide
Matthew D. Jones, ^a Xujun Wu,a,b Julian Chaudhurib,c Matthew G.
Davidson, ,a and Marianne J. Ellis *,b
a Department of Chemistry, University of Bath, Bath, BA2 7AY,
UK.
b Department of Chemical Engineering, University of Bath, Bath, BA2
7AY, UK. Tel: +44 (0) 1225 384484;
c Present address: School of Engineering & Informatics,
University of Bradford, Bradford BD7 1DP, UK.
*E-mail:
[email protected]; ^For catalysis:
[email protected]
KEYWORDS. Biomedical; Initiator; Polylactide; Tissue Engineering;
Zirconium.
ABSTRACT
Here a zirconium amine tris(phenolate) is used as the initiator for
the production of polylactide for biomedical applications, as a
replacement for a tin initiator (usually tin octanoate). The ring
opening polymerization (ROP) was carried out in the melt at 130 ºC.
The zirconium-catalyzed PLA (PLA-Zr) required 30 min, resulting in
a polydispersity index (PDI) of 1.17, compared to 1h and PDI=1.77
for tin-catalyzed PLA (PLA-Sn). PLA-Zr and PLA-Sn supported
osteosarcoma cell (MG63) culture to the same extent (cell number,
morphology, extracellular matrix production and osteogenic
function) until day 14 when the PLA-Zr showed increased cell
number, overall extracellular matrix production and osteogenic
function. To conclude, the reduction in reaction time, controllable
microstructure and biologically benign nature of the zirconium
amine tris(phenolate) initiator shows that it is a more effective
initiator for ROP of polylactide for biomedical applications
1. Introduction
Linear aliphatic polyesters are routinely used in biomedical
research.[1, 2] Ring-opening polymerization (ROP) of cyclic
monomers (i.e. lactide or glycolide) is a commonly used method to
synthesize linear aliphatic polyesters as it provides good control
over polymer molecular characteristics such as predictable
molecular weight, end group and polymer stereochemistry control.[3]
ROP requires an initiator, and tin compounds are the most widely
used in industry as they have the advantages of being cheap,
soluble in common organic solvents and relatively easy to
handle.[4] Concerns have been raised regarding the toxicity of tin
initiators for the synthesis of polymers for biomedical
applications.[5, 6] Tin compounds tend to accumulate in lung tissue
and brain, which may cause neurotoxicity and slow penetration into
blood circulation systems.[5, 7] To avoid these potential
side-effects, the amount of tin residue in a standard biomedical
polymer is limited to below 20 ppm according to the US Food and
Drug Administration[8]; it is impossible to completely remove the
residual tin from the resulting polymers, so sophisticated
purification processes have to be used in order to minimize the
metal residue.[8] Continuous efforts have been made to develop
initiators based on more biologically benign metals as alternatives
to tin initiators, for example aluminum[9-12], magnesium[13],
zinc[13-18] and calcium[19, 20] for production of biomedical
polymers. In addition, Group 4 metal complexes, have also shown
great promise to give polymers with desired properties.[21-24] The
polymerization has been shown to proceed via a traditional
coordination insertion mechanism, this has been proven via end
group analysis (MALDI-ToF mass spectrometry and NMR
spectroscopy).[25] It has been shown that an unusual combination of
high stereoregularity and reactivity can be achieved using a
zirconium initiator supported by amine tris(phenolate) ligands for
ROP of lactide under solvent-free conditions.[25] The application
of solvent-free conditions eliminates the hazard of using a solvent
to carry out polymerisation; furthermore it has been previously
noted that a solvent-free method is necessary for industry and
allows for a highly pure product[25]. In addition to this,
zirconium compounds typically exhibit 10-20 times fewer toxic
effects compared with tin.[26] Reduced processing conditions, e.g.
temperature and time, will help minimize manufacture cost which is
favorable for mass production, as is a reduction in batch-to-batch
variation which it can be hypothesized to be achieved by a more
controlled polymerization mechanism. Despite the great success in
developing relatively biocompatible initiators for well-controlled
ROP, to date, it is unclear how cells will respond to polymers
prepared from these initiators compared to the commercial tin
initiator. In this study, PLA prepared from the zirconium-based
amine tris(phenolate) initiator[25] (Zr) and the commercial
tin-based initiator, namely tin octanoate (Sn), were characterized.
A comparative investigation of the osteoblast-like osteosarcoma
MG63 cell line attachment, proliferation and differentiation on
smooth PLA films was performed to establish the effects of
initiator residue on cell behavior.
