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Available online at www.sciencedirect.com
2212-8271 © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the scientifi c committee of The Second CIRP Conference on Biomanufacturing
doi: 10.1016/j.procir.2015.11.019
ScienceDirect
The Second CIRP Conference on Biomanufacturing
PLLA Synthesis and Nanofibers production: Viability by Human Mesenchymal Stem Cell from
Adipose Tissue
Xavier, M.V.1 *, Macedo, M.F.1,4, Benatti, A. C. B.1,5 , Jardini, A.L. 1,2 , Rodrigues A.A. 1,2, Lopes
M.S.1,2, Lambert C.S.1,2, Filho, R.M. 1,2 , Kharmandayan, P. 1,5
1 – Institute of Biofabrication - School of Chemical Engineering, University of Campinas, CEP 13081-970, Campinas-SP, Brazil
2 – School of Chemical Engineering, University of Campinas, CEP 13081-970, Campinas-SP, Brazil
3 - Institute of Physics “Gleb Wataghin”, Departament of Applied Physics, University of Campinas, CEP 13083-859, Campinas-SP, Brazil
4 – School of Mechanical Engineering, University of Campinas, CEP 13083-860, Campinas – SP, Brazil
5 - School of Medical Sciences, Surgery Department, University of Campinas, CEP 13083-970, Campinas-SP, Brazil
* Corresponding author. Tel.: +55 19 4108-0014; fax: +55 19 3521-3965. E-mail address: maryvitelo@hotmail.com
Abstract
The absorbable polyacid is one of the most used and studied materials in tissue engineering. This work synthesized a
poly (L-lactic acid) (PLLA) through ring-opening polymerization and produced with it nanofibers by the
electrospinning process. The PLLA was analyzed by FTIR and its cytotoxicity was evaluated by the MTT assay and
Live/Dead® (Molecular Probes). The tests were performed in contact with human mesenchymal cells at varying times.
The high rates of viability and proliferation of cells in contact with the PLLA shown by MTT and Live/Dead® tests
demonstrate that this PLLA is a biocompatible material. There was also the successful production of electrospinning
nanofibers, which can be converted for specific biomedical applications in the future.
© 2015 The Authors. Published by Elsevier B.V.
Peer-review under responsibility of the scientific committee of The Second CIRP Conference on Biomanufacturing.
Keywords: biomaterials; material synthesis; PLLA; cell culture; MTT test; tissue-engineering; biofabrication.
1. Introduction:
For the past few years, there had been a growing interest in
biomaterials and tissue engineering. This interest is a result of
the indispensable role of those in the medical field.
Biomaterials are biocompatible and bioactive, with the intent
to interact with biological systems, as well as restore functions
of living tissues and organs on human or animal individuals [1-
4].
The polymers compose a very broad class of biomaterials, and
there had been development in the biomedical field for use of
© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the scientifi c committee of The Second CIRP Conference on Biomanufacturing
214 M.V. Xavier et al. / Procedia CIRP 49 ( 2016 ) 213 – 221
those polymeric materials. These biomaterials must show
appropriate mechanical properties as well as be biocompatible,
to be used in the human body. Thus they shouldn´t present any
local or systemic adverse biological response. The material
should be then non-toxic, non-carcinogenic, non-antigenic and
non-mutagenic. In blood applications, they must also be non-
thrombogenic [5].
The assigning of biomaterials appropriate for the development
of implants requires meeting certain criteria, so that its effects
on the surrounding tissues are decreased. The most pertinent
variables should be studied in detail, such as biocompatibility,
biodegradability, bioabsorbable capacity, degradation rate,
surface finish (pore diameter and porosity), etc. [6].
Biomaterials may show a low, medium or high plausible risk
to human safety, conditional on the kind and length of patient
contact with it. One of the suggested and appropriate steps for
the biological assessment of the medical devices is the in vitro
measurement of the cytotoxicity of the biomaterial [7].
Cytotoxicity assays are essential for biocompatibility analysis
of a biomaterial. These tests will be determined the cytotoxicity
effect of the material, depending on the time and contact of it
with the body, blood, bone and other tissues [8]. Therefore, the
in vitro cytotoxicity evaluation of biomaterials is an important
aspect in the research for tissue engineering.
