A recent publication by Dr. Sachin Kadam, CTO of Advancells, sheds light on natural osteoinductive bio-compatible scaffolds for effective clinical applications on musculoskeletal disorders. These scaffolds can enhance the efficiency of stem cell transplantation, and thus improve healthcare manifold.

1. https://doi.org/10.1177/0883911519894667
Journal of Bioactive and
Compatible Polymers
1­–17
© The Author(s) 2020
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DOI: 10.1177/0883911519894667
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JOURNAL OF
Bioactive
and
Compatible
Polymers
Layer-by-layer decorated herbal
cell compatible scaffolds for
bone tissue engineering: A
synergistic effect of graphene
oxide and Cissus quadrangularis
Shivaji Kashte1
, RK Sharma2
and Sachin Kadam1,3
Abstract
Among various bone regenerative and repair methods, use of osteoinductive scaffold as bone
grafts/substitute has gained wide importance worldwide. To develop such osteoinductive scaffold
that is more natural and which spontaneously stimulates osteoblast formation without any
differentiation media, we prepared electrospun poly ε-caprolactone scaffold which is further
modified by means of layer-by-layer method using Cissus quadrangularis callus culture extract
and graphene oxide (PCL-GO-CQ). The modified PCL-GO-CQ scaffold was compared with
plain poly ε-caprolactone scaffold and poly ε-caprolactone coated only with graphene oxide.
Physical properties, such as roughness, wettability, yield strength and tensile strength, of PCL-
GO-CQ scaffold were found to be superior. Also, PCL-GO-CQ scaffold showed more in vitro
cell compatibility with enhanced cellular proliferation on its surface. Presence of graphene oxide
and Cissus quadrangularis callus in scaffold helped in the differentiation of human umbilical cord
Wharton’s jelly-derived mesenchymal stem cells into osteogenic lineage without any differentiation
media in less than 20 days. The synergistic effect of Cissus quadrangularis callus extract and
graphene oxide in PCL-GO-CQ scaffold enhanced osteoblastic differentiation, osteoconduction
and osteoinduction potential of scaffolds making them highly potential in bone regeneration and
bone tissue engineering applications.
894667JBC0010.1177/0883911519894667Journal of Bioactive and Compatible PolymersKashte et al.
research-article2020
Original Article
1
Department of Stem cell and Regenerative medicine, Center for Interdisciplinary Research, D. Y. Patil Education
Society (Deemed to be University), Kolhapur, India
2
D. Y. Patil Medical College, D. Y. Patil Education Society (Deemed to be University), Kolhapur, India
3
Advancells Group, NOIDA, India
Corresponding author:
Sachin Kadam, Advancells Group, NOIDA, Uttar Pradesh 201301, India
Email: kadamsachin@gmail.com

2. 2 Journal of Bioactive and Compatible Polymers 00(0)
Keywords
Scaffolds, graphene oxide, Cissus quadrangularis, human umbilical cord Wharton’s jelly-derived
mesenchymal stem cells, bone tissue engineering
Introduction
Bone is a complex connective tissue that plays a
vital role in providing a structural framework,
mechanical support and flexibility to the body. It
is also involved in mineral storage, homeostasis,
and blood pH regulation.1,2
Bone defects and its
repair are the most common problems encoun-
tered worldwide.3
Bone is the second most
transplanted tissue after blood.4
As a matter of
fact, bone tissue engineering has become one of
the mainstream researches in regeneration,
repair or restructuring of bone tissues. Various
research methodologies have been flourished
with biomaterials and their applications in the
field of tissue engineering and regenerative
medicine. The various polymers, such as poly
ε-caprolactone (PCL), poly-d, l-lactic acid
(PDLLA),5
poly(L/DL-lactide) (PLDL),6
poly-
L-lactide (PLLA),7
poly(DL-lactic-co-glycolic
acid) (PLGA),8
have shown to be potent in terms
of their mechanical properties, such as tensile
strength, elastic modulus, biocompatibility,
higher cellular properties and even bone forma-
tion in rat model.4
PCL is a biocompatible, bio-
degradable and FDA-approved material and
used extensively in tissue engineering applica-
tions. PCL along with hydroxyapatite (HA),9
PCL-gelatin hybrid nanofibrous membranes,10
PCL-poly(1,4-butylene adipate-co-polycaprol-
actam (PBAPCL)–HA composite scaffold,11
are
used in bone tissue engineering.