2. Experimental Section
2.1 Polymerization
D,L-Lactide was purchased from Sigma-Aldrich and was purified by
recrystallisation from toluene and dried under vacuum overnight.
Tin octanoate (Sigma) was used as received. The zirconium amine
tris(phenolate) initiator was prepared according to the method
reported previously.[25] Polymerisations were performed at a
temperature of 130 ºC under solvent-free conditions with a monomer
to initiator (Zr or Sn) ratio [M]/[I] of 200:1. In all cases 3 g of
D,L-lactide were used. After the reaction time (0.5 or 1 h) an
excess of methanol was used to quench the reaction and the
resulting solid was dissolved in dichloromethane. The solvents were
then removed under vacuum. The resulting solid was further purified
by washing with methanol and dried in vacuo overnight. The
conversions were determined by 1H NMR spectroscopy (Bruker 300 MHz
spectrometer, solvent: CDCl3), and molecular weights (Mn) and
polydispersity index (PDI) of the polymers were determined by gel
permeation chromatography (GPC, Polymer Laboratories GPC 50,
solvent: THF, 1 mL/min flow rate at 35 ºC and referenced to
polystyrene standards). Three polymers were made: two using the tin
initiator and a reaction time of 0.5 h or 1.0 h, denoted as
PLA-Sn-0.5 and PLA-Sn-1.0, and one using the zirconium initiator
and a reaction time of 0.5h, denoted as PLA-Zr-0.5.
2.2 Preparation and Characterization of PLA films
5 % (w/v) PLA was first dissolved in chloroform. 200 μL of the
solution was placed onto a cover slip using 1 mL syringe at room
temperature (20 ºC) and allowed to spread. A thin layer of
transparent polymer film was obtained after complete evaporation of
solvent. The thin film was then washed with excess of distilled
water and further dried in vacuo overnight. The Zr and Sn initiator
metal residues in the PLA films prior to cell culture were measured
by inductively coupled plasma optical emission spectrometry
(ICP-OES, MEDAC Ltd, UK). The PLA films were sterilized in 1 %
antibiotic/antimycotic solution (Sigma) over night at 4 ºC prior to
cell culture 27 . After sterilization, the antibiotic/antimycotic
solution was removed and the PLA films washed with excess of
phosphate buffer saline (PBS). Metal content of the films was
measured by inductively coupled plasma optical emission
spectroscopy. To measure the water contact angle on the surface, 5
µl of distilled water was placed onto the film surface and the
measurement taken after 5 min using a goniometer (NRL
100-00).
2.3 Cell culture
The human osteosarcoma cell line MG63 was obtained from the
European Collection of Cell Culture (ECACC). The MG63 cells were
maintained at 37ºC and 5% CO2 in a standard cell culture medium
which contained Dulbecco’s Modified Eagle’s medium (DMEM, Sigma),
supplemented with 10% (v/v) fetal calf serum (FCS, Sigma), 1mM
sodium pyruvate (Sigma), 1% (v/v) antibiotic-antimycotic and 1%
(v/v) non-essential amino acids (NEAA, Sigma) for proliferation. To
differentiate MG63 cells, the standard cell culture medium was
switched to an osteogenic medium after 3 days which contained 10 mM
β–glycerophosphate (Sigma), 100 nM dexamethasone ( Sigma) and 50
μg/ml ascorbic acid-2-phosphate (Sigma). 28 The medium was
refreshed every 2-3 days. The PLA films were placed in a 24-well
tissue culture treated plates (NuclonTM Δ Surface) and secured with
customized silicon rings. MG63 cells were seeded onto the samples
with a density of 20,000 cells/ cm2 and maintained at 37ºC and 5%
CO2. Tissue culture polystyrene (TCPS) was used as control. MG63
cells were cultured for 21 days on the smooth PLA-Zr and PLA-Sn,
and TCPS. They were assessed in a standard culture medium up to 3
days then an osteo-differentiation medium up to 21 days.
2.4 Cell number
Cell number was determined using the PicoGreen assay kit
(Invitrogen Ltd, Paisley UK) as per the manufacturer’s
instructions. Briefly, samples were prepared by rinsing three times
with PBS then lysing with 0.05 % Triton X-100 and freeze-thawed at
-80 ºC for 2-3 cycles. The fluorescence was read at excitation and
emission 485/528 nm (BioTek Synergy HT) and readings compared to a
standard curve to calculate cell number.
