J. Joseph, R.P. Kumar, A. John, A. Abraham / Trends Biomater. Artif.

Organs, 34(3), 75-83 (2020)

Original Article www.sbaoi.org/tibao

Phytochemical Incorporated Spunned Nanofibrous

Polycaprolactone Scaffold for Stem Cell Loading and Drug
Delivery Applications
Josna Joseph1,2, R. Pradeep Kumar1,3, Annie John1, Annie Abraham1*
1
Advanced Centre for Tissue Engineering, Department of Biochemistry, University of Kerala, Thiruvananthapuram 695581, India
2
Christian Medical College, Vellore 632004, Tamil Nadu, India
3
Marshall Institute for Interdisciplinary Research, Marshall University, West Virginia 25755, Unites States

Received 6 April 2020 The merging of traditional medicine with modern technology in the building up of novel scaffold devices, is of
Accepted 11 May 2020 additional relevance in this era of regenerative medicine. Hence, the aim of this study is to fabricate and test the
Published online 15 July 2020 safety and efficacy of the flavonoid Naringenin, incorporated Polycaprolactone (Nar-PCL) electrospun nano
fibrous scaffolds intended for drug delivery and as stem cell loading stations. Electro spun mats of 5 % and 20%
wt/wt Naringenin-PCL was fabricated and physico-chemically characterisated by FTIR, NMR spectroscopy, XRD
analysis and SEM. In vitro drug release of Naringenin from spun mats were studied in PBS for 3, 6, 12 days
spectrophotometrically at 292 nm. L929 fibroblasts were grown over Nar-PCL and control PCL scaffolds and Cell
adhesion was demonstrated by SEM,viability by Calcien AM- Ethidium homodimer staining, and proliferation by
MTT and LDH assay. Simultaneously, viability and adhesion of isolated rat Adipose derived Mesenchymal stem cells
(ADMSCs) on different scaffolds were also assessed. Antibacterial activity of Naringenin against Psuedomonas Aeruginosa
and Staphylococcus aureus was analysed by Kirby-Bauer method. Finally, the combination product of 5% wt/wt Nar-
PCL portrayed better cytocompatible, regenerative and antibacterial properties of Naringenin, proving as a prospective
candidate for stem cell loading and drug delivery.

© (2020) Society for Biomaterials & Artificial Organs #20039120

Introduction could easily enable the generation of tissue-engineered products.

Drug delivery scaffolds incorporating synthetic stimulants such as
growth factors and cytokines for stem cell growth and tissue
induction are used in regenerative medicine. The high cost and
side effects of these growth factors [1] and mitogens necessitate
the need for the search of alternatives like clinically well-proven,
indigenous Phyto-factors in tissue engineering [2]. Their expected
role is chemotactic recruitment of endogenous stem cells and their
further differentiation in special microniches like phytochemical
incorporated novel scaffold systems, which is a leap in the realm
of tissue regeneration.
Phytochemicals used in traditional medicine are treasure houses
which have a wide range of biochemical and functional activities
that are yet to be explored and if tagged to innovative technologies
*Coresponding author
E-mail address: annieab2013@gmail.com (Prof. Annie Abraham, Advanced Centre for
Tissue Engineering, Department of Biochemistry & Director of Research, University of
Kerala, Kariavattom, Thiruvananthapuram, Kerala, India)
Various well-standardised phytochemicals have been used for
medical applications. Naringenin (4, 5, 7- trihydroxyflavanone,
NAR), a natural flavonoid aglycone of naringin, is widely
distributed in citrus fruits, tomatoes, cherries, grapefruit and cocoa
[3] and has well-known antioxidant bioactivity. Reports prove
that Naringin, which is the main component of the Rhizoma
Drynariae extract, enhanced the proliferation and osteogenic
differentiation of BM-derived hMSCs [4]. It has also been
extensively investigated for its pharmacological activities, including
antioxidant [5], antitumor [6], anti-inflammatory [7] and
hepatoprotective [8] effects. Utilising the anti-inflammatory, anti-
microbial and cholesterol-lowering [9] effects of bioflavonoids
like Naringenin, novel phytochemical incorporated constructs
could be synthesised for tissue engineering applications to aid
ailing tissues/organs.
