Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy
Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy
Ernesto Doncel-Pérez 0 1 2 4
Gary Ellis 0 1 2 4
Christophe Sandt 0 1 2 4
Peter S. Shuttleworth 0 1 2 4
Agatha Bastida 0 1 2 4
Julia Revuelta 0 1 2 4
Eduardo García-Junceda 0 1 2 4
Alfonso Fernández-Mayoralas 0 1 2 4
Leoncio Garrido 0 1 2 4
0 Synchrotron SOLEIL, L'Orme des Merisiers , Saint Aubin BP 48, 91192 Gif-sur-Yvette , France
1 Instituto de Ciencia y Tecnología de Polímeros, Consejo Superior de Investigaciones Científicas (ICTP-CSIC) , Juan de la Cierva 3, 28006 Madrid , Spain
2 Grupo de Química Neuro-Regenerativa, Hospital Nacional de Parapléjicos, Servicio de Salud de Castilla La Mancha (SESCAM) , 45071 Toledo , Spain
3 Leoncio Garrido
4 Instituto de Química Orgánica General, Consejo Superior de Investigaciones Científicas (IQOG-CSIC) , Juan de la Cierva 3, 28006 Madrid , Spain
Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I-II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds.
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); Electrospinning; Neural progenitor cells; FTIR spectroscopy
Ernesto Doncel-Pérez, Gary Ellis, and Leoncio Garrido contributed
equally to this work.
The intrinsic characteristics of the central nervous system
(CNS) are a major impediment to its spontaneous recovery
in response to injury and, as a consequence, lesions cause
permanent functional deficits that depend on their location
and extent. In fact, complete functional repair of a spinal cord
injury (SCI) cannot generally be achieved without precise and
significant aid [
]. Thus, SCI is a global problem that not only
affects the physical and psychological well-being of patients
and their families, but also places an enormous burden on the
economic resources of developed countries and increases the
mortality rates in developing nations [
]. To give an example,
the incidence of traumatic SCI in Western Europe was recently
reported to be between 218 and 316 cases (around half of
which are due to traffic accidents) per million habitants,
whereas for North America (the US and Canada), the mean
is over three times that [
]. In Canada alone, the annual cost of
SCI in 2012 was reported to be over 2 billion €, at least 32% of
which was ascribed to attendant care. In 2017, the National
Spinal Cord Injury Statistical Center in the USA estimated
that, depending on the severity of the SCI, the average lifetime
cost of treatment and care for a person who suffers a SCI at the
age of 25 was 1.4–4.3 M€ [
]. Hence, there is a great deal of
interest in improving this situation.
The multifactorial nature of this type of injury severely
limits therapeutic options, and the regeneration of injured
axons demands a timely, structured strategy that is able to
present multiple signals in a favorable environment [
Among the diverse options that are being explored to repair
neural tissue are a variety of tissue engineering approaches
that are designed to facilitate the regeneration of axons using
anisotropic scaffolds with biochemical cues [
include fibers of natural and synthetic materials that mimic the
spinal cord extracellular matrix and could be combined with
growth factors and different types of exogenous uncommitted
cells  or homing factors to stimulate endogenous stem cell
migration towards the injury site [
The development of fibrous substrates resembling the
extracellular matrix to support cell adhesion, differentiation, and
proliferation has drawn great interest from researchers
involved in tissue engineering [
] since the renaissance of
], a technique that allows the preparation
of nonwoven fibers with diameters ranging from tens of
nanometers to a few microns. While the tissue engineering of
electrospun scaffolds has shown some limitations (i.e., it is
difficult to fabricate complex three-dimensional structures
and cell infiltration is poor due to small pore sizes) [
substrates offer the possibility of exploring cell behavior on
exposure to a variety of chemical and physical cues in a
reproducible manner, and they facilitate the translation of new
and innovative strategies to in vivo preclinical research.
Scaffolds based on PHB [poly(3-hydroxybutyrate)] and its
copolymers with other β-hydroxy acids have been employed
in experimental models of CNS lesions [
scaffolds offer several advantages in relation to their intended
applications: they possess a reproducible and well-defined
polymer microstructure, amenable to processing by a wide
variety of methods, and they are both biodegradable and
biocompatible. However, as is often the case for many synthetic
polymers, the intrinsic surface properties of these materials do
not facilitate interaction between the scaffold and the cells.