Nowadays, many biomaterials are used in the medical area. We
can find bioabsorbable screws and pins, biodegradable
peripheral stents and suture materials. Frequently these
materials are made of poly(L-Lactide) (PLLA) and others
compounds which contains that polymer.
Polylactides in overall and in specific poly (L-lactide) (PLLA)
may be seen as second-generation biomaterials[1-4]. Different
variants of temporary devices have been used in the medical
field, and the most used are polyesters composed of derivatives
of α-hydroxy acids such as poly (L-lactic acid) (PLLA), poly
(D-lactic acid) (PDLA), poly (DL-lactic acid) (PDLLA), poly
(glycolic acid) (PGA) and polycaprolactone (PCL). The
polymer is degraded through simple hydrolysis, breaking the
molecule into small units, and so its products can then be
eliminated from the body through natural metabolic pathways,
such as citric acid cycle, or through renal excretion [9]. The
hydrolysis initially promotes the fall of the molar mass, mainly
in the amorphous regions. As the water diffuses into the device
and fragmentation occurs to decrease in mechanical strength
and further reduced weight due to hydrolysis and enzymatic
attack [10].Thus, no surgical removal of the materials is
necessary and over the time, the new tissue can be shaped
substituting the mechanical purpose of the implant itself.
PLLA has gained great attention because of its excellent
biocompatibility and mechanical properties. It also presents a
diversification of applications, since simple changes in their
physical and chemical structure may make it useful in different
areas. Depending on your application and your final
destination, you can get different products using specific
polymerization routes. However, its long degradation times
coupled with the high crystallinity of its fragments can cause
inflammatory reactions in the body. In order to overcome this,
PLLA can be used as a material combination of L-lactic and D,
L-lactic acid monomers, being the latter rapidly degraded
without formation of crystalline fragments during this process
[11].
Prior works done by our group [12, 13] have shown that the
PLLA can be obtained using different routes (Figure 1). In
general, there are three methods that can be used to produce
high molecular mass PLLA of about 100,000 Daltons: (a)
direct condensation polymerization; (b) azeotropic dehydrative
condensation and (c) polymerization through lactide formation,
the ring-opening polymerization [14]. Currently, direct
condensation and ring-opening polymerization are the most
used production techniques.
Figure 1. PLLA different synthesis methods [14]
While there are different ways to manufacture PLLA, its
production is not an easy task. The synthesis of PLLA demands
severe control of conditions such as temperature, pressure and
pH, just as well as the use of catalysts and long polymerization
time, which lead to a high-energy consumption to achieve the
final product.
PLLA not only is a biodegradable and biocompatible material,
it also presents a mechanical response similar to collagen, as
well as a semi crystalline structure, with an excellent elastic
capacity, characteristics that makes the polymer a perfect
candidate for electrospinning [15-18]. Electrospinning of
polymer solutions has been extensively used in the last few
years to produce polymeric fibers of Nano-dimensions [19].
The electrospinning fibers have exceptional properties due to
the biomimetic features dwelling on the fiber diameter, with
less than 1000 nanometers in diameter, in the Nano range as
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M.V. Xavier et al. / Procedia CIRP 49 ( 2016 ) 213 – 221
well as due to the high surface area/volume ratio [20]. In the
electrospinning process, a capillary tube or syringe and a
metallic electrode are used, associated to a grounded high
voltage supply in the polymeric solution. An electrical tension
is applied and when the electrostatic forces overcome the
solution's surface tension, the hemispherical surface of the
capillary's drop (Taylor's cone) elongates and an electrically
charged jet of polymeric solution is generated. During the jet's
trajectory to the collector, the solvent evaporates; thus solid
nanofibers are formed and collected in the form of non-woven
mats on the metallic collector. The variables of this process are
many: solutions concentration (which determines the solutions
viscosity), solvent type, applied electrical field, ionic salts
addition (which can increase the solution's electrical
conductivity), flow rate, temperature and others [19]. This
work will present evidence about the properties and synthesis
of PLLA scaffolds and nanofibers, as well as evaluate the
biocompatibility of it, through cytotoxicity assays.