Though various scaffold manufacturing processes are practised,
electrospinning has proved to be a simple and useful technique
for fabricating nanofibers from a rich variety of materials [10].
Electrospun fibres possess a high surface area to volume or mass
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 J. Joseph, R.P. Kumar, A. John, A. Abraham / Trends Biomater. Artif. Organs, 34(3), 75-83 (2020)
ratio, high porosity and vast possibilities for surface
functionalization [11]. Several research groups have formulated
electrospun scaffolds from various natural and synthetic polymers,
for potential use in human healthcare [12.13]. The high cost of
growth factor laden scaffolds necessitates the search for cost-effective
indigenous phytofactors added scaffolds for tissue engineering
applications. In the pioneering research, [14] Suganya et al. have
successfully synthesised Aloe vera incorporated PCL nanofibrous
scaffolds incorporated with silk fibroin-hydroxyapatite by
electrospinning method for bone tissue engineering applications
[15]. Similarly, phytochemical extracts with known osteogenic
properties like Cissus quadrangularis[16] have been loaded in chitosan/
carboxymethylcellulose scaffolds. Again, curcumin eluting PCL
nanofibers possessing osteogenic properties have been
electrospinned [17] too. Recently, Carica papaya loaded poly (vinyl
alcohol)-gelatin nanofibrous scaffold were used for wound healing
applications [18].
Also, Polycaprolactone (PCL) is a biodegradable and biocompatible
FDA approved polymer used in several products for clinical use
[19]. Hence, this study aims to fabricate Naringenin incorporated
PCL (Nar-PCL) electrospun scaffold for stem cell loading and drug
delivery for tissue regeneration applications.
Materials and Methods
Naringenin, ≥ 95% and polycaprolactone, avg.mn.80,000 (Sigma-
Aldrich), dimethylformamide (Spectrochem), tetrahydrofuran
(Spectrochem), dulbecco’s modified eagle’s medium (Himedia),
foetal bovine serum (Gibco), penicilillin-streptavidin (Invitrogen),
Ezcount MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide) assay kit, Himedia, LDH (Lactate De-
hydrogenase) cytotoxicity kit, Cayman Ltd., live-dead kit, Molecular
Probes.
Fabrication of phytochemical incorporated scaffold
An initial screening of various phytochemicals was carried out and
Naringenin, a natural flavonoid found widely in citrus fruits was
selected as the phytochemical of choice due to its pharmacological
activity. 10% wt/v PCL solution was prepared in Dimethyl
formamide (DMF) and Tetrahydrofuran (THF) at 4:1 ratio reported
accordingly by Remya et al. [20]. Then, 5 % wt/wt and 20% wt/wt
Polycaprolactone-Naringenin (weight of Naringenin concerning the
weight of PCL) solution was prepared in this solvent mixture. The
mixture was stirred in an RB flask at 200 rpm for 2 hours on a
magnetic stirrer. 20ml of this solution was electrospun at a flow
rate of 1ml/h at 12 kV from a spinneret (20 ml BD BioSciences
syringe, internal diameter ~19 mm) and the fibres were collected
on a rotating drum, rotating at a speed of 500 rpm (Holmarc Pvt
Ltd, Kochi). Neat-PCL electrospun mats (10%PCL) were fabricated
using similar spinning parameters. The electrospun mats were dried
at room temperature and dipped in 70% alcohol and UV sterilised
for further experiments. Similarly, control PCL electrospun bare
mats alone was also synthesised at the same flow conditions. The
spinning conditions were optimised after following five different
sets of spinning conditions as shown in table 1.
Table 1: Optimisation conditions of Electrospinning of Nar-PCL and PCL mats
Condi tion I Condition II Condition III
Flow rat e 1 ml/hr 1 ml/hr 1 ml/ hr
Voltage 10 kV 10 kV 13 kV
Tip to c ollector distance 13 cm 16 cm 16 cm
Speed of rotating drum 500 rpm 500 rpm 500 rpm
76
Physico-chemical characterisations of the Scaffold
Morphology and chemical composition of the scaffold was
characterised by Scanning Electron Microscopy (SEM), Fourier
Transform Infra-Red (FTIR) spectroscopy, X-ray Diffraction
Analysis (XRD) and Nuclear Magnetic resonance (NMR)
spectroscopy analysis respectively.