Thus, the functionality of the scaffolds must be modified by
the physical adsorption or covalent binding of specific
chemical moieties and macromolecular fragments to its surface.
An interest in developing effective strategies to
experimentally promote the repair of neural tracts in the CNS led us to prepare
functional and highly anisotropic scaffolds of
poly(3hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] via
electrospinning. Since the physical and chemical characteristics
of polymer scaffolds play an important role in the modeling of
cellular behavior during growth and proliferation [
response of neural precursor cells (NPCs) to stimuli induced by
P(HB-co-HHx) scaffolds must be characterized in order to guide
and optimize approaches to SCI functional repair. In particular,
studying the biochemical profile of cells and their local
environment using vibrational microspectroscopy could highlight
distinctive spectral markers associated with a specific cell response.
Fourier-transform infrared (FTIR) spectroscopy and
(particularly) high-resolution synchrotron-based infrared
microspectroscopy (SIRMS) have been successfully
employed to characterize many biological tissues [
and to explore several aspects of cell biology, such as cell
], changes in cell physiological state
], and the effects of exogenous agents on cell
biochemical profiles [
]. The brightness of the synchrotron IR
source provides diffraction-limited spatial resolution,
allowing high-quality spectra to be obtained through small
apertures that can be closely matched to the size of the cells
or features of interest in the sample. The study described in the
present paper applied SIRMS to search for spectral markers
that allow the roles of the scaffold morphology and surface
modification in the tuning and control of neural progenitor cell
response during cell differentiation and subsequent
proliferation to be evaluated. Spectra were obtained from NPCs
cultured for up to 48 h on electrospun P(HB-co-HHx) scaffolds
impregnated with poly-L-lysine (PLL) and laminin (L).
PolyL-lysine is a polypeptide commonly used in CNS-originated
cell cultures to facilitate cell adhesion to culture plates, and
laminin is a protein present in the extracellular matrix of the
CNS. Samples obtained at three time points were analyzed to
assess the evolution of the cell profile. We monitored
variations in the extracellular matrix composition using IR
spectroscopic biomarkers, particularly those associated with lipid and
protein contents, during the adhesion, differentiation, and
proliferation of NPCs on the scaffolds [
]. The spectroscopic
findings were correlated with the results of immunochemical
studies of cell morphology and membrane markers of
differentiating and/or differentiated cells in culture.
Materials and methods
[P(HB-coH H x ) ] w i t h a m o l a r c o m p o s i t i o n o f 1 4 . 3 % i n 3
hydroxyhexanoate, molecular weight Mw = 1.9 × 105 g mol−1
and polydispersity index Mw/Mn = 1.47, was supplied by
Professor Guo-Qiang Chen (Tsinghua University, China).
Prior to use, the copolymer was washed with ethanol under
constant stirring overnight, filtered, and vacuum dried to a
constant weight. Dichloromethane supplied by Carlo Erba
Réactifs-SdS (Val de Reuil, France) and methanol, ethanol,
and 1,1,1,3,3,3-hexafluoro-2-propanol purchased from
Sigma–Aldrich (Steinheim, Germany) were used as supplied.
Neurobasal medium and B27 supplement were purchased
from GIBCO (Paisley, UK); human bFGF and EGF were from
Peprotech (Rocky Hill, NJ, USA); L-glutamine,
L-glutamate, penicillin, streptomycin, bisBenzimide H 33258,
and a fragment of laminin containing the IKVAV
sequence were from Sigma–Aldrich; and fungizone was
from Invitrogen (Madrid, Spain). Rabbit polyclonal
antiGFAP was provided by Acris (Herford, Germany) and
monoclonal antibody for nestin was from Santa Cruz
Biotechnology (Dallas, TX, USA); antibody goat
antirabbit IgG conjugated to Alexa Fluor 488 and antibody
goat anti-rabbit IgG conjugated to Alexa Fluor 594 were
provided by Molecular Probes (Eugene, OR, USA).
Preparation of P(HB-co-HHx) fiber scaffolds
The fiber scaffolds were prepared by electrospinning
P(HBco-HHx) solutions in a mixture of solvents. Briefly, 1 g of
copolymer was dissolved in 3.5 mL of a mixture of
dichloromethane (3.3 mL) and 1,1,1,3,3,3-hexafluoro-2-propanol
(0.2 mL) at room temperature for 12 h with constant stirring.