2. Materials and Methods
2.1 Poly-lactic acid synthesis
The synthesis of PLLA was conducted by bulk polymerization
by adding L-lactide monomer into a glass reactor containing
the catalyst Sn (Oct) 2 (Sigma). The proportion
monomer/catalyst was 0.5%. The mixture was immersed in an
oil bath at 140°C for 2 hours under nitrogen flow. The produced
polymer was dissolved in chloroform, CHCl3 (Merck),
precipitated in ethyl alcohol and dried in a vacuum oven at
60°C for 12 hours.
2.2 Fourier Transform Infrared Spectroscopy (FTIR)
The functional groups of the produced PLLA were analyzed by
Fourier Transform Infrared Spectroscopy (FTIR), and
paralleled with standard commercial polymer, PLLA
(PURAC). The samples were evaluated by ATR accessory
SMART mode OMNI-SAMPLER in an infrared Fourier
transform spectrometer Thermo Scientific Nicolet 6700. The
spectra were analyzed in the mid-infrared range 4000-675cm-1,
obtained from the intensities absorption bands of molecules
expressed in transmittance.
2.3 Isolation of mesenchymal stem cells derived from adipose
tissue
The human adipose tissue was obtained by liposuction
procedure. It was washed with phosphate-buffered saline
(PBS) to remove any connective tissue and red blood cells
present. The adipose tissue was added to a 50-ml Falcon tube,
followed by digestion for 30 minutes at 37 °C with 20 mg
collagenase type 1A (Sigma, St. Louis, MO, EUA), 200 mg of
bovine serum albumin (BSA), 20 ml of Dulbecco's Modified
Eagle's Medium Low Glucose (DMEM-LG) and 10 μl of
gentamicin. After tissue digestion, 10 ml of fetal bovine serum
(FBS) was added to neutralize the enzymatic activity, and the
cells were centrifuged at 1500 rpm for 15 minutes, resuspended
in 10 ml DMEM with 10% FBS, seeded into culture plate and
incubated at 37 °C with 5% atmospheric CO2. After 24 hours,
the medium was changed every three days until the cells
reached 70% of confluence. After four passages, the Adipose
tissue-derived stem cells (ADSCs) (1x103 cells/ml) were
characterized and seeded into a 96-well plate and incubated
with DMEM-LG containing 10% FBS at 37 °C for 24 hours.
2.4 MTT test for cytotoxicity evaluation
The PLLA samples (2mm x 5 mm) were incubated with the
ADSCs for 24 hours. DMEM-LG containing 0.5% phenol was
used as the positive control for toxicity (CT+), whereas
DMEM-LG containing 10% FBS was used as the negative
control for toxicity (CT-). The cells were cultured for 24, 48
and 72 hours.
The modified MOSMANN method [21] was chosen to perform
the MTT assay. After the chosen incubation time, the scaffolds
were removed, and the wells were washed with 200 µl of PBS
and 200 µl of DMEM-LG. Next, 200 µl of thiazolyl blue
tetrazolium bromide solution (MTT, Sigma) in DMEM-LG
(0,5 mg/ml) was added, and the plate was incubated in the dark
for 4 hours at 37 °C. The MTT solution was withdrawn, and
200 µl of dimethyl sulphoxide (DMSO) was added to
determine the absorbance values, at an absorption curve of λ=
595 nm (FilterMax F5 Multi-Mode Microplate reader,
Molecular Probes).
The absorbance values of the results were expressed as optical
density (OD) as the mean ± standard deviation. The comparison
between the values was made with the method Least
Significance Difference (LSD) test of Fisher and parametric
data analysis One-way ANOVA. Analysis with p <0.05 were
considered significant. Analyzes were performed using
StatView software (SAS Institute Inc., Cary, NC, USA).