The spun scaffolds were gold coated and surface observed under
the scanning electron microscope (FEI, Nova NanoSEM 450). The
average fiber diameters from five different view fields (magnification
-50,000x) of three different scaffolds, namely PCL (control scaffold),
Nar-PCL (5% wt/wt) (T5) (B), Nar-PCL (20% wt/wt) (T20) were
measured using the software XT Microscope.
The surface functional groups of PCL scaffolds with and without
drug incorporation was studied by NICOLET 5700 FTIR in the
range of 400- 4500 cm-1 at room temperature in ATR mode. Change
in the spectral pattern was noted.
The XRD pattern of the samples was recorded on Bruker D8
Advanced X-Ray Diffractometer with CuKα radiation at an angular
incidence of 10-80o (2θ angle range). The analysis of the samples
was carried out by a computer software MDI-JADE and MAUD
(Material Analysis using Diffraction).
Also, a single pulse 1H nuclear magnetic resonance spectro-
photometer (1H NMR, AVANCE III, Bruker) was used to study
the chemical integrity of Naringenin in the PCL solution before
electrospinning. Deuterated chloroform (CDCl3) was used for
dissolving the polymer.
Contact angle and mechanical strength testing
The water contact angle measurement was made using a standard
contact angle analysis system in the air surrounding with a water
drop. Contact angle values indicate the hydrophobic/hydrophilic
nature of the biomaterials. The greater the water contact angle the
greater the hydrophobic nature of the material. The scaffolds (n =
6) were cut to the dimensions 4 cm × 1.5 cm. The contact angle was
determined in water using the Sessile drop technique using KSV
Sigma 701 tensiometer. The volume of the water drop was 10 µl.
Six measurements from each sample were recorded, and the average
of consecutive three values from each sample was taken.
The mechanical properties of scaffolds were assessed using Tritec
2000B (Triton Technology Limited, UK). Rectangular specimens
with dimensions (10 mm × 4 mm) were tested under tensile mode
at a frequency of 1 Hz and a temperature range from -100°C to
40°C.
In vitro drug release study
In vitro drug release was carried according to ISO 10993-13:2010
[21] (ISO 10993 13:2010) which provides general requirements for
the design of tests in a simulated environment for identifying and
quantifying degradation products from finished polymeric medical
devices ready for clinical use. Drug release was studied by incubating
Condition IV Condition V
1 ml/ hr 1 ml/hr
11 kV 12 kV
13 cm 16 cm
500 rpm 500 rpm
 J. Joseph, R.P. Kumar, A. John, A. Abraham / Trends Biomater. Artif. Organs, 34(3), 75-83 (2020)
the 5% wt/wt Nar-PCL mat (1 x 1cm) in 10 ml phosphate buffered
saline (PBS) at a pH of 7.4. at 37oC incubator for 3, 6, 12 days. At
specific periods, the PBS was collected and the nanofibrous sample
was replenished with fresh PBS. The UV absorbance of Naringenin
in PBS was determined using a UV spectrophotometer (Biotek,
USA) at λmax=292 nm. The rate of drug release over the different
period was inferred from the absorbance curve
Cellular adhesion on scaffolds by SEM studies
Cells (L929 fibroblasts) were seeded over the scaffolds at a cell
density of 1x104 /ml and grown in DMEM with 10% FBS, 1%
Penicillin-Streptavidin. Then the culture supernatant was removed
and cells were fixed with 4% paraformaldehyde. After overnight
fixation at 4oC, the cells were washed with phosphate buffered
saline and dehydrated in ascending grades of ethanol (50%, 70%,
90%, 100%) and isoamyl acetate, critical point dried and sputter
gold-coated before viewing under the scanning electron microscope.
Cytocompatibility evaluation- L929 fibroblasts
MTT Assay [22]
Cells were seeded over Nar-PCL (T5, T20) and PCL at 1*104/ml in
24 well plates and incubated for 24 hours and examined under a
phase-contrast microscope. Cells treated with 10mM concentration
of Cisplatin were taken as a positive control. After incubation, the
medium was removed and stored at -200C for further LDH assay.