The copolymer solutions were immediately electrospun in
a home-built apparatus consisting of a high-voltage power
supply (30 kV 600 W, SL Series, Spellman, Hauppauge,
NY, USA), a blunt tip needle (0.584 mm i.d.) connected to
the positive pole of the power supply, and a grounded 7 cm
diameter rotatable drum as collector. In addition, the flow
(0.2 mL/h) of the solution through the needle and into the
electric field was controlled with an infusion pump (100
Series, KD Scientific, Holliston, MA, USA). Usually, the
distance between the needle tip and the rotating drum was 16 cm,
the rotation speed was ~1200 rpm, and the applied voltage
was 11.5 kV. The temperature in the chamber during
electrospinning was maintained at around 22 °C and the
relative humidity varied between 20 and 24%. Fibers were
collected for 3.5 h on aluminum foil affixed to the rotating drum
to obtain mechanically stable scaffolds with a mean thickness
of approximately 35 μm. After vacuum drying them
overnight, the deposited fibers were cut into disks (∅ = 14 mm)
and stored in a dry environment at 4 °C until used.
The experiments followed the European Parliament and
Council Directive (2013/63/EU) and the Spanish regulation
(RD 53/2013) on the protection of animals for experimental
use. This study was approved by our institutional animal
use and care committee for animal welfare (Hospital
Nacional de Parapléjicos, registered as SAPA001). Wistar
rat embryos (E15) were obtained from previously
anesthetized pregnant rats by cesarean section. The rats were bred
and maintained at the animal house of the Hospital
Nacional de Parapléjicos in Toledo.
Striata from E15 rats were dissected and mechanically
dissociated into individual cells [
]. This cell suspension was
incubated in neurobasal medium and B27 supplement
containing human bFGF (10 ng/mL) and EGF (20 ng/mL) as well as
L-glutamine (0.5 mM), L-glutamate (25 mM), penicillin
(100 U/mL), streptomycin (0.1 mg/mL), and fungizone (2.5
lg/mL). After about 7 days in this BNB27^ medium, floating
neurospheres were formed and collected by low-speed
centrifugation, and were washed free of glutamate by suspension in
PBS and further centrifugation. Neurospheres were
dissociated by mild trypsinization, passed through a 25-gauge needle,
and expanded every 3–4 days.
Neural precursor cell differentiation
After four expansion passages, NPCs grown as neurospheres
for cell expansion were suspended in a 1:1 mixture of NB27
and DMEM incorporating 10% bovine serum. The
neurosphere suspension was plated onto a 24-well cell culture
plate that contained 14 mm ∅ disks of P(HB-co-HHx) fiber
substrates treated with 50 μg/mL poly-L-lysine and/or 20 μg/
mL of a laminin fragment containing the IKVAV sequence.
ZnS IR windows coated with laminin were used as a control.
The cells were incubated for 3 h to allow cell attachment and
24 and 48 h for neurosphere differentiation. After treatment,
the cells were fixed for immunocytochemistry and
characterized by SR-FTIR microspectroscopy.
The treated cells on PLL- and/or laminin-coated substrates
were fixed in 2% paraformaldehyde/sucrose in PBS
(12 min, 25 °C), washed with PBS, and immunostained
as follows. They were incubated for 30 min, at 25 °C, in
PBS containing 1% normal goat serum with 0.1% Triton
X100. The cells were then incubated (16 h at 4 °C) in the
same mixture containing the primary antibody. After
repeated washing with PBS, the cells on the substrates were
treated with the secondary antibody (45 min, 25 °C) H
33258, 10 g/mL for 10 min at 25 °C, washed three times
with PBS, mounted on a new 24-well plate with glycerol/
PBS (1/1), and examined on a fluorescence microscope
(DMI 6000B, Leica, Wetzlar, Germany). Mouse
monoclonal anti-nestin (2Q178, Santa Cruz Biotechnology) and
rabbit polyclonal anti-GFAP at 1/500 dilution were used
for immunocytochemistry and revealed by secondary
antibodies: goat anti-rabbit IgG conjugated to Alexa Fluor 594
or goat anti-mouse IgG conjugated to Alexa Fluor 488,
respectively, at a dilution of 1/1000.