Easily electrospinnable synthetic polymers,
such as PCL and polylactic acid (PLA), are pri-
marily hydrophobic. For its application in tissue
engineering, hydrophobicity of nanofibrous
mesh is required to be modified to become
partly hydrophilic; this can be achieved by sur-
face modification either by flame treatment,
corona discharge treatment, plasma modifica-
tion or surface graft polymerization.12
Surface
modification is one of the crucial factors identi-
fied to be responsible for cellular attachment
and enhanced cellular proliferation. Surface
modification also helps in the improvement of
biological properties of scaffolds. This interac-
tion between tissue and foreign surface largely
depends upon surface properties of materials,
such as wettability, roughness or topography,
surface charge and chemistry.13,14
Layer-by-layer method is a simple, rela-
tively fast, environmentally benign and poten-
tially an economic process15
that prepares
uniform multilayer films on substrates. The
deposition is fast and irreversible, with con-
trollable deposit thickness and uniform surface
coverage.16
Layer-by-layer deposition signifi-
cantly improves wettability and mechanical
strength of scaffold.15
The transformation of
hydrophobic electrospun block copolymer to
hydrophilic mesh using layer-by-layer method
has shown significant improvement in cell
viability and cell attachment of epidermal
cells.12,17,16
In this study, electrospun PCL
scaffolds were modified using graphene oxide
(GO) and Cissus quadrangularis (CQ) plant
callus extract.
GO has gained attention in bone tissue engi-
neering due to its large surface area, low bio-
logical toxicity and osteoinductive nature.18
Studies have found that GO has more hydro-
philic groups and easy dispersion ability.19
GO
along with HA and chitosan functionalized gra-
phene nanoplatelets (GNP) reinforced with
polyvinyl alcohol,20
GO with PLA and HA,21
GO-doped PLGA scaffolds19
and GO-poly-L-
lysine composites22
have applied for bone tissue
engineering. GO is the most fascinating materi-
als of today and their interaction with stem cells
revealed cellular compatibility and ability to
differentiate stem cells into osteoblasts, chond-
roblasts and neuronal lineages.23–25
CQ, also known as ‘Edible Stemmed Vine’, is
known for its medicinal properties which is
described in the ancient Ayurveda book Bhava
Prakash Samhita. Along with antimicrobial and

3. Kashte et al. 3
antioxidant activity,26
methanolic extract of CQ
has proven to be useful for bone fracture heal-
ing27,28
due to its high calcium ions (4% weight)
and phosphorus content. Active constituents of
CQ may promote proliferation and differentiation
of mesenchymal stem cells (MSCs) into osteo-
blasts and bone formation via wnt-LRp5-β-
catenin or mitogen-activated protein kinase
(MAPK)-dependent pathway.29
Therefore, results
show that PCL-CQ-HA nanofibrous scaffolds
have appropriate surface roughness for osteoblast
adhesion, proliferation and mineralization com-
pared to other scaffolds, making them potential
biocompatible material for bone tissue engineer-
ing.30
CQ callus extract accelerates fracture heal-
ingandearlyremodellingoffracture.Aphytogenic
isolated steroid is believed to be the main constitu-
ent in CQ. It has been observed that CQ acts by
stimulation of metabolism like an increased
expression of osteopoietin and increased uptake
of minerals, such as calcium, sulphur and stron-
tium, by osteoblasts in fracture healing.31
Here, porous PCL electrospun scaffolds
were modified with GO and CQ using layer-by-
layer method. These composite scaffolds help
to demonstrate morphological, physical and
biological characteristics that were suitable for
bone tissue.
Materials and methods
Materials
PCL (average molecular weight: 80,000 g/mol)
and GO (particle size: 100–1000 nm, prepared
by modified Hummers’ method) were supplied
by Sachin Kochrekar, Department of Chemistry,
Defence Institute of Advanced Technology,
Girinagar, Pune. All the chemicals were pro-
cured from Sigma–Aldrich (USA) and cell
culture growth media and supplements from
Invitrogen (USA) unless specified.
Methods
CQ callus culture. CQ plant was identified and
collected from Kolhapur region of Maharash-
tra, India, and used for further studies. For
callus culture of CQ, Murashige and Skoog
(MS) medium no. 6 (2.26 g/L; HiMedia, India)
along with α-naphthalene acetic acid (NAA;
2.5 mg/L; HiMedia), 6-benzylaminopurine
(BAP; 0.5 mg/L; HiMedia), sucrose (40 g/L;
HiMedia) and agar (10 g/L; HiMedia) were
used as callus induction medium. pH of the
medium was adjusted to 5.6 and sterilized by
autoclaving, followed by which the stem of
CQ plant was washed for 10 min under run-
ning tap water. These washed stem surfaces
were then sterilized by 70% ethanol (v/v) for
5 min and stem explants were immersed in
0.01% HgCl2 (mercuric chloride) solution
for 2 and 3 min, respectively. Finally, stem
explants were washed five times using sterile
distilled water and cut into 10–13 mm pieces.