2.5 Viability by Live/Dead®
A Live/Dead fluorescence assay kit (Molecular Probes) was
used to qualify the ADSC viability. In a qualitative test for
biocompatibility Live Dead®. The cells were seeded (3 x 106
cells/ml) into a 96-well plate and incubated with DMEM-LG
containing 10% FBS at 37 °C for 24 h. After this period, the
PLLA scaffolds (2mm x 5 mm) were incubated with the cells
for 24 h. A Live/Dead fluorescence assay kit (Molecular
Probes) was used to qualify the cells viability. After 24 h, the
cells were washed with 200 µl of PBS and treated with a
solution of Calcein AM and Ethidium homodimer-1 according
to the manufacturer’s instructions. The cells were incubated at
37 °C for 30 min and then washed and maintained in PBS. The
216 M.V. Xavier et al. / Procedia CIRP 49 ( 2016 ) 213 – 221
cells were observed by inverted fluorescence microscopy
(Nikon E800) with a specific program (Image Pro-Plus
software).
2.6 Electrospinning process
For the production of the PLLA nanofibers, the polymer was
dissolved with acetone and chloroform. The solution was then
loaded in a 10ml syringe, connected to a polyamide cylinder,
attached to a 0,7mm hypodermic needle as a nozzle. The flow
rate of the jet (8ml/h) was managed using a syringe pump. To
charge the solution, a 15kV high voltage power-source was
used. The distance between the needle and the collector plate
was of 17cm. Meaning to enhance the experimental settings, a
default electrospinning setup was used as shown in Figure 2.
Figure 2. Diagram of the Electrospinning apparatus 1. Metallic Target;
2.Needle; 3.Nanowire; 6.Nanofiber mat; 7, Syringe Infuser;
8.Polymer; 9.High Voltage Source.
2.7 PLLA nanofiber morphology
To analyze the nanofiber morphology, images of the PLLA
nanofibers were documented in the Scanning Electron
Microscope (SEM) (LEO Electron Microscopy 440i). The
nanofiber mat was sputter coating with gold, and then analyzed
by SEM. The images were then processed with an image
software (Image Pro Plus; Media Cybernetics Inc., USA).
3. Results and Discussion
Poly-lactic acid synthesis
The PLLA was synthesized by opening of the cyclic dimer of
L-lactide in order to obtain high molecular weight polymer.
The synthesis temperature was maintained at 140 °C thus
avoiding, high temperatures, which lead to a depolymerization
process which allows the decrease of the molecular weight of
the polymer [24,25]. The obtained polymer had the PLLA
molecular weight 86.93 g/mol.
Fourier Transform Infrared Spectroscopy (FTIR)
FTIR analyses were made to determine the functional groups
of the products obtained in order to understand more deeply
what happens in the polymerization of Poly (lactic acid).
The standard PLLA and the synthesized by our group, were
structurally analyzed by spectroscopy in the infrared (FTIR).
The infrared analyzes were performed to determine the
functional groups of the product obtained, compared with the
standard and verified the formation of the polymer. The
spectrum of synthesized PLLA and standard PLLA is presented
in Figure 3.
Fig. 3. FTIR spectrum of PLLA synthesized compared with PLLA
standard.
In Figure 3 for the standard commercial PLLA, vibrations were
observed symmetrical and asymmetrical valence of 1130.42
and 1044.92 cm-1, related to C-O the grouping COO; stretching
of the C-H at 2999.47 and 2948.81 cm-1; COO stretching to 872
cm-1; valence vibration of C = O of COO at 1754 cm-1 and CH
bending vibrations at 1387.86 and 1452 cm-1.
For PLLA obtained in the synthesis, were observed symmetric
and asymmetric vibration valence 1130.53 and 1040.14 cm-1,
related to C-O the grouping COO; stretching of the C-H bond
at 2989.63 and 2946.87 cm-1, COO stretching to 870.76 cm-1,
valence vibrations of C=O of COO at 1750 cm-1 and C-H
bending vibrations at 1374.56 and 1345.14 cm-1.
The results demonstrated the similarity of the peaks relating to
the absorption bands of each sample. Which shows the
formation of the synthetized polymer PLLA through the
studied pathway. The bands of functional groups are the same
as those obtained in the standard sample and also those found
in the literature shown by Nikolic [26], Motta and Duek [27].
And Lasprilla [28], confirming the formation of the polymer.