Scanning electron microscopy
The polymer scaffolds were analyzed prior to cell culture
using scanning electron microscopy (SEM). Briefly, the
scaffolds were coated with approx. 5 nm Au/Pd and observed on a
Philips (Eindhoven, Netherlands) XL30 scanning electron
microscope at ambient temperature using the parameters
indicated in each micrograph.
Preparation of imprints for FTIR microspectroscopy measurements
To acquire IR spectra of cell cultures on polymer fibers,
imprints of the substrates with cells were prepared [
stored fixed samples were moistened with two drops of
distilled water, placed between two 1-mm-thick ZnS IR
windows, and lightly compressed for 30 min to allow the transfer
of cellular material from the polymer scaffold onto the
window in contact with the cell-seeded surface. After this time,
the press was carefully opened and the window with the
imprint was exposed to air and dried overnight at room
temperature (see Fig. S1 in the BElectronic supplementary material,^
ESM, for the methodology).
Cell cultures on ZnS windows were observed as prepared;
no imprints were made.
ZnS windows were chosen for imprinting purposes due
to their wide transparency domain in the mid-infrared and
their relatively good mechanical properties, and because
they are biocompatible, allowing cell growth (they show
a cell viability of >90%, and cell morphologies are
similar to those obtained in standard polystyrene culture
Synchrotron radiation FTIR microspectroscopy (SIRMS)
Spectra of imprints and cell clusters were recorded at the
biological endstation of the SMIS beamline at Synchrotron
SOLEIL, employing both bending and edge radiation from a
bending magnet at a constant current (top-up mode) of around
400 mA. The spectra were obtained on a Continuum XL
(Nicolet (Thermo Scientific), Waltham, MA, USA)
microscope equipped with a liquid-nitrogen-cooled MCT/A
detector, a 32×/NA0.65 Schwarzschild objective, a motorized
knife-edge aperture, and an xyz motorized stage, which was
coupled to a Nicolet 5700 FTIR spectrometer (Thermo Fisher
Scientific, Villebon-sur-Yvette, France). The microscope was
operated in dual aperture mode with a 15 × 15 μm2 spatial
aperture. A spectral resolution of 4 cm−1 was achieved and
128 scans were accumulated at each data point to obtain a high
signal to noise ratio.
The acquired spectra were first visualized using OMNIC 9.2
(Thermo Scientific, Madison, WI, USA) in order to predefine
the spectral regions of interest and to eliminate spectra
exhibiting strong Mie scattering effects.
The spectra of cells and imprints were analyzed by
multivariate pattern recognition techniques using The Unscrambler
X, version 10.3 (Camo Software AS, Oslo, Norway). Two
spectral regions, 3050–2800 cm−1 and 1800–1400 cm−1,
corresponding mainly to lipid and amide I–II bands, respectively,
were selected. These regions were preprocessed before
analysis [third-degree polynomial, 7/7 point smoothing prior to
calculation of the second derivative (third-degree polynomial,
7/7 points) and unit vector normalization]. Principal
component analysis (PCA) was carried out using preprocessed,
mean-centered data on the two spectral regions combined, as
well as on the amide I–II bands. Four to seven principal
components (PCs) were calculated using the singular-value
decomposition (SVD) algorithm and leverage correction. 2D
score plots and 1D loading plots were examined, respectively,
to identify any spectral clustering and to obtain information on
the spectral bands leading to those clusters. Unsupervised
cluster analysis was performed by considering the k-means
and Euclidean distance.
Neural precursor cell differentiation: immunostaining
Scaffolds of P(HB-co-HHx) with highly aligned fibers were
prepared via electrospinning as shown in Fig. S2 (see the
ESM). After coating with either laminin (L) or
poly-L-lysine-laminin (PLL/L), these scaffolds were used as cell
supports in cultures of E15 rat neurospheres that were isolated and
cultured for up to 48 h, as described in the BMethods^ section.
