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Citation: Yoshikawa, O.; Basoli, V.;
Boschetto, F.; Rondinella, A.; Lanzutti,
A.; Zhu, W.; Greco, E.; Thieringer,
F.M.; Xu, H.; Marin, E. Simple
Electrospinning Method for
Biocompatible Polycaprolactone
β-Carotene Scaffolds: Advantages
and Limitations. Polymers 2024,16,
1371. https://doi.org/10.3390/
polym16101371
Academic Editor: Young-Sam Cho
Received: 15 April 2024
Revised: 3 May 2024
Accepted: 7 May 2024
Published: 11 May 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
polymers
Article
Simple Electrospinning Method for Biocompatible
Polycaprolactone β-Carotene Scaffolds: Advantages
and Limitations
Orion Yoshikawa 1, Valentina Basoli 2, Francesco Boschetto 3,4 , Alfredo Rondinella 5, Alex Lanzutti 5,
Wenliang Zhu
1
, Enrico Greco
6,7
, Florian Markus Thieringer
2,8
, Huaizhong Xu
9
and Elia Marin
5,10,11,12,
*
1Ceramic Physics Laboratory, Faculty of Materials Science and Engineering, Kyoto Institute of Technology,
Sakyo-ku, Matsugasaki, Kyoto 606-8585, Japan; m2672032@edu.kit.ac.jp (O.Y.); wlzhu@kit.ac.jp (W.Z.)
2Medical Additive Manufacturing Research Group (Swiss MAM), Department of Biomedical Engineering,
University of Basel, Hegenheimermattweg 167C, 4123 Allschwil, Switzerland;
valentina.basoli@unibas.ch (V.B.); f.thieringer@unibas.ch (F.M.T.)
3Center for Excellence in Hip, Scottish Rite for Children, Dallas, TX 75219, USA; boschetto.cesc@gmail.com
4Department of Orthopedic Surgery, UT Southwestern Medical Center, Dallas, TX 75390, USA
5Polytechnic Department of Engineering and Architecture, University of Udine, 33100 Udine, Italy;
alfredo.rondinella@uniud.it (A.R.); alex.lanzutti@uniud.it (A.L.)
6Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127 Trieste, Italy;
enrico.greco@units.it
7National Interuniversity Consortium of Materials Science and Technology (INSTM), Trieste Research Unity,
Via G. Giusti 9, 50121 Firenze, Italy
8Clinic of Oral and Cranio-Maxillofacial Surgery, University Hospital Basel, 4031 Basel, Switzerland
9Department of Biobased Materials Science, Kyoto Institute of Technology, Sakyo-ku, Matsugasaki,
Kyoto 606-8585, Japan; xhz2008@kit.ac.jp
10 Biomaterials Engineering Laboratory, Kyoto Institute of Technology, Sakyo-ku, Matsugasaki,
Kyoto 606-8585, Japan
11 Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Matsugasaki, Kyoto 606-8585, Japan
12 Materials Innovation Laboratory, Kyoto Institute of Technology, Sakyo-ku, Matsugasaki,
Kyoto 606-8585, Japan
*Correspondence: elia-marin@kit.ac.jp; Tel.: +81-757247554
Abstract: In this study, electrospun scaffolds were fabricated using polycaprolactone (PCL) loaded
with varying concentrations of
β
-carotene (1.2%, 2.4%, and 3.6%) via the electrospinning technique.
The electrospinning process involved the melting of PCL in acetic acid, followed by the incorporation
of
β
-carotene powder under constant stirring. Raman spectroscopy revealed a homogeneous distri-
bution of
β
-carotene within the PCL matrix. However, the
β
-carotene appeared in particulate form,
rather than being dissolved and blended with the PCL matrix, a result also confirmed by thermo-
gravimetric analysis. Additionally, X-ray diffraction analysis indicated a decrease in crystallinity with
increasing
β
-carotene concentration. Mechanical testing of the scaffolds demonstrated an increase
in ultimate strain, accompanied by a reduction in ultimate stress, indicating a potential plasticizing
effect. Moreover, antimicrobial assays revealed a marginal antibacterial effect against Escherichia coli
for scaffolds with higher
β
-carotene concentrations. Conversely, preliminary biological assessment
using KUSA-A1 mesenchymal cells indicated enhanced cellular proliferation in response to the scaf-
folds, suggesting the potential biocompatibility and cell-stimulating properties of
β
-carotene-loaded
PCL scaffolds. Overall, this study provides insights into the fabrication and characterization of
electrospun PCL scaffolds containing
β
-carotene, laying the groundwork for further exploration in
tissue engineering and regenerative medicine applications.
Keywords: electrospinning; polycaprolactone;
β
-carotene; scaffolds; tissue engineering; cellular
proliferation; antibacterial
Polymers 2024,16, 1371. https://doi.org/10.3390/polym16101371 https://www.mdpi.com/journal/polymers
Polymers 2024,16, 1371 2 of 20
1. Introduction
Tissue engineering holds tremendous promise in regenerative medicine by offering
innovative solutions for repairing and replacing damaged tissues and organs [
1
]. Central
for this field is the development of scaffolds [
2
], which serve as three-dimensional templates
to support cell adhesion [
3
], proliferation [
4
], and tissue regeneration [
5
]. Among various
scaffold fabrication techniques, electrospinning is notable for its versatility in producing
nanofibrous structures that mimic the extracellular matrix (ECM) of natural tissues [6].
Electrospinning, a technique initially developed in the early 20th century, has gained
prominence in recent years for its ability to fabricate nanofibrous scaffolds with high surface
area-to-volume ratios and controllable fiber diameters [
7
]. The process involves the use
of an electric field to draw a polymer solution or melt from a syringe tip, resulting in the
formation of ultrafine fibers as the solvent evaporates or solidifies. Electrospinning offers
flexibility in material selection, with various polymers, including synthetic polymers like
polycaprolactone (PCL), as well as natural polymers such as collagen and gelatin, being
compatible with the process [8].
PCL has emerged as one of the most favored polymers for electrospinning due to
its excellent processability, biodegradability, and biocompatibility [
9
]. However, PCL has
limitations such as poor cellular adhesion [
10
] and a lack of inherent bioactive properties,
which may hinder its effectiveness in promoting tissue regeneration. To overcome these
drawbacks, researchers have explored surface treatments [
10
] and the incorporation of
bioactive substances into PCL scaffolds to enhance their biological functionality [11–14].
Carotenoids, a diverse group of naturally occurring pigments found abundantly in
fruits [
15
], vegetables [
16
], and certain microorganisms [
17
], possess a remarkable array of
bioactive properties that hold potential in various biomedical applications. Carotenoids
offer significant protection against free radical damage due to their ability to scavenge
reactive oxygen species (ROS) [
18
], which is crucial for maintaining cellular health and
function and potentially aids in preventing chronic diseases like cardiovascular disor-
ders [
19
], cancer [
20
], and neurodegenerative conditions [
21
]. Studies have shown their
effectiveness in reducing inflammation associated with various conditions like arthritis [
22
],
asthma [
23
], and inflammatory bowel disease [
24
]. Specific carotenoids also exhibit the
ability to stimulate cell proliferation and differentiation, essential processes in tissue re-
generation and wound healing, making them promising candidates for promoting tissue
repair and growth in various medical contexts. Among different carotenoids,
β
-carotene
was chosen due to its well-established biological activities, readily available source, and
established safety profile, making it a promising candidate for incorporation into scaffolds
for biomedical applications.