2.5 Cell morphology
Cell morphology on the PLA films was observed by f-actin staining
and scanning electron microscopy (SEM). For cytoskeletal
organization, FITC-phalliodin (Sigma) was used to label f-actin
filaments. Cells were washed with PBS, fixed with 3.7 %
formaldehyde in PBS for 5min and permeabilized with 0.1 %
TritonX-100 for 10min before staining with 50 µM FITC-phalloidin
for 40min at room temperature. Cells were observed with a Leica
DMI4000B fluorescent microscope. For SEM, at each time point, the
culture medium was removed and the samples were fixed in 2.5 %
glutaraldehyde for 2 h. The samples were then washed with serum
free culture medium and post-fixed in 1 % osmium tetroxide in PBS
buffer for 1 h. After thoroughly washing with serum free culture
medium, the samples were stained with 2 % aqueous uranyl acetate in
water for 1 h in dark. Then the samples were freeze-dried
over-night. A thin layer of gold was sputter-coated onto the
samples before examining by SEM (JOEL JSM 6480LV SEM).
2.6 Osteogenic function
The alkaline phosphatase (ALP) activity of cells was measured using
the p-nitrophenyl phosphate reduction method. Cells were lysed
using 0.05 % Triton X-100 solution then the samples were frozen and
thawed at -80 ºC for 2-3 cycles, and sonicated for 15 mins. 50 μL
of samples were added into 50 μL of p-nitrophenyl phosphate
substrate in a 96-well plate and incubated for 1 h at 37 ºC. The
reaction was stopped by the addition of 50 μL of 1 N NaOH and
absorbance measured at 405 nm. The ALP activity of cells was
normalized to the cell number measured by the Picogreen assay. The
osteocalcin (OC) solid phase Enzyme Amplified Sensitivity
Immunoassay (EASIA, Invitrogen, UK) was used to quantify OC
production as per the manufacturer’s instructions. Standards and
samples were pipetted into 96-well multi-plate and reacted with
pre-coated monoclonal antibody labeled with horseradish peroxidase
(HRP). Wash solution was used to remove unbound enzyme labeled
antibody after 2 h incubation. The bound enzyme labeled with
antibody can be detected by conducting a chromogenic reaction for a
further 30 min. The absorbance measurements were performed at 450
nm and OC production calculated using a standard curve.
Mineralization was analyzed using Alizarin Red S to detect the
deposited calcium ions on the PLA films and TCPS as described
previously. 29 Briefly, MG63 cells were fixed in 10 % (v/v)
formaldehyde and washed with PBS for three times. Samples were
immersed in 40 mM Alizarin Red S (pH=4.2) solution for 20 mins,
with gentle shaking before being washed with PBS for 5 mins.
Insoluble red colour calcium ion-Alizarin Red S complex was
observed microscopically (Leica DMI4000B) followed by using Image J
analysis software (http://rsb.info.nih.gov/ij) to analyze the total
area of mineralization stained by Alizarin Red S.30
2.7 Statistical analysis
Statistical analysis was conducted using one way or two-way ANOVA
with Bonferroni post tests using Prism 4.0 (GraphPad Software Inc.,
San Diego CA, USA).
3. Results and Discussion
3.1 Polymerization
PLA-Zr has a significant heterotactic bias compared to PLA-Sn.
Under the same reaction conditions of 130 ºC and 0.5 h, PLA-Zr
showed higher conversion compared with the PLA-Sn, while
maintaining a relatively low polydispersity index (PDI) (Table
1).
Table 1. GPC measurements for the PLA prepared by the Zr and Sn
initiators at 130 ºC with [M]/[I]=200:1.
PLA
PLA-Sn
0.5
68
23,500
1.53
PLA-Sn
1.0
90
33,900
1.77
PLA-Zr
0.5
96
33,500
1.17
[a] Conversion of PLA was calculated by 1H NMR [b] Molecular weight
(Mn) and polydispersity index (PDI) were determined by GPC,
relative to polystyrene standards.
The PLA produced using the Zr(IV) initiator has a Pr value
(probability of racemic enchainment) of 0.9 whilst the Sn(IV)
produced PLA with a Pr value of 0.6. To achieve comparable
molecular weight for the PLA polymerized using both initiators, the
reaction time for the Sn-initiated polymerization was extended to 1
h. At this extended reaction time a higher conversion and molecular
weight for the PLA-Sn-1.0 (from here PLA is named according to
initiator-time of polymerisation (hours)) was achieved compared to
PLA-Sn-0.5, but a lower conversion and higher PDI compared to the
PLA-Zr-0.5 at 0.5 h. The zirconium amine tris(phenolate) complex
was chosen as an initiator in this study because it offers high
reactivity for ROP[25] in the synthesis of PLA with a defined
structure, and is potentially less toxic than the tin alternatives.