In addition to the cultures on P(HB-co-HHx) fibers, zinc
sulfide (ZnS) IR windows coated with laminin were used as a
positive control for NPC differentiation. The cells were
incubated in the presence of serum to favor cell adhesion and cell
differentiation, and were fixed for immunocytochemistry after
the specified time had elapsed. The results are illustrated in
Fig. 1. At 3 h, aggregated NPCs that were effectively attached
to substrates were observed, with a low expression of GFAP
(green signal) and a high expression of nestin (red signal; see
Fig. 1a, d, g). Nestin is an intermediate filament protein that is
widely considered a biomarker of neural progenitor cells [
Therefore, during the initial stages of NPC culture on the
polymer substrates, high expression is anticipated, but this
expression is expected to reduce as the culture and cell
differentiation progresses. At 24 h, the NPCs had migrated and
colonized the substrates as individual cells, but higher
Fig. 1a–i Differential nestin and
GFAP expression of neural
precursor cells on P(HB-co-HHx)
substrates at specified times. The
subfigures show the results of
immunostaining with antibodies
for nestin (red) and GFAP (green)
and DNA fluorescence images of
cell nuclei (blue). The highest
number of GFAP+ cells was
observed at 48 h in fibers coated
with PLL/laminin (f), and the
lowest number in laminin-coated
fibers (i) at 48 h. Scale bars =
expression of nestin than GFAP was still observed (Fig. 1b, e,
h). The highest number of GFAP+ cells was obtained for
substrates coated with PLL/laminin, contrasting with the low
GFAP signal seen for laminin-coated substrates, as
demonstrated for the P(HB-co-HHx) substrates by Fig. 1f and i,
respectively (taken at 48 h). The intracellular expression of
nestin and GFAP intermediate filaments detected with the
specified antibodies revealed the presence of cells with
precursor and astroglial phenotypes, respectively. The cell
populations had blue-stained nuclei.
The measured sample sets consisted of laminin-coated ZnS
IR windows and P(HB-co-HHx) fibers coated with only
laminin (L) or with both poly-L-lysine and laminin (PLL/
L) for culture times of 3, 24, and 48 h. Initial attempts to
measure the cells grown on the fiber scaffolds using ATR
microspectroscopy through a ZnSe hemisphere [
were hampered by two fundamental problems: significant
distortion in the IR spectral background was observed due
to the Mie scattering effect generated by the cells due to the
irregular topography of the fiber substrates, and the
overwhelmingly strong IR absorption bands associated with the
polymer fibers. To overcome these issues, imprints of the
cells grown on the polymer scaffolds were made to transfer
the cells from the polymer scaffolds to ZnS infrared
transparent substrates, as described in the BMethods^ section
(and in Fig. S1 of the ESM), and these were subsequently
measured by transmission FTIR microspectroscopy. A
similar approach has been successfully employed by Das
et al.  to study fresh tissue using FTIR. Figure 2 shows
a characteristic imprint corresponding to a sample of NPCs
on P(HB-co-HHx) fibers coated with PLL/L, which was
obtained 24 h after seeding NPCs. The dark areas contain
the cellular material that was pressure-transferred from the
scaffold to the IR window. Although it is possible that the
material was incompletely transferred from the polymer
fibers to the ZnS windows, the variation in the outcome
of the spectroscopic measurements for imprints of a given
experimental setting was insignificant, or at least far
smaller than the variations caused by changing the culture
IR spectra were collected at specified locations by mapping
the imprinted area and, for the cells cultured directly on IR
windows for 3 and 24 h, at the positions where cell clusters
were clearly observed. Samples of cells on IR windows at 48 h
could not be measured due to insufficient material (the spectra
acquired showed poor signal-to-noise ratios), as the cells
became detached from the window during the processing
performed for the measurements, most likely because the high
proliferation of cells led to the formation of a detachable sheet
due to the relatively low contact surface area compared to the
fibers. For each sample, at least 30 spectra were acquired and
the entire data set for all samples included over 850 spectra.
Figure 3 presents the average spectra obtained for all substrate
types and at various culture times in the spectral interval
between 3750 and 850 cm−1. In order to compare the results
obtained for each group, their corresponding
secondderivative spectra were calculated after performing smoothing,
Fig. 2 Mosaic of bright-field
micrograph images (approx.
1.4 mm × 1.2 mm) corresponding
to NPCs on P(HB-co-HHx) fibers
coated with poly-L-lysine and
laminin, obtained after 24 h of
vector normalization, and PCA over the spectral range 3050–
2800 cm−1, where bands corresponding mainly to lipids are
observed, and the 1800–1400 cm−1 region, which mainly includes
protein and amide I–II bands. The use of the second-derivative
spectrum makes it easier to interpret the results since it has a flat
baseline, so the positions of the negative peaks observed can be
precisely correlated with the bands and shoulders observed in the
absorption spectrum. For small cells, the signal-to-noise ratio
advantage obtained with the synchrotron source makes this
chemometric approach feasible.