Cytotoxicity by MTT
Cytotoxicity test was performed to study the polymer
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M.V. Xavier et al. / Procedia CIRP 49 ( 2016 ) 213 – 221
biocompatibility. During the last few years, the interest of in
vitro tests has increased, as an alternative to animal
experimentation. The cytotoxicity is the harmful in vitro effect
induced in the cell culture system by the presence of a certain
substance or material, such as a biomaterial for instance. In this
study we used the MTT assay (a direct and suitable assay for
the quantitative in vitro biocompatibility evaluation), on which
the metabolic activity and the rate of cell growth have indicated
the degree of PLLA cytotoxicity in the cell culture.
MTT [29] is a yellow salt which is reduced by mitochondrial
dehydrogenase activity of the enzyme resulting in a formazan
salt purple. This reduction occurs only in living cells. Thus, cell
viability can be determined by the intensity of purple color,
which is proportional to the amount of formazan crystals
formed.
After performing the MTT test, the absorbance values that were
obtained, generated the curves below (Figure 4). The curves
show the cytotoxicity of mesenchymal cells in contact with the
PLLA after 24, 48 and 72 hours.
Figure 4. Kinetic curves of cell proliferation as measured by optical
density (OD) from the MTT assay for mesenchymal cells cultured
with PLLA, negative control for toxicity (CT-) and positive control
for toxicity (CT+) for 24 hours, 48 hours e 72 hours.
According to the ANOVA test, there are no statistically
significant differences between the PLLA and the (CT-) after
24 hours and 48 hours of culture (p > 0.05). However after 72
hours, the cells cultured with PLLA showed higher
proliferation when compared to (CT-), considered statistically
significant (p < 0.05).
These results show that the synthetized PLLA doesn’t
negatively affect the mesenchymal cell viability in the
evaluated periods. This indicates that the material does not
present cytotoxic behavior after 24, 48 and 72 hours, in
accordance with the MTT studies of Sarasua (2011) [30] and
WU (2014) [31]. In the MTT tests made by Niu (2015) [32] and
Liu (2014) [33], they evaluated polymers that showed no in
vitro cytotoxicity as well, demonstrating correlation with our
results.
Viability by LIVE/DEAD®
The Live/Dead® assay shows in a qualitatively matter the
polymer biocompatibility when in contact with the cells. The
cells were cultured with the biomaterial in three different times:
24, 48 and 72 hours. There was also the presence of positive
control for toxicity (CT+) and negative controls of toxicity
(CT-). The live cells reacted with the fluorescent marker
SYTO® 9, staining the viable cells with green. On the other
hand, un-viable cells were stained red, showing cell death.
Figure 5 shows the results obtained by the Live/Dead® method
by inverted fluorescence microscopy.
Figure 5. Images show cell viability on Live/Dead® test after 24
hours, 48hours and 72hours. A- Shows the negative control for
toxicity (CT-), live cells in green fluorescence. B- PLLA, the cells
present green fluorescence as negative control (CT-). C- Positive
Control for toxicity (CT+), dead cells shown in red fluorescence.
Figures 5A1, 5A2 and 5A3 show the negative control for
toxicity (CT-), on different times, where the viable cells with
intact cellular membrane, were stained fluorescent green. On
Figures 5B1, 5B2 and 5B3 show the cells in contact with PLLA
for 24 hours, 48 hours and 72 hours respectively. The images
of the cells in contact with PLLA appeared normal morphology
on all times, just like the ones of (CT-) (Fig 5A and 5B). On the
other hand, Figures 5C1, 5C2 and 5C3 show the positive
control for toxicity (CT+), where it was stained red on all times,
showing dead cells, with presence of debris and cell fragments.
The results obtained in this assay shows that the cells in contact
with new PLLA demonstrated the same green fluorescence that
the Negative Control (CT-), which reassure that the
mesenchymal cells are alive and proliferating.
The work of Bernstein (2012) [34], tested PLLA with different
formats, such as screws and pins, and through his
LIVE/DEAD® test, no cytotoxicity was detected, which
corresponded with our results.