These surface-sterilized explants were inocu-
lated into centre of sterile MS media contain-
ing culture tubes. Culture tubes were incubated
in dark at room temperature of 28°C. These
culture tubes were observed daily for callus
formation for 3–4 weeks.
CQ callus culture extraction. Fully grown callus
from 4–5 weeks grown culture was selected,
dehydrated, dried and fine powder was made.
Crude extract was prepared using a Soxhlet
apparatus with ethanol. Thus, the obtained
extract was partitioned using petroleum ether
followed by which pure form of extract was
prepared. This extract was checked for the pres-
ence of phytosterol using Salkowski test.30
Briefly, a small quantity of extract was dis-
solved in 1 mL chloroform. Few drops of con-
centrated sulphuric acid were added along the
walls of test tube containing mixture. The for-
mation of brown ring at the bottom of test tube
indicates the presence of phytosterol.
Preparation of scaffolds by electrospinning. PCL
solution (10% w/v) was prepared by dissolv-
ing PCL in tetrahydrofuran (THF): methanol
(3:1) for 30 h of magnetic stirring. PCL scaf-
folds were fabricated by electrospinning with
parameters of flow rate of 0.8 mL/h, voltage
12 kV. Thus, distance between the tip of
syringe and collector was adjusted to 12.5 cm.

4. 4 Journal of Bioactive and Compatible Polymers 00(0)
PCL scaffolds were then used for further sur-
face modifications.
Modification of scaffolds using layer-by-layer
method. The solutions of GO (1 mg/mL) and
CQ (1 mg/mL) were prepared separately by
dispersing components in distilled water
through sonication. These solutions were used
to modify surface of electrospun PCL scaf-
folds. PCL-GO scaffold was prepared by sim-
ply dipping PCL scaffold repetitively in GO
solution for 2 min followed by air drying. Simi-
larly, PCL-GO-CQ scaffold was prepared by
dipping PCL scaffold first in GO solution and
then in CQ solution, alternatively for 2 min
with intermediate air-drying cycles. To deter-
mine the most effective surface coating, 30, 60
and overnight coating cycles were used for sur-
face modification.
Leaching study was carried for adhesion of
GO as per ISO-10993-12. The leaching was
least for overnight dipped samples followed by
60 cycles as compared to 30 cycles of air drying
after overnight dipping. This could be due to
more interaction between PCL and GO in 60
dipped cycles. However, 60 cycles over 30
cycles were selected with overnight dipped
samples because of more uniform deposition as
well as lesser leaching. It was concluded that
the leaching study was not significant as very
small amount of GO was observed in leached
solution (see Supplemental Material).
Characterization of scaffolds. These prepared
scaffolds were characterized for morphological,
physical, mechanical and biological properties.
Morphological analysis. The fibre diameter of
electrospun PCL and surface-modified scaf-
folds was examined by field emission scanning
electron microscope (FESEM; Carl Zeiss, Ger-
many) at an accelerating voltage of 15 kV. In
case of FESEM, scaffolds were cut into
5 × 5 mm2
, mounted on to sample stubs and
sputter-coated with gold using SC 7640 sputter
coater (Quorum Technologies Ltd, UK). The
coated GO was then analysed on electrospun
fibres of PCL. From FESEM micrographs,
scaffold fibre diameter was measured using
image analysis software (ImageJ; National
Institutes of Health, USA).
Surface morphology of PCL and surface-
modified scaffolds was analysed by atomic
force microscopy (AFM; Asylum Research,
USA) using tapping mode. The scaffolds were
cut into small pieces and were stuck on a glass
slide using cellophane tape. Scan rate of 1.0 Hz
and scan area of 10 µm were used for imaging.
Physical analysis. Fourier-transform infrared
(FTIR) spectra were recorded for all scaffolds
(FTIR; Bruker, Germany). The spectra were
obtained with 30 scans per sample ranging from
3000 to 500 cm−1
.
Wetting properties. Water contact angle was
determined by sessile drop method using drop
shape image analysis software. A droplet of
pure water is deposited vertically on to the sur-
face and contact angles were measured by con-
tact angle goniometer (KRUSS, Germany)
using an optical subsystem. Therefore, angle
formed between solid–liquid interface and
liquid–gas interface is determined as liquid
contact angle. Contact angle measurement of
liquid droplets on a solid substrate (n = 3) was
used to characterize surface wettability and
hydrophilic/hydrophobic nature of surface.