β
-carotene, a natural antioxidant and precursor of vitamin A, has garnered signifi-
cant attention for its potential biomedical applications, including antioxidant [
25
], anti-
inflammatory [
26
], and cell-stimulating properties [
27
,
28
]. Incorporating
β
-carotene into
PCL scaffolds represents a promising strategy to imbue them with bioactive characteristics,
thereby enhancing their suitability for tissue engineering applications [29–32].
In this context, the present study aims to fabricate
β
-carotene-loaded PCL scaffolds
using the electrospinning technique and investigate their potential for biomedical applica-
tions, particularly for tissue regeneration. This research, based on a very simple material
production method, explores the influence of varying
β
-carotene concentrations on the mor-
phology, physicochemical properties, mechanical characteristics, and biological responses
of the scaffolds. Based on the literature on similar composite materials, it is hypothesized
that the incorporation of
β
-carotene into PCL scaffolds could not only improve their bio-
compatibility and promote cell interaction but also preserve the mechanical integrity of
the matrix, rendering them versatile platforms for tissue engineering and regenerative
medicine endeavors.
Polymers 2024,16, 1371 3 of 20
2. Materials and Methods
2.1. Electrospinning Process
For the production of the base material, 400 mg of PCL (Mw ~80,000, Sigma Aldrich,
St. Louis, MO, USA) was added to 2 mL of acetic acid (Nacalai Tesque, Kyoto, Japan) and
stirred for 12 h. For the composites, 5 mg, 10 mg, and 15 mg (about 1.2%, 2.4%, and 3.6%,
respectively) of
β
-carotene powder (1
µ
m average particle diameter, Nacalai Tesque, Kyoto,
Japan) were added to the solution and stirred for an additional hour.
Printing parameters were chosen from a previous work [
33
]: 1 mL of solution was
added to a syringe with a 23G needle and injected at a rate of 0.2
µ
m/s (flow rate:
3.5 ×10−3mm3/s
) under an applied voltage of 10 kV while keeping the metal needle
at a fixed distance of 5 cm from the target aluminum foil cathode. The samples were
produced under controlled environmental conditions (T = 25 ◦C and RH = 25–35%).
2.2. Characterization Techniques
2.2.1. Confocal Imaging
Micrographs of the samples’ surfaces were taken using a 3D laser-scanning microscope
(VKX200K series, Keyence, Osaka, Japan) with magnifications ranging from 10
×
to 150
×
and a numerical aperture between 0.30 and 0.95. Surface maps obtained from the merging
of different images could be acquired by using a dedicated automated
xy
stage combined
with the autofocus function for the zaxis.
Phase-contrast and fluorescence images of cell viability and morphology were acquired
using a Nikon AXRGH confocal microscope (Nikon, Japan) equipped with a CCD camera
at magnifications of 10×(0.16) and 20×(water immersion objective 0.8).
2.2.2. Scanning Electron Microscope
An SM-700 1F Scanning Electron Microscopy (JEOL, Tokyo, Japan) device was em-
ployed to capture high-magnification images of the samples both pre- and post-biological
testing. Prior to observation, the samples underwent sputter-coating with a platinum layer
(approximately 2 nm thick) and were subsequently examined at an accelerating voltage
of 10 kV.
2.2.3. Spinnability Test
The spinnability of the solutions was evaluated with a simple pull-out (pull-up) test,
where a polyethylene cylindrical probe (2 mm diameter) was immersed in the electro-
spinning solution to a depth of 1 cm and then pulled out from the solution at a speed of
500 mm/min
. A fiber will then form between the meniscus of the solution and the tip, and
the maximum length of this fiber depends on the cohesive forces between the molecules of
the solution and its adhesion to the probe. The length of the fiber at the breaking point can
then be used to qualitatively estimate the ability of the solution to form fibers.
The devices used for these assessments (separate from the electrospinning one), to-
gether with test parameters, were designed through an optimization process. Polyethylene
was chosen as the material for the probe because of its adhesion to the studied solutions, so
that the pull-out test could be carried out. The probe’s chosen diameter allows the forma-
tion of fibers with a cross-section that can withstand tension without immediate rupture, so
the differences between the cohesive forces of different solutions can be appreciated. The
depth, on the other hand, allows material to be taken from the core of the solution, in order
to have a homogeneous composition. The pull-out speed was chosen to make the influence
of gravity on the test results non-significant.
2.2.4. Raman Spectroscopy
Raman imaging was conducted utilizing a confocal Laser Raman microscope (RA-
MANtouch, Nanophoton Co., Ltd., Osaka, Japan) with excitation sources at 532 nm and a
nominal power of 200 mW. To mitigate the risk of sample burning, the power output was
Polymers 2024,16, 1371 4 of 20
regulated by adjusting a dedicated ND filter. The micro-probe employed lenses ranging
from 5×to 100×magnifications, with numerical apertures spanning from 0.5 to 0.23.
Imaging involved the acquisition of linear arrays (
x
axis) consisting of 400 points,
which were subsequently combined into a bi-dimensional map (yaxis).
The average spectra for each material were then analyzed and deconvoluted using
dedicated software (Labspec 5.0, Horiba, Kyoto, Japan).
2.2.5. X-ray Diffraction
XRD analyses were performed on a Rigaku Ultima IV (Rigaku Corporation, Tokyo,
Japan), using CuKa radiation. Diffraction patterns were acquired in the range of 5
◦
–50
◦
with a step size of 0.02 at a rate of 3
◦
/min. The penetration depth of the XRD probe was in
the order of 1 mm. For PCL, the peaks at about 22
◦
[110] and 24
◦
[200] [
34
] were considered
representative of the crystalline phase, while the main peaks related to
β
-carotene could be
found at about 24
◦
, 24.5
◦
, 25.5
◦
, 28.5
◦
, and 29.5
◦
[
35
]. The crystallinity index was calculated
as the ratio between the integrated intensity of the crystalline peaks and the total integrated
intensity after subtraction of the β-carotene contribution, using the following equation:
Xc=IPCL
Itotal −Iβ
×100
where
Xc
is the crystallinity index,
IPCL
is the sum of the intensity of the two main peaks
related to PCL,
Iβ
is the cumulative intensity of the peaks related to
β
-carotene, and
Itotal
is
the total integrated intensity.
2.2.6. Differential Scanning Calorimetry
Differential Scanning Calorimetry (DSC) was performed using a DSC 2920 (TA Instru-
ments, New Castle, DE, USA) at a heating rate of 10
◦
C min
−1
in a nitrogen atmosphere.
Spectra were analyzed using commercial software (Origin 8.5, Originlab Corp., Northamp-
ton, MA, USA).