The PLA polymerized from the zirconium amine tris(phenolate)
possessed a high degree of heterotactic enrichment and molecular
weight in agreement with theoretical values, and a narrow molecular
weight distribution.[25] These factors are closely related to
polymer properties such as degradation, hydrophilicity and
mechanical strength, which ultimately affect cell adhesion and
function. 30
The PLA-Zr-0.5, and PLA-Sn-1.0 (with similar molecular weight to
PLA-Zr-0.5) were selected for the fabrication of PLA smooth films,
used so topographical effects were comparable. The water contact
angle and metal residue in the resulting PLA-Zr-0.5 and PLA-Sn-1.0
films were also comparable: 80.5±1º and 81±1º, 0.21 wt% (Zr) and
0.23 wt% (Sn), respectively.
3.2 Cell number and morphology
Cell attachment was quantified six hours after seeding. 80% of
cells were attached to the PLA-Zr-0.5 and PLA-Sn-1.0 films, and
tissue culture polystyrene (TCPS) which was used as the control. An
increase in cell number was observed for all the samples,
indicating that MG63 cells can proliferate on the PLA-Zr and PLA-Sn
films irrespective of initiator. Over the course of the first three
days, the cell number on all the samples was comparable to each
other and no significant difference (p>0.05) was observed (Fig.
1).
Figure 1. MG63 cell attachment and proliferation on the PLA-Zr,
PLA-Sn films. PLA-Zr-0.5 showed significantly higher cell number at
day 14 and 21 compared with PLA-Sn-1.0. Tissue culture polystyrene
was used as the control and cell number was quantified using the
Picogreen reagent. PLA-Zr; PLA-Sn; TCPS. Asterisks indicate a
significant difference (*p<0.05 and **p<0.01) between groups
at the same condition.
The cell number continued to increase between three and 21 days
(now in osteogenic medium). The cell number was comparable for
PLA-Zr-0.5 and PLA-Sn-1.0 films and TCPS at day seven; however, the
cell number on the PLA-Zr-0.5 was significantly higher than the
PLA-Sn-1.0 at day 14 and 21 with p<0.05 and p<0.01,
respectively. There was no significant difference in cell number
between the PLA-Zr-0.5 and the TCPS control at day 14 and 21.
After six hours, cells started to spread and attach to the surface
and there were still some round cells observed on both PLA-Zr-0.5
and PLA-Sn-1.0 (Fig. 2 a, c, e, g). The cells were flattened,
stretched their pseudopodia and interacted with each other at 24 h
(Fig. 2 b, d, f, h). After three days, the PLA-Zr-0.5 and
PLA-Sn-1.0 surfaces were covered by a confluent cell monolayer.
There were no obvious differences in cell morphology for the cells
on the PLA-Zr-0.5 and PLA-Sn-1.0 at three days. Between seven and
21 days the MG63 population became more tightly packed.
Figure 2. MG63 cell morphology at six hours and 24 h. (a – d)
cytoskeleton organization demonstrated by FITC-phalloidin staining
of f-actin; (e – h) scanning electron microscopy images. Scale bars
are 50 m for (a) to (d) and 10 m for (e) to (h).
Cell number increased over 21 days of culture for the PLA-Zr-0.5,
PLA-Sn-1.0 and TCPS substrates. In this study, the properties of
the PLA-Zr-0.5 and PLA-Sn-1.0 smooth films were comparable for
molecular weight, hydrophilicity, topography and initiator metal
residue content. It is known that Sn compounds are more cytotoxic
than Zr compounds.[5, 7, 26, 27] The lower number of cells on the
PLA-Sn-1.0 from day 14 may have been caused by the material
degradation and release of tin compounds. The findings in this
study are in line with the findings from Czajkowska and co-workers
who cultured Saos2 cells on poly(L-lactide-co-glycolide) (lactide
to glycolide ratio of 85:15) prepared from zirconium
acetylacetonate(IV) initiator, and found 85% viable cells on
polymer prepared from the Zr(IV) initiated and 65% viable cells on
the polymer prepared from the Sn(Oct)2 initiator, relative to the
control on day seven. 32
3.3 Osteogenic function
From day three osteogenic media was used to differentiate the
cells, and osteogenic function was observed from day seven. Trends
of alkaline phosphatase (ALP) activity differed between the films
and the TCPS (Fig. 3); the level of ALP activity for PLA-Zr-0.5 was
significantly higher
Figure 3. Alkaline phosphatase activity of the MG63 cell function
on the smooth PLA-Zr-0.5, PLA-Sn-1.0 and TCPS at day 7, 14 and 21.
Osteogenic media was added from day three. Asterisks indicate a
significant difference (p<0.001).