In an initial statistical analysis of the spectral data, the
culture time was the only experimental variable
considered, and a clear division of the spectra into three groups
was observed. In Figure 4a, the average second-derivative
spectra of NPCs 3, 24, and 48 h after seeding on the three
types of substrates are shown. It can be seen that the
intensities of the bands associated with the asymmetric and
symmetric stretching modes of mainly lipid methylene
moieties at 2922 and 2852 cm−1, respectively, initially
increase but then decrease, such that they have dropped
significantly at 48 h. The same pattern of change in relative
intensity is also observed for lipid ester carbonyl bands at
1741 cm−1 and lipid methylene deformation modes at
around 1468 cm−1 (Fig. 4b).
The most characteristic bands identified in the
secondderivative spectra of NPCs cultured on the P(HB-co-HHx)
Fig. 3 Average IR spectra (3700–
850 cm−1) from all data sets of
imprints and ZnS IR windows
with cells after different culture
(P(HB-co-HHx)/PLL/L and P(HB-co-HHx)/L) after seeding, illustrating
the differences between the spectra in the a lipid region (3050–
2800 cm−1) and b the amide I–II region (1800–1400 cm−1)
fibers and ZnS IR windows, as well as their assignments, are
summarized in Table 1.
A detailed review of the spectral profiles from cells on each
type of substrate (P(HB-co-HHx)/PLL/L, P(HB-co-HHx)/L,
and ZnS/L) at given culture times showed only minor
fluctuations in the lipid band intensities (data not shown).
When IR spectra were grouped according to culture time, in
the region between 1700 and 1600 cm−1 (amide I band, Fig. 4b),
a decrease in the intensity of the band attributed to α-helix
structure (1657 cm−1) was observed at 48 h [
]. A shift of more than
15 cm−1 in the band associated with β-sheet structure [
1638 to 1622 cm−1, and an increase in the band intensity at
1693 cm−1 were also observed; the latter is generally assigned
to β-turns and antiparallel β-sheet structures [
]. Also, these
changes appear to be correlated with those observed for the
amide II [
] peak at 1516 cm−1.
An assessment of the amide I band for cells on each type of
substrate at the studied culture times showed that the presence
of α-helix structures is favored on ZnS substrates, while
βsheet structures are more likely to be formed on
P(HB-coHHx)/L fibers (see Fig. S3a and b in the ESM). The FTIR cell
profiles on P(HB-co-HHx)/PLL/L initially exhibit an
intermediate behavior, whereas all cells grown on the polymer fibers
show similar spectra at 48 h (see Fig. S3c in the ESM).
Multivariate analysis was implemented to identify possible
trends in the changes observed in the entire spectral data set.
Principal component analysis (PCA) was performed on the
second-derivative spectra calculated from all of the spectra
acquired, considering the spectral regions 3050–2800 cm−1
and 1800–1400 cm−1 in the model. Two-dimensional score
plots of PC-1 vs. PC-2 were found to provide adequate
clustering of spectra from the cells as a function of cluster time
(see the upper part of Fig. 5). Unsupervised cluster analysis of
these data yielded classification percentages of 93.7, 97.2 and
91.1% of all samples at 3, 24, and 48 h of culture time,
respectively. A closer examination of the corresponding loading
plots of PC-1 (Fig. 5, bottom) revealed that the main bands
Characteristic FTIR bands observed from neural progenitor cells during adhesion and differentiation on P(HB-co-HHx) fibers and ZnS IR
Band max., 2nd derivative, cm−1
PC-1, 3 h vs 24 h, cm−1
PC-1, 24 h vs 48 h, cm−1
contributing to cluster formation were the methylene lipid
bands at 2922 and 2852 cm−1, the amide I bands at 1695
and 1628, and the amide II bands at 1545 and 1516 cm−1.
The outcome of PCA for pairwise comparisons of culture
times (3 h vs. 24 h and 24 h vs. 48 h) are illustrated in Fig.