Then the cells were added with 500µl MTT solution (50 µl reagent
+ 450 µl serum-free media) and incubated for 3hours at dark. The
yellowish MTT is reduced to dark coloured formazan by viable cells
only. The formazan crystals formed were solubilized with MTT
lysis buffer. The colour developed was quantitated with ELISA
plate reader (BioRad systems, USA) measuring at wavelength 570
nm.
The cells survival (CS) expressed as a percentage was calculated as
follows.
CS = (OD drug exposed cell / Mean OD control wells) x 100
The graph was plotted by taking percentage viability in the y-axis
and different sample groups in the x-axis. The results were analysed
statistically by one way ANOVA.
Lactate Dehydrogenase (LDH) Assay
This is a colourimetric assay that quantitatively measures LDH, a
stable cytosolic enzyme that is released into the culture medium
upon cell damage or lysis occurs during both apoptosis and necrosis
[23]. LDH catalyses the reduction of NAD+ to NADH and H+ by
oxidation of lactate to pyruvate, which in turn catalyse the reduction
of a tetrazolium salt to a coloured formazan and the absorbance
of the formazan developed can read at 490 nm. The amount of
formazan produced is proportional to the amount of LDH released
into the culture medium.
Table 2: Antibacterial effect of Naringenin on Staphylococcus aureus and
Psuedomonas aeruginosa (inhibition zone is indicated in mm in the table)
Zone of inhibition (mm) against Zone of inhibition (mm) against
Drug Conc. Staphylococcus aureus MTCC 96 Pseudom onas aeruginosa MTCC 96
Plate I Plate II Plate III Plate I
Naringenin
R R R 17
(10 mg/ml)
Naringenin
R R R 15
(5 mg/ml)
Naringenin
R R R 10
(2.5 mg/ml)
Amoxyc illin (Am)
19 20 18 20
30 mcg/disc
From the media collected from cells seeded over Nar-PCL (T5,
T20) and PCL, 100 µl of the supernatant was added to 96 well cell
culture plates. Then added LDH assay buffer and incubated plates
with gentle shaking for 30 minutes at room temperature. Read the
absorbance at 490 nm using a plate reader (BioRad, USA). LDH
activity was determined by plotting a graph against absorbance in
the y-axis and different experimental groups in the x-axis. The
results were analysed statistically by one way ANOVA.
Cell viability: Live-Dead Staining
Cell viability on the scaffold was analysed by Calcein AM- Ethidium
homodimer Live Dead staining. Cell seeded constructs were
incubated in DMEM containing 4mM Calcein-AM and 2mM
Ethidium homodimer-1 for 30 min. Live cells are permeable to the
nonfluorescent Calcein AM which is converted into fluorescent dye
Calcein by the cytoplasmic esterase present in living cells. On the
other hand, Ethidium homodimer-1 enters the cell if there is
damage to the membrane integrity and is fluorescent when bound
to the nucleic acid. The imaging was done using a Zeiss fluorescence
microscope.
Cytocompatibility-adipose-derived mesenchymal stem cells
(ADMSCs)
Isolation of ADMSC
Adipose tissue was collected aseptically from cadaver rats (IAEC
approval No. IAEC-KU-2/2016-17-BC-AA (49)). About 2g of
adipose tissue retrieved from the suprascapular subcutaneous site
of animal model, retrieved aseptically, was minced manually and
mixed with an equal volume of 0.2% collagenase (Invitrogen)
solution, digested at 37ºC for 30 mins and filtered. The digest was
centrifuged at 15º C for 10 mins at 2500 rpm & pellet re-suspended
in DMEM – HG, seeded into T25cm 2 flask (NUNC) and
maintained in culture with DMEM - HG containing 10% fetal
bovine serum (FBS) and 50 units/ml of penicillin and 50 mg/ml
of streptomycin (Gibco, Anti – Anti Mix) at 37ºC under 5% CO2
atmosphere. The medium was changed every three days until
confluency. On confluency, the cells were trypsinised by adding
0.25% Trypsin EDTA in the culture dish for 5 minutes. Fresh
medium was added to stop the action of trypsin, the trypsinised
cell suspension was then centrifuged at 2500 rpm for 5 minutes to
collect the pellet. The pellet was re-seeded again (P1) and further
cultured and passaged, with medium renewal alternate day, until
the desired passage is reached.