2.2.7. Mechanical Testing
The mechanical properties of the different scaffolds were measured using a tensile
tester (STA-1150, ORIENTEC, Tokyo, Japan). The samples, specifically prepared for me-
chanical testing, featured two reinforced clamping regions on both sides and had an initial
testing length of 20 mm. The specimens were then stretched at a tensile rate of 10 mm/min,
at room temperature. The tensile strength, elongation at break, and tensile modulus were
given by the tensile tester after obtaining the stress–strain curve, while the toughness was
calculated using commercial software (Origin 8.5, Originlab Corp., Northampton, MA,
USA). The measurements were repeated six times for each group of samples.
2.3. Biological Testing
2.3.1. Cell Culture
The BJ-1 cell line was subcultured maintaining a seeding density of 3
×
10
3
cells
·
cm
−2
in DMEM (Minimum Essential Medium Eagle-alpha modification, Gibco, Thermo Fisher,
Zürich, Switzerland) with 10% FBS (fetal bovine serum, Gibco, Thermo Fisher, Zürich,
Switzerland), 100 U
·
ml
−1
penicillin, and 100
µ
g
·
ml
−1
streptomycin (Gibco). The medium
was changed every second day. BJ-1 cells were maintained at 37
◦
C in a 5% CO
2
humidified
atmosphere. This culture was used to address the biocompatibility of
β
-carotene/PCL
composites before electrospinning.
KUSA-A1 cells (JCRB, Osaka, Japan) were first cultured and incubated in a medium
consisting of 4.5 g/L of glucose DMEM (D-glucose, L-glutamine, phenol red, and sodium
pyruvate, Nacalai tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum. The
various samples were previously sterilized upon exposure to ethanol and put in a 24-well
plate one by one. The cultured cells were then deposited on the samples in the well at a
seeding concentration of 10
5
cells/well. Cells were concentrated into 50 mL of solution and
Polymers 2024,16, 1371 5 of 20
gently deposited on the samples, then incubated for an hour. Then, 1 mL of culture medium
was added to each well. An osteogenic medium was used after 24 h. The medium consisted
of Dulbecco’s modified Eagle medium (DMEM) supplemented with nominal amounts of
the following constituents: 50 mg/mL ascorbic acid, 10 mM b-glycerol phosphate, 100 mM
hydrocortisone, and 10% fetal bovine serum. All samples were incubated at 37
◦
C for up to
10 days. The medium was changed a total of three times.
2.3.2. Cell Testing
BJ-1 cells were tested on PCL-modified material. In order to avoid the adhesion of
cells on the tissue culture plate, TC24wells were precoated with 200
µ
L Agarose 1.5%.
After the solidification of agar, PCL+ curcumin samples were located in the wells. Re-
spectively, 250,000 cells in 300
µ
L were seeded on no-prewet scaffolds (directly on the
surface) or on scaffolds that had been prewet for 1 h in medium solution (prewet 1 h).
All samples were prepared in triplicates. BJ-1 cells were co-stained with 5
µ
M Calcein
(Sigma-Aldrich, St. Louis, MO, USA, #17783) and 0.625 µg/mL Ethidium Homodimer for
30 min to discriminate between live and dead cells.
For KUSA-A1, the cytotoxicity of the substrates was assessed and compared by analyzing
the samples using the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. This colorimetric assay utilizes MTT, a yellow tetrazolium salt, which is
metabolized by metabolically active cells into a purple formazan product. The conversion
of MTT to formazan is directly proportional to cell viability. The formazan solutions were
then analyzed using microplate readers (EMax, Molecular Devices, Sunnyvale, CA, USA)
at an absorbance wavelength of 570 nm. The resulting optical density (OD) values served
as an indicator of cell viability and cytotoxicity.
Osteogenic differentiation was assessed after 10 days in the osteogenic medium by
staining cultured KUSA-A1 cells with the green osteocalcin stain (Osteocalcin Mouse anti-
Human, PE, Clone, Fisher Scientific, Hampton, NH, USA) following the manufacturer’s
instructions. Fluorescence microscopy was used to visualize osteocalcin localization and
quantify the stained area (with respect to the total area of the image) using image analysis
software (Fiji, release 2.9.0 [
36
]). This approach avoids potential interference from the red
color of
β
-carotene, allowing for a more accurate assessment of bone formation compared
to other common protocols, such as ALP activity assays. As
β
-carotene is also known
to emit fluorescence in the green region, the results had to be post-treated with analysis
software (Fiji, release 2.9.0 [
36
]) to remove the weaker background fluorescence. The test
was performed at 10×.
2.3.3. Bacteria Culture
Escherichia coli (25922
®
ATCC™) (simply E. coli, henceforth) was cultured at 37
◦
C at
Kyoto Prefectural University of Medicine using brain heart infusion (BHI) agar (Nissui,
Tokyo, Japan). Starting from an initial 1.0
×
10
9
CFU/mL, the concentration was diluted
with phosphate-buffered saline (PBS) at physiological pH and ionic strength. Subsequently,
100
µ
L of the bacterial suspension at a density of 1
×
10
8
CFU/mL was spread onto a BHI
agar plate. The samples were sterilized by UV and pressed into the bacteria on BHI agar
for inoculation.
2.3.4. Bacteria Testing
After 24 and 48 h of incubation, the samples were washed with PBS and bacterial
viability was assessed using a colorimetric assay (Microbial Viability Assay Kit-WST,
Dojindo, Kumamoto, Japan). This assay employed a colorimetric indicator (WST-8), which
produces a water-soluble formazan dye upon reduction in the presence of an electron
mediator. The amount of the formazan dye generated is directly proportional to the
number of living microorganisms. Solutions were analyzed using microplate readers (EMax,
Molecular Devices, Sunnyvale, CA, USA) through optical density at 600 nm (OD600).
Polymers 2024,16, 1371 6 of 20
2.4. Statistical Analysis
To assess the statistical significance of the differences observed among the groups, we
employed a one-way analysis of variance (ANOVA). This approach enabled us to determine
whether any of the mean values for the measured outcomes varied significantly across the
experimental groups. For each test, we utilized a sample size of n = 5 specimens, ensuring
adequate statistical power for the analysis.
3. Results
Figure 1shows the surface morphology of the samples, as obtained at low magnifica-
tions using a confocal laser microscope. When the PCL/acetic acid solution is electrospun
without any additional additive (Figure 1a), the deposition results in a large amount of
elongated droplets, a common defect already observed in a previous work [
33
], associated
with electrospinning that usually indicates an excessive polymer concentration, high vis-
cosity, or a non-optimized electrical field. The quality of the deposition seems to improve
progressively with an increase in the concentration of
β
-carotene, with almost no visible
droplets for solutions containing 3.6% (Figure 1d) of β-carotene.
Polymers 2024, 16, x FOR PEER REVIEW 6 of 20
of living microorganisms. Solutions were analyzed using microplate readers (EMax, Mo-
lecular Devices, Sunnyvale, CA, USA) through optical density at 600 nm (OD
600
).
2.4. Statistical Analysis
To assess the statistical significance of the differences observed among the groups,
we employed a one-way analysis of variance (ANOVA). This approach enabled us to de-
termine whether any of the mean values for the measured outcomes varied significantly
across the experimental groups. For each test, we utilized a sample size of n = 5 specimens,
ensuring adequate statistical power for the analysis.