Our findings with the quantitative cytotoxicity assay MTT and
qualitative cytotoxicity test LIVE/DEAD® showed that the
mesenchymal cells in contact with the synthetized PLLA
proliferated just as the ones of the negative control, keeping the
normal morphological characteristics of the cell. That
218 M.V. Xavier et al. / Procedia CIRP 49 ( 2016 ) 213 – 221
demonstrates that our synthetized PLLA is a biocompatible
material.
PLLA Electrospinning
The electrospinning process was performed after the PLLA
synthesis and the following realization of the cytotoxicity tests,
and evaluation of it biocompatibility. PLLA nanofibers were
produced, forming a mat as shown in Figure 6.
Figure 6. SEM image of the PLLA nanofibers mat. 1000x.
The nanofiber mat contains fibers that are extremely thin, and
yet that keep their morphological structure. Those have a
diameter of less than 1µm, as shown in Figure 7. To repair or
restore the function of damaged or diseased tissue it is
necessaire many complex methodes , employing a mix of
knowledge and techniques of engineering, chemistry and cell
biology, the use of PLLA and nanofibers to create a more
efficient three-dimensional structure to assist it, is a well
accepted method in the scientific community..
The nanofiber scaffold has a high surface area per unit volume,
as well an extremely interconnected pore network and fiber
diameters that mimic the extracellular matrix environment.
Figure 7. SEM image of the PLLA nanofiber mat. 10.000x.
With intention to show that the electrospinning process to
produce the nanofibers wouldn´t be prejudicial to cell growth,
the calorimetric assay of MTT [21] was performed with the
PLLA nanofibers. The cytotoxicity of the nanofibers are shown
in Figure 8.
Figure 8. Kinetic curves of cell proliferation as measured by optical
density (OD) from the MTT PLLA Nanofibers assay, negative control
for toxicity (CT-) and positive control for toxicity (CT+) for 24 hours
and 48 hours.
The graphics show the cytotoxicity of the mesenchymal cells
in contact with the PLLA nanofibers after 24 hours and 48
hours. According to the ANOVA test, there are no statistically
significant differences between the PLLA and the negative
control of cytotoxicity (CT-) after 24 hours and 48 hours of
culture (p > 0.05). Though, the cells cultured with the PLLA
nanofibers showed higher proliferation when compared to the
positive control of cytotoxicity (CT+), considered statistically
significant (p < 0.05). Thus, the data shows that the
electrospinning process wasn´t harmful to the cells.
The nanofibers are known for mimic the natural extracellular
matrix conditions, and because of this characteristic, they help
promote a higher cell adhesion, migration and proliferation.
This leads to a higher cell growth and so tissue regeneration.
Thus, making it an extremely interesting device to be used in
tissue engineering. Considering the presented studies, it is
possible to use the knowledge on the following tissue
engineering (TE) tests with this material for clinical
application, it is essential to fabricate autologous TE skin
substitutes, with sufficient mechanical strength for handling
and suturing during surgical implantation and effective
functionality for facilitating wound closure [35]. Composite
scaffolds can also be created using electrospinning. For
example, by sequentially spinning different polymer solutions,
a scaffold with layers can be constructed. Each layer can be
tailored for specific cell adhesion and could be potentially
beneficial for zonal articular cartilage or arterial vessel repair
[36].
There are many applications of the electrospinning technology
in the biomedical field. The reasons are quite evident such as
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M.V. Xavier et al. / Procedia CIRP 49 ( 2016 ) 213 – 221
the simplicity of the procedure in generating the large surface
area- to- volume ratio of the material and the mechanical
stability of the fibres that allows for its use in the biomedical
field [37]. The improvement of PLLA production and
electrospinning process generate new TE possibilities to be
tested.
4. Conclusion
This study has proved that the ring-opening polymerization is
a viable process for the production of PLLA derivation from
the lactic acid in these studied conditions. It also shows that this
PLLA is a non-toxic polymer to the cell, hence being then
considered biocompatible. Thus, PLLA can be employed to the
manufacture of nanofibers by the electrospinning process,
which can be used for different purposes in the medical field.
Acknowledgments
The authors wish to acknowledge the financial support
provided by the Scientific Research Foundation for the State of
São Paulo (FAPESP Process 2008/57860-3) and National
Council for Scientific and Technological Development (CNPq
Process 573661/2008-1)..
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