Characterisation by Immunofluorescence
P3 ADMSCs, which were isolated as mentioned above, was
observed and photographed by Phase Contrast Microscope for its
fibroblast-like morphology and confirmed by immunofluorescent/
immunocytochemical staining with FITC labelled CD 90 antibody
(ab226, Abcam). To evaluate the morphology of isolated ADMSCs,
the cells in the P3 passage were cultured on sterile coverslips for 24
Plate II Plate III
16 18
14 16
9 11
18 22
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Figure 1: Scanning Electron Micrographs of PCL (A) Nar-PCL (5% wt/wt) (B), Nar-PCL (20% wt/wt)(C). Morphology of
Nano fibrous electro spun mats (magnification-800x) along with their respective higher magnification (D, E, F 50,000x).. The
mean diameter of the fibres depict their nano-submicron range size and show significant difference(p÷d”0.01),a-PCL and T20,
b-T5 and T20, with their respective Contact angle measurement (G) PCL, (H) 5% wt/wt and (I) 20% wt/wt

Figure 2: FTIR Spectra of standard Naringenin, Nar-PCL (5% wt/wt) and PCL
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Figure 3: NMR Spectra of Naringenin-PCL solution prior to electro spinning of fibres showing the incorporation of drug to
PCL

hours and then fixed in 3.7% paraformaldehyde. The cell-seeded SEM revealed the random aligned fibrous nature of the neat-PCL
coverslips were given three washes of PBS for 5 minutes before (figure 1A) scaffold whereas there were precipitates (likely of the
chemical fixation and also before fluorescent staining. The fixed drug, Naringenin) over the Nar loaded PCL (figure 1B,1C,1F). There
cells were then permeabilized using 0.1% Triton (Sigma chemicals, was a significant increase in the diameter of 20%wt/wt Nar-PCL,
India) for 5 minutes and protein blocked for 5 minutes and treated ~840 nm (figure 1C, F) with a considerable increase in the precipitates
with FITC labelled CD 90 primary antibody overnight. After PBS of Naringenin over the fibres when compared to neat-PCL.
washing three times, the stained coverslips were then mounted
using fluorescent mounting media (Dako Ltd.) and viewed under The contact angle of the different scaffold surfaces, measured by a
fluorescent microscope TCS SP8 (Leica, Germany). goniometer revealed the increase in hydrophilicity of the
phytochemical incorporated group ((132.3ο(T5) 103.3ο(T20)) in
Anti-bacterial activity of Naringenin comparison to the neat-PCL (137.8ο) (figure 1).
Antibacterial activity of Naringenin against Pseudomonas aeruginosa Physico-chemical characterisation of the phytochemical
and Staphylococcus aureus was analysed by Kirby- Bauer disc diffusion incorporated scaffold
method. Briefly, overnight bacterial cultures were diluted in the
Nutrient broth to obtain a bacterial suspension of 108 CFU/ml. FTIR Spectroscopy
Filter paper discs of Whatman no.1 (6 mm diameter) saturated The characteristic peaks of Naringenin indicating C-OH stretching,
with extract of Naringenin (50, 100µg) was placed on bacterial C-Br strong bond at 1054, 718 cm-1 (benzene derivative), was found
suspension smeared plate. The 70% ethanol solution was placed in Nar-PCL scaffold (1048, 730 cm-1 ) with a relative shift (figure 2)
as control. The plates were incubated at 37 °C for 24 h. The
antibacterial activity was assessed by measuring the zone of
inhibition around the drug incorporated disc.
Statistics
All the quantitative tests were evaluated statistically by one way
ANOVA test using the vasserstat online statistical program. The
results are represented as mean ± SD.
Results
Morphological analysis of Phytochemical incorporated
scaffolds by SEM
Naringenin incorporated Polycaprolactone mats consisting of nano-
to submicron-sized fibres were synthesised (figure 1). Electron
micrographs showed that the electrospun mats (5% wt/wt) were
composed of nano-sized fibres (~290 nm) (figure 1B,1E) and is
thinner than the fibres in the PCL mats (~310nm)(figure 1A,1D), Figure 4: In vitro drug release profile from 5% wt/wt Nar-
which is ideal for better cell attachment and proliferation. PCL at pH7.4. The results are mean ± SD, n=4, p < 0.05
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Figure 5. Cell adhesion by SEM evaluation of L929 cells grown over PCL (A) T5(B) and
T20(C) after 24 hours of culturing (magnification-3000x)

and confirmed the incorporation of Naringenin compound to the
PCL scaffold. PCL shows the characteristic peaks at 1744 cm-1 (C=0
ester bond), 1188 cm-1 (C-O-C stretching) and 2917 cm-1 (C-H
stretch).