3. Results
Figure 1 shows the surface morphology of the samples, as obtained at low magnifi-
cations using a confocal laser microscope. When the PCL/acetic acid solution is electro-
spun without any additional additive (Figure 1a), the deposition results in a large amount
of elongated droplets, a common defect already observed in a previous work [33], associ-
ated with electrospinning that usually indicates an excessive polymer concentration, high
viscosity, or a non-optimized electrical field. The quality of the deposition seems to im-
prove progressively with an increase in the concentration of β-carotene, with almost no
visible droplets for solutions containing 3.6% (Figure 1d) of β-carotene.
Fiber fusion at the contact points, another common structural defect for electrospun
scaffolds, caused by the evaporation of the solvent, can also be observed on all scaffolds,
independently of the presence of β-carotene.
Figure 1. Laser microscope images of the samples at low magnifications: (a) pure PCL, (b) 1.2% β-
carotene, (c) 2.4% β-carotene, (d) 3.6% β-carotene.
Figure 1. Laser microscope images of the samples at low magnifications: (a) pure PCL,
(b) 1.2% β-carotene, (c) 2.4% β-carotene, (d) 3.6% β-carotene.
Fiber fusion at the contact points, another common structural defect for electrospun
scaffolds, caused by the evaporation of the solvent, can also be observed on all scaffolds,
independently of the presence of β-carotene.
A qualitative evaluation of the samples’ uniformity is presented in Figure 2. In these
four pictures, the images of Figure 1were transformed into binary black or white, split
Polymers 2024,16, 1371 7 of 20
into 9 x 12 regions, and processed in order to calculate the percentage of white pixels for
each region. For visualization purposes, the region with the highest density of electrospun
material (higher amount of white pixels) is marked in bright yellow and the lowest in
black. From a purely qualitative point of view, the brighter the color of the circle, the higher
the material density of the specific sub-region, and the more uniform the colored circles
across the surface, the more uniform the density. This simplified representation clearly
shows that the lowest average density is achieved with 1.2% of
β
-carotene, followed by the
2.4% sample.
Polymers 2024, 16, x FOR PEER REVIEW 7 of 20
A qualitative evaluation of the samples’ uniformity is presented in Figure 2. In these
four pictures, the images of Figure 1 were transformed into binary black or white, split
into 9 x 12 regions, and processed in order to calculate the percentage of white pixels for
each region. For visualization purposes, the region with the highest density of electrospun
material (higher amount of white pixels) is marked in bright yellow and the lowest in
black. From a purely qualitative point of view, the brighter the color of the circle, the
higher the material density of the specific sub-region, and the more uniform the colored
circles across the surface, the more uniform the density. This simplified representation
clearly shows that the lowest average density is achieved with 1.2% of β-carotene, fol-
lowed by the 2.4% sample.
Figure 2. Automatic, software-generated visualization of the fiber density and dispersion based on
Figure 1: (a) pure PCL, (b) 1.2% β-carotene, (c) 2.4% β-carotene, (d) 3.6% β-carotene.
A quantification of the fiber dispersion and density observed in Figure 2 is given in
Figure 3a, where 100% represents a purely white area (0% of porosity) and 0% a purely
black one (no visible fibers), confirming that the sample with 1.2% of β-carotene, which
has the darkest circles in Figure 2,
has the lowest average fiber density and a low relative
dispersion, comparable to that of the PCL reference. From the combined results of Figures
2 and 3a, it seems that the presence of β-carotene progressively increases the density of
the scaffolds, while decreasing the amount of droplets.
Figure 2. Automatic, software-generated visualization of the fiber density and dispersion based on
Figure 1: (a) pure PCL, (b) 1.2% β-carotene, (c) 2.4% β-carotene, (d) 3.6% β-carotene.
A quantification of the fiber dispersion and density observed in Figure 2is given in
Figure 3a, where 100% represents a purely white area (0% of porosity) and 0% a purely black
one (no visible fibers), confirming that the sample with 1.2% of
β
-carotene, which has the
darkest circles in Figure 2, has the lowest average fiber density and a low relative dispersion,
comparable to that of the PCL reference. From the combined results of
Figures 2and 3a
, it
seems that the presence of
β
-carotene progressively increases the density of the scaffolds,
while decreasing the amount of droplets.
Polymers 2024,16, 1371 8 of 20
Polymers 2024, 16, x FOR PEER REVIEW 8 of 20
Figure 3. (a) Average signal intensity (full) and statistical dispersion of the signal intensity (striped)
for the different samples, calculated on the 48 sub-regions of Figure 2. The statistical dispersion of
the histograms is based on 5 repetitions on different images. (b) Contact angles as measured on the
different scaffolds and (c) pull-out distance for the different samples. Results marked with “*” have
a p-value < 0.05 when compared with all others while those marked with “n.s.” had p-value > 0.05
and were nonsignificant.
Figure 3b shows the results of the water contact angle measurements performed on
the different samples. PCL is a hydrophobic polymer with a water contact angle usually
between 80 and 110°. For the reference scaffold, the value resulted to be about 97° and,
despite the large statistical dispersion caused by the inhomogeneity of the scaffold struc-
tures, the addition of β-carotene progressively increased the contact angle up to about
112° for a concentration of 3.6%.
Figure 3c shows the results of a pull-out test performed on the four different solutions
before electrospinning. This experiment is a semi-quantitative evaluation of the cohesive
forces between the molecules of the solution, which would affect both the spinnability and
the mechanical properties. It can be observed that the cohesive forces are higher for the
composite containing 1.2% β-carotene, followed by pure PCL and then the two composites
with higher concentrations of β-carotene.
Figure 4 shows representative images of the four scaffolds at 10 k magnifications. All
fibers appear to be smooth, with comparable average diameters. Given the average diam-
eter of β-carotene particles, these should be visible in SEM images, although they cannot
be distinguished. The Raman results in the following figures, however, will confirm the
incorporation of the powder into the scaffolds. Fiber fusion is clearly visible at all contact
Figure 3. (a) Average signal intensity (full) and statistical dispersion of the signal intensity (striped)
for the different samples, calculated on the 48 sub-regions of Figure 2. The statistical dispersion of
the histograms is based on 5 repetitions on different images. (b) Contact angles as measured on the
different scaffolds and (c) pull-out distance for the different samples. Results marked with “*” have a
p-value < 0.05 when compared with all others while those marked with “n.s.” had p-value > 0.05 and
were nonsignificant.
Figure 3b shows the results of the water contact angle measurements performed on
the different samples. PCL is a hydrophobic polymer with a water contact angle usually
between 80 and 110
◦
. For the reference scaffold, the value resulted to be
about 97◦and
, de-
spite the large statistical dispersion caused by the inhomogeneity of the scaffold structures,
the addition of
β
-carotene progressively increased the contact angle up to about 112
◦
for a
concentration of 3.6%.
Figure 3c shows the results of a pull-out test performed on the four different solutions
before electrospinning. This experiment is a semi-quantitative evaluation of the cohesive
forces between the molecules of the solution, which would affect both the spinnability and
the mechanical properties. It can be observed that the cohesive forces are higher for the
composite containing 1.2%
β
-carotene, followed by pure PCL and then the two composites
with higher concentrations of β-carotene.
Figure 4shows representative images of the four scaffolds at 10 k magnifications.