S4 (see the ESM). As indicated previously, a reduction in lipid
band intensity with increasing culture time was observed.
Also, the main changes observed in the amide I–II spectral
region are those associated with the increase in band
intensities mentioned above and the decrease in the intensity of the
band at 1660 cm−1 during the initial stages of culture.
A PCA performed on the second-derivative spectra of cells
grouped by type of substrate yielded improved clustering of the
biochemical profiles as a function of culture time, particularly for
the substrates P(HB-co-HHx)/PLL/L and ZnS/L (see Fig. S5a and
c in the ESM). However, the P(HB-co-HHx)/L scaffolds showed a
broader overlap (see Fig. S5b in the ESM). It was not possible to
distinguish between substrate types and coatings at each culture
time by analyzing the spectral regions (lipid and amide I–II bands
combined). Since, as indicated previously, all of the substrates
showed similar lipid profiles at a given culture time, an analysis
that only took into account the spectral region between 1800 and
1400 cm−1 was performed. Figure 6 shows selected 2D PCA score
plots for the amide I–II spectral region of NPCs at culture times of
3, 24, and 48 h on the three types of substrates studied:
P(HB-coHHx)/PLL/L (PC-1 vs PC-4), P(HB-co-HHx)/L (PC-2 vs PC-3),
and IR windows/L (PC-2 vs PC-5). Although a significant overlap
between the groups is apparent, particularly at 3 h, there is a
noticeable tendency to segregate according to the type of
substrate/coating. The loading plots in Fig. 6 show that these results
are mainly due to changes in the previously mentioned bands of
the NPC profiles: the amide I bands associated with β-turns
(~1690 cm−1), α-helices (~1660 cm−1), and β-sheets (1638–
1622 cm−1) of proteins, as well as the amide II band at
1512 cm−1, which is closely related to β-turns [
The FTIR spectrum of NPCs on P(HB-co-HHx) fibers
showed a marked decrease in lipid band intensity at the
longest culture time studied, 48 h. This observation is consistent
with previous observations which showed a reduction in lipid
content that was associated with a loss of stem cell pluri- or
]. However, there is still no clear consensus
about this, as the opposite behavior—an increase in lipid
production with cell differentiation—has also been reported [
Several outcomes may be anticipated, depending on the
differentiation path of the NPCs. NPC differentiation to an
oligodendrocyte-like cell could increase the intensity of the
lipid bands due to an increase in myelin. On the other hand, if
NPCs differentiate to neuron or astrocyte-like cells, a
reduction in the lipid signal intensity might be observed. In our case,
previous results have shown that on standard culture plates
with the culture medium used in this study, neurospheres
differentiate to an intermediate stage, maintaining their
differentiation potential to some extent [
The changes observed in the amide I band, corresponding
to an increase in the amount of β-sheets and β-turns present,
support the above statement. The immunochemistry results
show that the GFAP signal increased progressively with
increasing culture time, particularly on ZnS/L IR windows and
P(HB-co-HHx)/PLL/L fibers. The presence of the protein
nestin, a biomarker for NPCs, was more prominent during
the initial stages of cell culture.
An increase in the heterogeneity of the biochemical profiles
of cells with increasing culture time can be anticipated
circles), and ZnS/L (green triangles)]. The spectral region included in
the analysis was 1800–1400 cm−1
because, for the electrospun polyester fiber substrates, the
likelihood of finding cells at different stages of differentiation
would increase over time. This would lead to a broadening of
the spectral cluster determined by PCA. Although the
outcome of this report is influenced by the limited set of variables
studied and the duration of cell culture, it is worth pointing out
that the cells on fibers coated only with laminin exhibited the
greatest overlap between the three distinct populations
identified by FTIR microspectroscopy. On the other hand, the NPCs
on ZnS IR/L windows showed the narrowest distributions.
Thus, although the culture time is a major influence on the
cell IR biochemical profile, these observations suggest that the
type of substrate/coating present affects the cell differentiation
pathway, and could be adjusted to modulate the cellular
response appropriately. In this regard, given the cell profile
heterogeneity and the short exposure times to the biomaterials, the
observation that spectral clustering was influenced by the type
and coating of the cell scaffolds used is remarkable. These
findings are in agreement with results from other studies
showing that the surface chemistry and topography substantially
influence the outcome of NPC differentiation [
], and highlight
the potential of synchrotron FTIR microspectroscopy for
studying the interactions between cells and biomaterials.