XRD analysis
From the XRD pattern, the characteristic peak of PCL is presented
clearly (figure not included) at diffraction angles of 2θ, 21.65ο, 23.25ο,
47.49ο and 48.52ο respectively. Similar peaks were identified in Nar-
PCL scaffolds whereas PCL scaffold and Nar-PCL scaffolds did not
show much difference in the crystalline structure.
NMR Spectroscopy
FTIR spectroscopy combined with NMR spectroscopy has
confirmed the loading of the drug to PCL scaffolds with standard
peaks of Naringenin in Nar-PCL.
The chemical shifts of these are identical to that of Naringenin and
PCL suggesting that Naringenin is distributed in the PCL fibres.
The right-hand side of the spectrum shows peaks corresponding
to PCL (figure 3) with chemical shifts at δ 1.36-1.42 ppm and in the
range of δ 1.6-1.69 ppm are attributable to the -(CH2)3; δ 2.2-2.31
ppm is associated with the -CH2 protons adjacent to the carbonyl
group and δ 4.02 ppm corresponds to the methylene protons
adjacent to the oxygen moieties of the ester linkage, whereas the
left-hand side of the spectrum corresponds to characteristic peaks
of Naringenin.
In vitro drug release
In vitro drug release carried out according to ISO guidelines showed
that the maximum rate of Naringenin was released on 6th day (figure
4) when the drug release was studied in PBS- pH of 7.4 at 370C.
The absorbance of Naringenin was read at 292 nm based on the
earlier published study [24]. This data showed there was a continued
release of Naringenin even at 12 days as revealed by in vitro drug
release study warranting the sustained drug release potential of
these scaffolds.
Cell adhesion studies
L929 cells showed more attachment and spreading on Nar-PCL
scaffold (T5&T20) (figure 5B, 5C) than the control (figure 5A) after
an incubation of 24 hours. There was a change in the usual
morphology of L929 cells observed generally, from their normal
fusiform shape to round shape as seen in the SEM photographs.
In vitro cytotoxicity
MTT assay
The cell viability, inferred from MTT Assay at 24h was high (99%)
for Nar-PCL - 5%, T5 when compared to Nar-PCL - 20%, T20,
PCL and lowest (54%) on cisplatin added group (figure 6A). There
was a statistically significant difference between cisplatin (positive
control for cytotoxicity) and all other experimental groups(T5, T20,
PCL) and between the different experimental groups.

Figure 6: Cell proliferation of L929 fibroblasts grown over Nar-PCL(T5, T20), PCL and Cisplatin
control group analysed by A) MTT assay; B) LDH assay, n=3, *p < 0.001, mean ± SD
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Figure 7: Fluorescence Micrographs of cell viability over the different scaffolds demonstrated by Live- Dead
staining of ADMSC’s grown over A) neat PCL B) T5 and C) T20 (10 X magnification). Cell adhesion over the
scaffolds shown by SEM of ADMSC seeded over D) neat PCL E) T5 and F) T20 (Magnification 1000x)

LDH Assay
Similarly, the highest LDH release was observed on cisplatin added
group when compared to other groups (figure 6B), confirming the
observation from MTT assay. There was a statistically significant
difference between cisplatin (positive control for cytotoxicity) and
all other experimental groups(T5, T20, PCL) and between the
different experimental groups.
Cell viability – Live Dead staining.
The cellular viability evaluation by Calcein AM-Ethidium
Homodimer staining showed corroborating results with that of
MTT and LDH assays. More viable cells with normal phenotype
were observed on Nar-PCL-T5, when compared to the control
PCL scaffold alone and Nar-PCL-T20 (figure not shown).