All fibers appear to be smooth, with comparable average diameters. Given the average
diameter of
β
-carotene particles, these should be visible in SEM images, although they
cannot be distinguished. The Raman results in the following figures, however, will confirm
the incorporation of the powder into the scaffolds. Fiber fusion is clearly visible at all contact
Polymers 2024,16, 1371 9 of 20
points, but the fibers involved retain most of their original geometry. On the microscopic
scale, the addition of
β
-carotene up to 3.6% does not seem to affect the morphology of the
scaffold in any measurable way.
Polymers 2024, 16, x FOR PEER REVIEW 9 of 20
points, but the fibers involved retain most of their original geometry. On the microscopic
scale, the addition of β-carotene up to 3.6% does not seem to affect the morphology of the
scaffold in any measurable way.
Figure 4. High-magnification micrographs of the electrospun fibers: (a) pure PCL, (b) 1.2% β-caro-
tene, (c) 2.4% β-carotene, (d) 3.6% β-carotene.
The average Raman spectra acquired on the surface of the scaffolds are presented in
Figure 5, as compared to the average spectrum of the pure β-carotene powder. The main
bands of these spectra are listed in Table 1 together with their respective assignments. Due
to the high Raman cross-section of β-carotene, the bands of PLC are clearly visible only
on the reference sample, as the strongest band of β-carotene is about 1000× more intense
than the strongest band of PCL, when acquired under the same experimental conditions.
In reality, PCL bands are still visible on the three composite scaffolds, but their signal is
so low compared to β-carotene that they are easily confused with the background noise.
Apart from the main bands of β-carotene, two relatively strong fluorescence bands can
also be observed, between 1000 and 4000 cm
−1
. The sum of these bands, centered at about
595 nm and 625 nm, gives the Raman spectrum baseline the same fluorescence emission
previously reported in the literature for all-trans-β-carotene [37].
Despite the variations in absolute intensity, the relative intensity of the various β-
carotene bands observed in Figure 5 is constant across the three composite scaffolds and
the reference, meaning that there is no clear evidence of a chemical interaction between
the two phases. From the Raman results, it appears that the β-carotene particulate in the
scaffolds did not react with PCL and had the same structure as the original powder. The
reason why the β-carotene particles cannot be visualized in Figure 4 is probably due to a
change in particle size caused by the solvation and subsequent evaporation of the solvent.
Figure 4. High-magnification micrographs of the electrospun fibers: (a) pure PCL, (b) 1.2%
β
-carotene,
(c) 2.4% β-carotene, (d) 3.6% β-carotene.
The average Raman spectra acquired on the surface of the scaffolds are presented in
Figure 5, as compared to the average spectrum of the pure
β
-carotene powder. The main
bands of these spectra are listed in Table 1together with their respective assignments. Due
to the high Raman cross-section of
β
-carotene, the bands of PLC are clearly visible only
on the reference sample, as the strongest band of
β
-carotene is about 1000
×
more intense
than the strongest band of PCL, when acquired under the same experimental conditions. In
reality, PCL bands are still visible on the three composite scaffolds, but their signal is so low
compared to
β
-carotene that they are easily confused with the background noise. Apart
from the main bands of
β
-carotene, two relatively strong fluorescence bands can also be
observed, between 1000 and 4000 cm
−1
. The sum of these bands, centered at about 595 nm
and 625 nm, gives the Raman spectrum baseline the same fluorescence emission previously
reported in the literature for all-trans-β-carotene [37].
Polymers 2024,16, 1371 10 of 20
Polymers 2024, 16, x FOR PEER REVIEW 10 of 20
Figure 5. Average Raman spectra of the different samples.
Table 1. Assignment of the Raman bands presented in Figure 5, with relevant literature references.
Position
[cm−1] Vibrations Origin References
Position
[cm−1]
3045 2𝑣 β-carotene [38] 3045
2930 𝑣 PCL [39] 2930
2850 𝑣 PCL [39] 2850
2675 𝑣+𝑣
β-carotene [38] 2675
2525 𝑣+𝑣
β-carotene [38] 2525
2310 2𝑣 β-carotene [38] 2310
1525 𝑣 β-carotene [38] 1525
1470 𝛿 PCL [40] 1470
1440 𝛿 PCL [40] 1440
1415 𝛿 PCL [40] 1415
1305 𝜏 PCL [40] 1305
1285 𝜏 PCL [40] 1285
1155 𝑣 β-carotene [38] 1155
1110 𝑣 PCL [40] 1110
1065 𝑣 PCL [40] 1065
1035 𝑣 PCL [40] 1035
1005 𝑣 β-carotene [38] 1005
960 𝑣 PCL [40] 960
Despite the relative intensity of the β-carotene signal with respect to PCL, it is still
possible to observe signal fluctuations depending on the location. In Figure 6a, portions
of the scaffolds are analyzed by Raman imaging, using a high-point-density linear sensor.
Figure 5. Average Raman spectra of the different samples.
Table 1. Assignment of the Raman bands presented in Figure 5, with relevant literature references.
Position
[cm−1]Vibrations Origin References Position
[cm−1]
3045 2v1β-carotene [38] 3045
2930 vaCH PCL [39] 2930
2850 vsCH PCL [39] 2850
2675 v1+v2β-carotene [38] 2675
2525 v1+v3β-carotene [38] 2525
2310 2v2β-carotene [38] 2310
1525 v1C=Cβ-carotene [38] 1525
1470 δCH2PCL [40] 1470
1440 δCH2PCL [40] 1440
1415 δCH2PCL [40] 1415
1305 τCH2PCL [40] 1305
1285 τCH2PCL [40] 1285
1155 v2CC β-carotene [38] 1155
1110 vCC PCL [40] 1110
1065 vCC PCL [40] 1065
1035 vCC PCL [40] 1035
1005 v3CH β-carotene [38] 1005
960 vC−COO PCL [40] 960
Polymers 2024,16, 1371 11 of 20
Despite the variations in absolute intensity, the relative intensity of the various
β
-
carotene bands observed in Figure 5is constant across the three composite scaffolds and
the reference, meaning that there is no clear evidence of a chemical interaction between
the two phases. From the Raman results, it appears that the
β
-carotene particulate in the
scaffolds did not react with PCL and had the same structure as the original powder. The
reason why the
β
-carotene particles cannot be visualized in Figure 4is probably due to a
change in particle size caused by the solvation and subsequent evaporation of the solvent.
Despite the relative intensity of the
β
-carotene signal with respect to PCL, it is still
possible to observe signal fluctuations depending on the location. In Figure 6a, portions
of the scaffolds are analyzed by Raman imaging, using a high-point-density linear sensor.
By applying a high-pass filter to the
β
-carotene signals, so that the intensity is recorded
only if at least 100
×
stronger than that of PCL, we can observe a dispersion of red dots
on the otherwise green PCL polymeric fibers. These red dots, which are not visible in the
microscopic images of Figures 1and 4, represent the locations where
β
-carotene powder
particles are embedded into the PCL matrix. It can be observed that the average intensity
and the overall amount of red pixels in Figure 6a are dependent on the concentration
of
β
-carotene, as expected, suggesting that the filtering method used could effectively
discriminate between particulates actually present at the investigated location and Raman
signals from deeper regions of the sample.