(p<0.001) than PLA-Sn and TCPS at day seven and 14. However, ALP
activity was comparable for PLA-Zr-0.5, PLA-Sn-1.0 and TCPS at 21
days. Cells on TCPS showed a near-linear increase in ALP activity
between days seven and 21; cells on PLA-Sn-1.0 showed a continual
increase but the rate of increase was slower from day 14; cells on
PLA-Zr-0.5 showed a peak of ALP activity at 14 days, which was
significantly higher than both PLA-Sn-1.0 and TCPS. However at 21
days the ALP activity was comparable on all three substrates. A
possible explanation for this distinctive behavior of zirconium is
discussed below in conjunction with the other functional
analysis.
Osteocalcin (OC) expression followed the same trend for all three
substrates, with a slight increase at day 14 compared to day seven
and four times the amount of expression at day 21 compared to day
14 (Fig. 4). At day 21, the PLA-Zr-0.5 showed more abundant
extracellular matrix formation than PLA-Sn-1.0 (Fig. 5).
Figure 4. Expression of osteocalcin by the MG63 cells on the smooth
PLA-Zr-0.5, PLA-Sn-1.0 and TCPS at day 7, 14 and 21. Osteogenic
media was added from day three. PLA-Zr-0.5; PLA-Sn-1.0; TCPS. No
significant differences were seen.
Figure 5. Mineralized matrix formation analysis at 21 days. (a)
& (b) Light microscopic images of Alizarin Red staining for
bone nodules, indicated by the black arrow labeled ‘Ca’. Scale bar
50 µm. (c) Measured areas of staining using the ImageJ software,
n=10. Asterisks indicate a significant difference (*p<0.0001
when compared to TCPS).
The formation of mineralized nodules was enhanced when culturing
cells on the PLA films compared to TCPS. The average area of
Alizarin Red S (ARS) staining for the PLA-Zr-0.5 and PLA-Sn-1.0
were 2342 ± 202 µm2 and 2091 ± 117 µm2 respectively. For the TCPS
positive control, small calcium nodules (total area of 455 ± 41
µm2) were observed on the surface while no mineralized bone nodules
were observed on the TCPS negative control. The formation of
mineralized nodules on both the PLA-Zr-0.5 and PLA-Sn-1.0 was
significantly greater than the TCPS positive control (p<0.0001)
while being comparable to each other.
Theoretically, ALP activity increases with cell differentiation
until it reaches a maximum indicating a mature bone-forming
phenotype, then decreases suggesting production of extracellular
matrix and its mineralization. 33 This rise-fall pattern was
observed when MG63 cells were cultured on the PLA-Zr-0.5, while
PLA-Sn-1.0 and TCPS culture showed a continued increase until day
21, although the rate of increase was reduced for PLA-Sn-1.0. The
change in rate of OC expression, increasing from day 14, is
supported by the theory that ALP and OC are relatively early and
late biomarkers for an osteoblastic phenotype respectively. 34 When
cells became more mature, the ALP activity decreased and the OC
increased. The theory is further supported by the observation that
at day 21 the formation of collagenous extracellular matrix was
more pronounced on PLA-Zr-0.5 than on PLA-Sn-1.0 (Fig. 6); note
that this is total matrix and not per cell so it follows that, with
more cells on the PLA-Zr-0.5, there is correspondingly more
matrix.
Figure 6. Extracellular matrix formation. SEM images showing
collagenous extracellular matrix formation, indicated by the white
arrow ‘Col’.(a) PLA-Zr-0.5; (b) PLA-Sn-1.0. There is more matrix
deposition on PLA-Zr-0.5 than on PLA-Sn-1.0.
4. Conclusion
This paper has demonstrated that PLA-Zr-0.5 is a favorable
alternative to PLA-Sn-1.0 in terms of both polymerization
conditions, and cell response. While the presence of late-stage
osteogenic markers were comparable on all substrates, the
early-stage osteogenic marker ALP, and cell number was
significantly increased on the PLA-Zr-0.5 substrate. Since in this
study the properties of the PLA-Zr-0.5 and PLA-Sn-1.0 smooth films
were comparable for molecular weight, hydrophilicity, topography
and initiator metal residue content, the differences were
attributed to the different metals present. This is an important
consideration when an application requires cell expansion and,
alongside the advantageous polymerization conditions and polymer
properties, the findings suggest a zirconium-based catalyst for PLA
ROP when this is the case.
Acknowledgements
The authors thank the University of Bath for funding this work.
Inductively coupled plasma optical emission spectroscopy was
carried out by Medac GmbH
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