Adipose-Derived Stem cells- cell adhesion and viability over
scaffolds
Similar to L929 fibroblasts, when ADMSC’s (positive for CD90 &
CD105 markers-data not shown) were cultured over the different
electrospun scaffolds, cell viability was found high on T5 (figure
7B), when compared to ADMSC on T20 (figure 7C) and Neat PCL
(figure 7A). A similar trend was observed for the ADMSC adhesion
over the Nar-PCL scaffolds (figures 7E,7F) when examined by
SEM. Interestingly, more cellular adhesion and spreading of
ADMSCs were observed when compared to L929 fibroblasts when
grown over the PCL scaffolds. This is promising, paving way for
future endeavours of exploiting the proven osteoregenerative
potential of Naringenin for the creation of stem cell-seeded
osteoregenerative constructs for implantation in bone injury models.

Figure 8: Anti-bacterial activities of Naringenin against S. aureus MTCC 96 and P. aeruginosa MTTC 424
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Antibacterial activity of the phytochemical
The anti-bacterial effect of Naringenin against common skin
pathogens was also confirmed by disc-diffusion method (figure 8
and table 2). These results are significant to avoid iatrogenic
infections, which is a common type of infection, associated with
implantation procedures. Naringenin extract showed a zone of
inhibition against common skin pathogens such as Staphylococcus
aureus and Pseudomonas aeruginosa in comparison with that of the
standard antibiotic, streptomycin (table 2). Since Staphylococcus aureus
[25] is one of the most common implant-associated pathogens,
the potent antibacterial effect of Naringenin loaded scaffolds would
be beneficial in wound healing.
Discussion
Biologically derived matrices are preferred choices over synthetic
ones for tissue repair due to their biocompatible nature, tissue
integration abilities and their matching mechanical properties with
tissue [26,27]. The advent of stem cells, with their self-renewing
and differentiation characteristics, offer a therapeutic promise of
high potential. Replenishing stem cells from exogenous or
endogenous sources to the site of injury is the most challenging
task in tissue regeneration. The general practice in induced cellular
differentiation and proliferation is the usage of high-cost growth
factors of animal origin. This necessitates the search for an alternate
source of inducing factors and phytochemicals with inherent tissue
regenerative properties, less toxicity and affordable cost to emerge
as potential candidates. Well characterised Phyto-factors are treasure
houses yet to be explored, though used from time immemorial
for tissue regeneration in traditional medicine. The regulation of
β-catenin, which is involved in osteoblastogenesis via Akt (protein
kinase B) and AMPK (AMP-activated protein kinase) signalling,
has been demonstrated as a possible mechanism for the osteogenic
properties of Naringin [28]. The outcome of this study, if translated
to the clinical setting would help in the development of economic,
indigenous healthcare products like osteoinductive scaffolds.
Nanotechnology and advanced fabricating techniques like
electrospinning offer abundant possibilities in the development
of new generation biomaterials and tissue-engineered products
due to the varied biological responses to nano and micro
dimensional matrices. Further, electrospinning is a technique that
can be used to fabricate continuous fibres from micrometre to
nanometer diameter structures. Suitable polymer formulation with
or without drug incorporation fed through a spinneret and high
voltage is applied to the tip for producing a thin fibrous stream
[29, 30]. The simplicity of the electrospinning process itself can
also provide the ability to conveniently incorporate therapeutic
compounds into the electrospun fibres for preparing useful drug
delivery systems. Electrospun drug delivery systems combine the
ideal nanosize properties with physical entrapment of drug/
phytochemical moieties resulting in the continued release of drug
molecules to the target site. Thus, this study was carried out to
fabricate a polymer-phytochemical hybrid electrospun scaffold
incorporated with an osteogenic, anti-atherogenic, anti-inflammatory
natural flavonoid, Naringenin for tissue engineering applications.
Though various groups have formulated electrospun scaffolds from
different polymer sources, attempts to incorporate standardised
phytochemicals to electrospun scaffolds are very few and nil with
the flavonoid compound, Naringenin emphasising the relevance
of this study.
SEM images (Fig 1) and FTIR (Fig 2) spectroscopy have confirmed
the morphology and the drug incorporation into the scaffolds
respectively. The in vitro drug release also confirms the stability of
Naringenin drug released into PBS from the electrospun scaffold
82
as the typical peaks of Naringenin at 292 nm was observed clearly at
all measured period. The wettability studies by contact angle
measurement have revealed the increase in hydrophilicity of the
scaffold surface upon the incorporation of phytochemical (Fig 1).