When the amount of
β
-carotene increases, so does the uniformity of its distribution
on the fibers. For the sample containing 1.2% particulates, the red pixels are concentrated
in one area, on the left. For a concentration of 2.4%, the red pixels are in most areas, but
a few completely green fibers are still visible. When the concentration reaches 3.6%, the
distribution results in being relatively uniform across the whole image.
Figure 6b shows the distribution of
β
-carotene inside the composite, expressed as red
pixel area coverage. As the images of Figure 6a were acquired using an arbitrary filtering
method, these measurements are to be considered qualitative, but give an effective and
immediate way of comparing the three composites. Despite the amount of particulate being
increased linearly, the area coverage measured by Raman imaging appears to increase
exponentially. This effect can be due to irregularities in the distribution of the particulate
inside the polymeric matrix, but also a consequence of the filtering process.
Representative stress–strain curves for the four different samples are shown in Figure 7.
The addition of
β
-carotene to the PCL matrix leads to a progressive decrease in ultimate
strength, coupled with a slight increase in ultimate strain. These results suggest that
β
-
carotene might act as a plasticizer for the PCL matrix, but its effects are limited and not
dose dependent.
The main parameters associated with the mechanical properties of the scaffolds that
can be extrapolated from the stress–strain curves are resumed in Figure 7, in panels
from b to e.
As observed in Figure 7, the ultimate stress (Figure 7b) progressively decreases with
the amount of
β
-carotene particulates, while the elongation at break (Figure 7c) initially
increases by about 25% but then remains stable up to 3.6% particulates, as the results of
the three composites show no statistically significant difference between each other. The
Young’s modulus (Figure 7d) of the four materials also decreases, following the same
trend previously observed for the ultimate stress, with the composite containing 3.6% of
β
-carotene showing less than half the modulus of the pristine PCL reference. The increased
elongation at break of the composites also results in an increased toughness, but the results
appear to be affected by a large statistical dispersion, as the toughness is influenced by the
scattering of both ultimate stress and elongation at break.
Polymers 2024,16, 1371 12 of 20
Polymers 2024, 16, x FOR PEER REVIEW 11 of 20
By applying a high-pass filter to the β-carotene signals, so that the intensity is recorded
only if at least 100× stronger than that of PCL, we can observe a dispersion of red dots on
the otherwise green PCL polymeric fibers. These red dots, which are not visible in the
microscopic images of Figures 1 and 4, represent the locations where β-carotene powder
particles are embedded into the PCL matrix. It can be observed that the average intensity
and the overall amount of red pixels in Figure 6a are dependent on the concentration of
β-carotene, as expected, suggesting that the filtering method used could effectively dis-
criminate between particulates actually present at the investigated location and Raman
signals from deeper regions of the sample.
When the amount of β-carotene increases, so does the uniformity of its distribution
on the fibers. For the sample containing 1.2% particulates, the red pixels are concentrated
in one area, on the left. For a concentration of 2.4%, the red pixels are in most areas, but a
few completely green fibers are still visible. When the concentration reaches 3.6%, the dis-
tribution results in being relatively uniform across the whole image.
Figure 6. (a) Dispersion of
β
-carotene particulates into the PCL fibers, as observed by using Raman
imaging. The
β
-carotene signal has been filtered with a high-pass filter, so that the signal is at least
100
×
stronger than that of PCL. (b) Area coverage of the
β
-carotene Raman signal for
I1525 ≥
100
I3045
.
Results marked with “*” have a p-value < 0.05 when compared with all others.
Polymers 2024,16, 1371 13 of 20
Polymers 2024, 16, x FOR PEER REVIEW 13 of 20
Figure 7. (a) Representative stress–strain curves for the different samples, as obtained from tensile
testing. Main parameters extrapolated from the tensile testing experiments: (b) ultimate strength,
(c) ultimate strain, (d) Young’s modulus, and (e) toughness for the different samples. Results marked
with “*” have a p-value < 0.05 when compared with all others while those marked with “n.s.” had
p-value > 0.05 and were nonsignificant.
As observed in Figure 7, the ultimate stress (Figure 7b) progressively decreases with
the amount of β-carotene particulates, while the elongation at break (Figure 7c) initially
increases by about 25% but then remains stable up to 3.6% particulates, as the results of
the three composites show no statistically significant difference between each other. The
Young’s modulus (Figure 7d) of the four materials also decreases, following the same
trend previously observed for the ultimate stress, with the composite containing 3.6% of
β-carotene showing less than half the modulus of the pristine PCL reference. The in-
creased elongation at break of the composites also results in an increased toughness, but
Figure 7. (a) Representative stress–strain curves for the different samples, as obtained from tensile
testing. Main parameters extrapolated from the tensile testing experiments: (b) ultimate strength,
(c) ultimate strain, (d) Young’s modulus, and (e) toughness for the different samples. Results marked
with “*” have a p-value < 0.05 when compared with all others while those marked with “n.s.” had
p-value > 0.05 and were nonsignificant.
The decrease in mechanical strength and Young’s modulus suggests that
β
-carotene
weakens the structure of the PCL matrix, possibly due to the interruption of the polymeric
chains. This result suggests that the amount of additive should be kept as low as possible
to avoid compromising the mechanical properties and stability of the material.
In the DSC analysis, while the peak melting temperature remains relatively constant
across all samples (Figure 8a), a notable decrease in peak intensity is observed with in-
creasing
β
-carotene content. From pure PCL at ~60
µ
cal/s
2
, the intensity progressively
declines to ~45
µ
cal/s
2
in the 3.6%
β
-carotene composite. One possible explanation for
Polymers 2024,16, 1371 14 of 20
this phenomenon, as previously hypothesized, is that
β
-carotene acts as a plasticizer and
disturbs the crystal formation, increasing the amorphous fraction within the PCL matrix.
This increased amorphous content melts at a wider temperature range with lower enthalpy
compared to the crystalline portion, contributing to a smaller and broader peak in the
DSC curve.
Polymers 2024, 16, x FOR PEER REVIEW 14 of 20
the results appear to be affected by a large statistical dispersion, as the toughness is influ-
enced by the scattering of both ultimate stress and elongation at break.
The decrease in mechanical strength and Young’s modulus suggests that β-carotene
weakens the structure of the PCL matrix, possibly due to the interruption of the polymeric
chains. This result suggests that the amount of additive should be kept as low as possible
to avoid compromising the mechanical properties and stability of the material.
In the DSC analysis, while the peak melting temperature remains relatively constant
across all samples (Figure 8a), a notable decrease in peak intensity is observed with in-
creasing β-carotene content. From pure PCL at ~60 µcal/s
2
, the intensity progressively de-
clines to ~45 µcal/s
2
in the 3.6% β-carotene composite. One possible explanation for this
phenomenon, as previously hypothesized, is that β-carotene acts as a plasticizer and dis-
turbs the crystal formation, increasing the amorphous fraction within the PCL matrix. This
increased amorphous content melts at a wider temperature range with lower enthalpy
compared to the crystalline portion, contributing to a smaller and broader peak in the DSC
curve.
Figure 8. Representative (a) first derivative DSC curves and (b) XRD diffraction pattern for the dif-
ferent samples.