Nevertheless, further experiments incorporating different
surface chemistries and extended culture times are required to
attain a more robust response.
Neural progenitor cells were cultured on electrospun
P(HB-co-HHx) fiber substrates coated with either laminin
or poly-L-lysine/laminin. At different times after cell
seeding, the samples were fixed and the cellular material
on the fibers was partly transferred onto IR windows
using a modified touch imprint cytology method. This
strategy proved to be very effective and enabled the
biochemical profiles of the NPCs that evolved on the coated
polymer fibers to be determined with SIRMS. The
intensities of bands in the lipid and amide I–II regions were
observed to be influenced by the substrate type and
coating and the culture time. These results demonstrate the
important insights that SIRMS studies can provide into
the responses of NPCs to biomaterials (in this case to
the morphology and surface chemistry of polymeric
scaffolds), suggesting that such studies could be a
fundamental tool in the preparation and optimization of cellular
scaffolds for CNS tissue engineering and regenerative
Acknowledgements Financial support was provided by the Ministerio de
Economía y Competitividad (MINECO), projects FIS/ISCIII PI11/01436
and PI11/00592, MAT2015-65184-C2-2-R, and the CSIC. The research
leading to these results was funded by the European Community’s
Seventh Framework Programme (FP7/2007-2013) under grant agreement
no. 312284 (CALIPSO). Synchrotron measurements were performed at
the SMIS beamline of Synchrotron SOLEIL. PS is grateful to MINECO
for Ramón y Cajal postdoctoral fellowships (RYC-2014-16759). We
would like to thank all the beamline staff for their helpful support and
Compliance with ethical standards
Conflicts of interest The authors declare that they have no competing
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Ernesto Doncel-Pérez is Head of
the Neuroregenerative Chemistry
Group at The National Hospital
f o r P a r a p l e g i c s i n To l e d o
(Spain). His main research
interest is the recovery of the central
nervous system after a lesion. He
devised a chemical approach that
includes new drug development
and the study of biocompatible
and biodegradable polymers for
use as vehicles for drug delivery
or as substrates for neural
precursor cells used in transplants.
Gary J. Ellis is a senior research
scientist at the Instituto de Ciencia
y Tecnología de Polímeros, CSIC,
Madrid (Spain), and Head of the
Polymer Physics Group (http://
nanoparticleenhanced polymeric materials for
use in diverse technological fields.
He cultivates a special interest in
the application of vibrational
spectroscopy (including SIRMS) to a
wide range of problems in
Christophe Sandt is a beamline
scientist at the SMIS infrared
microspectroscopy beamline at
the SOLEIL synchrotron facility.
He has been working for several
years on the applications of
vibrat i o n a l m i c r o s p e c t r o s c o p i e s
(Raman and infrared) to
biomedical and biological applications.
Peter S. Shuttleworth is a
Ramón y Cajal fellow at the
Instituto de Ciencia y Tecnología
de Polímeros, CSIC, Madrid
(Spain). He has been working for
several years on the development
of biobased polymers and
nanocomposites as well as bioderived
mesoporous carbons for
applications in water purification and
electrical double-layer capacitors.
Agatha Bastida is Head of the
B i o o r g a n i c C h e m i s t r y
Department at the Instituto de
Q u í m i c a O rgá n i c a G e ne r a l ,
CSIC, Madrid (Spain), where she
applies modern organic chemistry
methodologies to the design,
synthesis, and characterization of
Julia Revuelta is a tenured
scientist at the Instituto de Química
Orgánica General, CSIC, Madrid
(Spain). Her research interests are
the synthesis of new
glycosaminoglycans and their incorporation
into biomaterials for treating
spinal cord injury.
Eduardo García-Junceda is
senior research scientist at the
Instituto de Química Orgánica
General, CSIC, Madrid (Spain).
He has recently been working on
the chemoenzymatic synthesis of
glycopolymer analogs of heparan
and chondroitin sulfate for
application to the repair of lesions in
the central nervous system.
is a research professor at the
Instituto de Química Orgánica
General, CSIC, Madrid (Spain).
His main research interest is the
synthesis and biomedical
applications of carbohydrates, and his
current research activities are
focused on heparan and chondroitin
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