In this study, we could observe that the L929 fibroblast cells showed
more adhesion, viability and proliferation on Nar-PCL scaffold
when compared to the neat PCL. The lack of expected spread
morphology might be attributed to two main factors, the shorter
incubation time as well as the wettability of the PCL scaffold. Poly
Capro Lactone scaffolds are hydrophobically proven by the
wettability measurements (data from contact angle). Also, some
earlier reports with L929 cells seeded PCL scaffolds showed similar
cell attachment records with the morphology of fibroblast cells
over the PCL scaffolds as round and spherical in contrast to the
fusiform shape. Hence, they have attempted to create co-blend
scaffolds with more hydrophilic polymers like PLGA and PVA [31,
32]. Similarly, in the current study, we have attempted to incorporate
a well-known anti-oxidant, anti-bacterial phytochemical, Naringenin
to PCL scaffold to enhance its wettability, cell adhesion, proliferation
properties (Fig 1, 5). This has been proved successful from the
increase in hydrophilicity of the Nar-PCL scaffold favouring more
cellular adhesion.
The isolated ADMSCs were positive for stem cell markers, CD90
and CD 105 (images not included), proving their stemness. In
contrast to the cytocompatibility studies with L929 fibroblasts,
ADMSCs showed better adhesion, viability and proliferation over
Nar-PCL scaffolds than on neat PCL (Fig 7). One plausible reason
is the MSCs acquiring an elongated spindle-shape morphology,
faster than fibroblasts when exposed to tumour secreted soluble
factors [33]. Further, this might also be attributed to the reported
proliferative effects of Naringenin on Mesenchymal stem cells [4].
Also, previous studies have proven the osteogenic differentiation
potential of mesenchymal stem cells when seeded over neat
polycaprolactone nanofiber scaffold [34] and PEG-PCL copolymeric
nanofiber scaffolds [35]. Thus, these synthesised drug delivery
scaffolds have novel multivariate applications like stem cell loading
for exogenous supply to the site of injury as well as the drug
delivery capability for attracting endogenous stem cells for further
initiation of the regenerative and reparative processes.
Earlier studies from our group confirmed that poly Vinyl
pyrrolidone (PVP) coated Naringenin nanoparticle exerts significant
antioxidant and anti-inflammatory potential, without altering any
biochemical and haematological parameters [24, 36]. Hence, through
this study, we have attempted to create a novel drug-loaded
electrospun scaffold that has great potential in tissue engineering
applications. Cell adhesion, proliferation and viability studies have
proved that 5 % wt/wt in comparison to 20% wt/wt PCL-
Naringenin electrospun scaffold showed better cytocompatibility
and this has been selected for further studies. Future in vivo studies
could further warrant the osteogenic regeneration potential of these
novel hybrid scaffolds. To our knowledge, this is the first attempt
to synthesise the well standardised phytochemical, Naringenin
incorporated electrospun scaffolds for stem cell loading and drug
delivery applications.
Conclusions
Novel electrospun biodegradable PCL scaffolds possessing
antibacterial effects of bioflavonoid, Naringenin proved to support
the cellular attachment, viability and proliferation of adipose-derived
stem cells and normal fibroblasts. The findings of this study could
elicit a future stream of investigations probing the usage of Phyto-
factors as safer, economic alternatives to synthetic growth factors
and mitogens. Considering the anti-atherogenic and osteoinductive
 J. Joseph, R.P. Kumar, A. John, A. Abraham / Trends Biomater. Artif. Organs, 34(3), 75-83 (2020)
properties of Naringenin, these electrospun scaffolds could be
further developed as cost-effective, indigenous and
osteoregenerative scaffolds.
Acknowledgement
The authors sincerely thank Dr Rahul Mahadevan Pillai for the
technical help. The authors would like to acknowledge the
Sophisticated Instrumentation & Computation Centre of the
University of Kerala and testing facility at BMT wing, SCTIMST,
Thiruvananthapuram for evaluation of the samples. The first
author acknowledges the financial assistance from YIPB project,
Kerala Biotechnology Commission, KSCSTE and Emeritus UGC
Fellowship for AJ.
Data Availability Statement
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
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