X-ray diffraction patterns (Figure 8b) further support the hypothesis of β-carotene
acting as a plasticizer, as the addition of 1.2% of reinforcement powder causes a drop in
the intensity of the peaks related to the (200) and (110) orientations [34]. When more β-
carotene is added to the composite, the intensity of the diffraction pattern further de-
creases (2.4%), before stabilizing (3.6%). These results further support the hypothesis that
β-carotene particulate lowers the amount of ordered phase in the PCL polymer.
The results of the biological testing performed on the four different scaffolds are sum-
marized in Figures 9 and 10.
Figure 8. Representative (a) first derivative DSC curves and (b) XRD diffraction pattern for the
different samples.
X-ray diffraction patterns (Figure 8b) further support the hypothesis of
β
-carotene
acting as a plasticizer, as the addition of 1.2% of reinforcement powder causes a drop in the
intensity of the peaks related to the (200) and (110) orientations [
34
]. When more
β
-carotene
is added to the composite, the intensity of the diffraction pattern further decreases (2.4%),
before stabilizing (3.6%). These results further support the hypothesis that
β
-carotene
particulate lowers the amount of ordered phase in the PCL polymer.
The results of the biological testing performed on the four different scaffolds are
summarized in Figures 9and 10.
Polymers 2024, 16, x FOR PEER REVIEW 15 of 20
Figure 9. BJ-1 cell line seeded directly on cell culture plates, PCL, or PCL with 3.6% of β-carotene.
The BJ-1 cells commonly used for toxicity assays following ISO 10993-5 [41] guide-
lines were directly seeded onto PCL or PCL modified with β-carotene. The control used
was a cell culture plate, obviously highly efficient for cell culture adhesion and prolifera-
tion. Therefore, our gold standard (CTR +) was established based on this control. Three
days after seeding BJ-1 cells on the constructs, previously placed in wells coated with 1.5%
agar to prevent adhesion to the well surface, live and dead staining was performed using
Ethidium Homodimer and Calcein. These markers indicate cell integrity and vitality (Cal-
cein) and cell death (Ethidium Homodimer), respectively, as they bind to the DNA of
damaged cells.
Figure 9. BJ-1 cell line seeded directly on cell culture plates, PCL, or PCL with 3.6% of β-carotene.
Polymers 2024,16, 1371 15 of 20
Polymers 2024, 16, x FOR PEER REVIEW 15 of 20
Figure 9. BJ-1 cell line seeded directly on cell culture plates, PCL, or PCL with 3.6% of β-carotene.
The BJ-1 cells commonly used for toxicity assays following ISO 10993-5 [41] guide-
lines were directly seeded onto PCL or PCL modified with β-carotene. The control used
was a cell culture plate, obviously highly efficient for cell culture adhesion and prolifera-
tion. Therefore, our gold standard (CTR +) was established based on this control. Three
days after seeding BJ-1 cells on the constructs, previously placed in wells coated with 1.5%
agar to prevent adhesion to the well surface, live and dead staining was performed using
Ethidium Homodimer and Calcein. These markers indicate cell integrity and vitality (Cal-
cein) and cell death (Ethidium Homodimer), respectively, as they bind to the DNA of
damaged cells.
Figure 10. Indirect test of toxicity of material immersed for 24 h in cell culture medium and then
transferred to BJ-1 cells. Toxicity was tested after 24 h and 72 h.
The BJ-1 cells commonly used for toxicity assays following ISO 10993-5 [
41
] guidelines
were directly seeded onto PCL or PCL modified with
β
-carotene. The control used was
a cell culture plate, obviously highly efficient for cell culture adhesion and proliferation.
Therefore, our gold standard (CTR +) was established based on this control. Three days
after seeding BJ-1 cells on the constructs, previously placed in wells coated with 1.5% agar to
prevent adhesion to the well surface, live and dead staining was performed using Ethidium
Homodimer and Calcein. These markers indicate cell integrity and vitality (Calcein) and
cell death (Ethidium Homodimer), respectively, as they bind to the DNA of damaged cells.
The results presented in Figure 10 highlight that the cells adhered to the plate and
showed no distress (CTR+), demonstrating cellular quality as a control. Interestingly,
after 3 days, cells on PCL appeared rounded compared to those on PCL modified with
β
-carotene. From a cellular perspective, this could be explained by the
β
-carotene modifica-
tion influencing the hydrophilicity of the material, promoting cell adhesion and subsequent
proliferation. Cells on PCL
β
-carotene, in fact, exhibited a more even distribution and
morphologically resembled the CTR, which, of course, showed higher density due to the
optimized surface.
However, it was intriguing to observe that, despite not performing a prewetting step,
which is typically necessary for PCL due to its hydrophobic nature, the modification with
carotene significantly enhanced cell adhesion. Therefore, it is plausible that this modifica-
tion, by augmenting both adhesion and proliferation, enables direct cellular attachment.
As observed in Figure 11a, the amount of KUSA-A1 cells, recorded at both
24 and 72 h
of culture, increases with the concentration of
β
-carotene and is always comparable if not
superior to the control (cell culture without samples) for all composite materials. This
indicates that the material is not cytotoxic, also suggesting that it potentially stimulates
cellular proliferation. The number of cells shows a small decrease going from 24 to 72 h
Polymers 2024,16, 1371 16 of 20
for concentrations of
β
-carotene equal or inferior to 1.2%, while the opposite is true for
higher concentrations.
Polymers 2024, 16, x FOR PEER REVIEW 16 of 20
Figure 10. Indirect test of toxicity of material immersed for 24 h in cell culture medium and then
transferred to BJ-1 cells. Toxicity was tested after 24 h and 72 h.
The results presented in Figure 10 highlight that the cells adhered to the plate and
showed no distress (CTR+), demonstrating cellular quality as a control. Interestingly, after
3 days, cells on PCL appeared rounded compared to those on PCL modified with β-caro-
tene. From a cellular perspective, this could be explained by the β-carotene modification
influencing the hydrophilicity of the material, promoting cell adhesion and subsequent
proliferation. Cells on PCL β-carotene, in fact, exhibited a more even distribution and
morphologically resembled the CTR, which, of course, showed higher density due to the
optimized surface.
However, it was intriguing to observe that, despite not performing a prewetting step,
which is typically necessary for PCL due to its hydrophobic nature, the modification with
carotene significantly enhanced cell adhesion. Therefore, it is plausible that this modifica-
tion, by augmenting both adhesion and proliferation, enables direct cellular attachment.
As observed in Figure 11a, the amount of KUSA-A1 cells, recorded at both 24 and 72
h of culture, increases with the concentration of β-carotene and is always comparable if
not superior to the control (cell culture without samples) for all composite materials. This
indicates that the material is not cytotoxic, also suggesting that it potentially stimulates
cellular proliferation. The number of cells shows a small decrease going from 24 to 72 h
for concentrations of β-carotene equal or inferior to 1.2%, while the opposite is true for
higher concentrations.
Figure 11. Biological testing results with KUSA-A1 and E. coli: (a) KUSA-A1 cell optical density, (b)
area coverage of the osteocalcin staining with respect to the investigated area of the sample, (c) E.
coli optical density. Results marked with “*” have a p-value < 0.05 when compared with all others.
At 10 days, the scaffolds containing β-carotene are all partially covered by newly
formed bone tissue, as indicated by the presence of osteocalcin (Figure 11b). The amount
Figure 11. Biological testing results with KUSA-A1 and E. coli: (a) KUSA-A1 cell optical density,
(b) area
coverage of the osteocalcin staining with respect to the investigated area of the sample,
(c)E. coli
optical density. Results marked with “*” have a p-value < 0.05 when compared with
all others.
At 10 days, the scaffolds containing
β
-carotene are all partially covered by newly
formed bone tissue, as indicated by the presence of osteocalcin (Figure 11b). The amount of
osteocalcin grows with the concentration of
β
-carotene, suggesting that the reinforcement
also influences bone tissue formation. This can be caused either by an increased cellular
adhesion to the substrate or by a direct metabolic effect of the
β
-carotene. When compared
to the positive control (in this case, a disc of titanium grade 5 alloy), the PCL reference shows
a decrease in the osteocalcin content, and even an addition of 1.2%
β
-carotene does not bring
the composites on par with the titanium reference. The sample containing
3.6% β-carotene
,
on the other hand, has almost double the area coverage of the positive control.
When tested with E. coli for 24 and 48 h of culture (Figure 11c), the scaffolds show
a decrease in optical density (OD) with increasing
β
-carotene concentration, suggesting
reduced bacterial colonization and proliferation. The maximum observed reduction in
OD reaches 20% at a concentration of 3.6%. This implies a potential weak antibacterial or
bacteriostatic effect associated with the presence of β-carotene within the scaffolds.
4. Discussion
Morphological and chemical analyses performed on the electrospun scaffolds indicate
that PCL fibers incorporating up to 3.6% of
β
-carotene can be effectively produced with
minimal effort. Still, thermogravimetric analysis and X-ray diffraction indicate that the
presence of
β
-carotene particulate adversely affects the ordered fraction of the PCL structure,
in particular at elevated concentrations.
Polymers 2024,16, 1371 17 of 20
Despite incorporating
β
-carotene particulate into the PCL solution prior to electro-
spinning, significant chemical bonding between these molecules might not have been
achieved due to several factors. Firstly, the utilization of acetic acid as the solvent might
not have facilitated strong interactions between the highly hydrophobic
β
-carotene and
the semi-crystalline PCL, owing to their differing polarities. Secondly, the relatively short
processing time and low temperatures associated with electrospinning might not have
provided sufficient energy or activation for potential covalent bond formation, even if
suitable functional groups were present. Additionally, the physical form of the
β
-carotene
(powder) could have influenced its dispersion within the PCL matrix, limiting potential
contact points for interaction.
While weaker van der Waals forces or
π
-
π
stacking interactions might still be present, ex-
ploring alternative solvents or processing techniques in future studies could promote stronger
interactions and potentially increase the mechanical properties of the electrospun fibers.
For what concerns the biological response,
β
-carotene presents a complex interplay
between its antioxidant and pro-oxidant behavior, influenced by factors like concentration,
cell type, and environmental conditions [
42
]. While its antioxidant properties might protect
cells from harmful reactive oxygen species (ROS), excessive
β
-carotene can have pro-
oxidant effects at higher concentrations or under specific conditions.
Interestingly, in this research, the addition of
β
-carotene to PCL scaffolds promoted
both cellular proliferation and bone formation. Such observations align with previous stud-
ies [
32
,
43
–
45
], but the topic is still controversial. At moderate concentrations,
β-carotene’s
antioxidant activity might protect cells from ROS-induced damage, creating a more fa-
vorable environment for proliferation.
β
-carotene has also been reported to enhance the
expression of Runx2, ALP, and osteopontin mRNA [
46
], increase early osteoblastic differen-
tiation, and reduce bone loss [
47
] by regulating osteoclastogenesis, but higher doses can
also result in cytotoxicity, in particular for cells with altered metabolic pathways, an effect
that has been extensively studied for the treatment of tumors [
48
,
49
]. Further studies have
demonstrated that oxidized
β
-carotene is cytotoxic and inhibits the mitochondrial function
in various cell lines [50].
The bioactive effects reported in this research are in line with results previously
obtained on fibers containing up to 4% of
β
-carotene [
32
], suggesting that these amounts
are under the threshold for potential cytotoxicity in human cells. Moreover, it has been
demonstrated that
β
-carotene stimulates mineralization as OCP and TCP are produced
near the areas where
β
-carotene exists. Through the production of HA precursors, HA
seed crystals are generated and mature into hard tissues by taking Ca
2+
and PO
43-
from the
extracellular fluid [51].
For what concerns the antibacterial effect observed in Figure 11c, the reduction is
much less pronounced than what was previously observed in the literature [
52
]. This is
partially due to the relatively low concentration of
β
-carotene inside the scaffolds, but
it should be noted that the best results from the literature were observed in carotenoids
extracted by plants [
53
], bacteria [
54
], and animals [
55
], which potentially contain other
bioactive substances that might play a synergic effect. There is no reason to believe the
chemical structure and/or the biological activity of naturally extracted carotenoids to be
different from laboratory standards.
5. Conclusions
Fibers of PCL functionalized with up to 3.6%
β
-carotene could be easily electrospun
from an acetic acid-based solution. This method yields fibers morphologically comparable
with those of the pristine PCL polymer, but with an increased plasticity, a lower ultimate
strength, and a decreased elastic modulus.
The loss of mechanical properties may be attributable to the composite structure, in
particular a sharp reduction in PCL crystallinity, suggesting that
β
-carotene disrupts the
structure of the polymeric matrix without forming strong bonds.
Polymers 2024,16, 1371 18 of 20
The composites demonstrate enhanced bioactive properties, notably in cell prolifer-
ation and bone tissue formation, along with a limited bacteriostatic effect when tested
against E. coli.
These findings indicate that PCL composites containing
β
-carotene hold promise
for biomedical applications. Nonetheless, it is crucial to carefully control the
β
-carotene
concentration, as elevated levels may manifest cytotoxic effects. Further research should
explore methods to improve the bonding between PCL and
β
-carotene. This could involve
exploring alternative solvents or the incorporation of bonding molecules to create a more
cohesive and mechanically robust composite.
Author Contributions: Conceptualization, E.M. and H.X.; methodology, H.X., V.B., A.L. and F.B.;
software, E.M.; validation, F.M.T., W.Z., A.L. and E.G.; formal analysis, F.B. and A.R.; investigation,
O.Y., V.B. and F.B.; resources, E.M., F.M.T. and H.X.; data curation, E.M. and W.Z.; writing—original
draft preparation, E.M.; writing—review and editing, E.G., F.B., V.B. and A.R; visualization, E.M.;
supervision, A.L., E.M., F.M.T. and H.X.; project administration, E.M., V.B.; funding acquisition, E.M.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the KIT Alumni Association/KIT Young Researchers Support
Project of the Kyoto Institute of Technology, 2022.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors on request.
Acknowledgments: The authors are thankful to Saburo Hosokawa and Ryusuke Sugimoto of the
Faculty of Materials Science and Engineering, Kyoto Institute of Technology, for the X-ray analyses.
Conflicts of Interest: The authors declare no conflicts of interest.
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