Mechanical properties. Tensile properties were
calculated at room temperature using universal
tensile machine (STS 248; Star Testing Systems,
India). Scaffolds were cut into cylinders (n = 3)
and tested. The maximum loading capacity was
100 N with strain rate of 5 mm/min. Accordingly,
the resulting stress–strain curves, yield strength
and tensile strength were calculated.30,32,33
Isolation and culture of human umbilical cord
Wharton’s jelly-derived mesenchymal stem cells
Collection of human umbilical cords. Human
umbilical cords were collected from caesarean
deliveries with a proper patient consent. These
collected cords were then transported to cell
culture lab in L15 transport medium. Collected
cords were washed with phosphate-buffered

5. Kashte et al. 5
saline (PBS) to remove cord blood and blood
clots. Surface disinfection of these cords was
carried out using 10% betadine solution fol-
lowed by sterile PBS washes. The cleaned
cords were used to isolate human umbilical
cord Wharton’s jelly-derived mesenchymal
stem cells (hUCMSCs).
Isolation and expansion of hUCMSCs. In cell
culture lab under strict sterilization, blood ves-
sels were removed from cleaned cord and cord
was chopped into pieces of 1–2 mm length
using a sterile surgical blade. The chopped cord
tissue was digested with a cocktail of enzymes,
Collagenase Type IV: Dispase II (7:1 v/v), for
30 min at 400 r/min and at 37°C on magnetic
orbital shaker. After 30 min, digested tissue was
exposed to Trypsin (0.05%) and EDTA (0.02%)
for further 20 min digestion. The homogenate
was then filtered through a sterile muslin cloth
and centrifuged at 1500 r/min for 10 min to
isolate pellet. Pellet containing cells were cul-
tured in Dulbecco’s Modified Eagle’s Medium
(DMEM): Ham’s F12 (DMEM:HF12, 1:1)
medium supplemented with 10% serum penicil-
lin (100 units/mL) and streptomycin (100 µg/mL).
The cells were incubated for 48 h at 37°C, 5%
CO2. Hence, the medium was changed after
every 48 h and first passage was carried out
after 8 days followed by every 4 days. The iso-
lated hUCMSCs were cryopreserved using 10%
dimethyl sulfoxide (DMSO) with standard pro-
tocol34
for further applications.
In vitro studies
Cell seeding. Scaffolds were cut appropri-
ately to fit into 48-well plates. These scaffolds
were washed with PBS thrice and then steri-
lized by ethylene oxide (EtO). At ~80% con-
fluency, hUCMSCs were trypsinized and were
seeded on 48-well plates containing scaffold
at cell 1.0 × 104
cells/mL. These cell-seeded
plates were incubated at 37°C, 5% CO2 for 1, 4
and 7 days to study cell attachment, cytotoxicity
and cell proliferation activity.
Cell attachment and proliferation study. After
1, 4 and 7 days in culture, cell-seeded scaffolds
were fixed using 4% paraformaldehyde (PFA).
These PFA-fixed scaffolds were then analysed
for cellular attachment of proliferation over
scaffold surface by various methods, including
FESEM.
Confocal microscopy imaging. Cell-seeded
scaffold post 1, 4 and 7 days of incubation were
fixed with 4% PFA; cells were permeabilized
using permeabilization buffer. Non-specific
binding sites on scaffolds were blocked using
bovine serum albumin (BSA) and cells on
scaffolds that were stained using nuclear stain
4′,6-diamidino-2-phenylindole (DAPI). Slides
were mounted on mounting media, and images
were captured with a confocal microscope (Carl
Zeiss). The images were processed with Zen
software (Carl Zeiss). Analysis with ImageJ
software was performed for cell count.
MTT cell viability and proliferation assay. The
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) was prepared in DMEM
for final concentration of 5 mg/mL (pH = 7.4),
filter sterilized through 0.2 µm filter to sterile
light-protected container. And 50 µL MTT solu-
tions were added into each well of them hav-
ing cell-seeded scaffolds. After incubation of
3 h, DMSO was added to each well to dissolve
formazan crystals. The quantity of a purple-
coloured formazan was measured at 570 nm
with a reference of 650 nm using a plate reader
spectrophotometer (Hitachi, Japan).35
The same
procedure was followed for 4th and 7th day.
Osteoblastic differentiation
Alizarin Red S staining for calcium. The hUC-
MSCs were seeded on scaffolds for 14 and
21 days in serum-free growth media. The con-
trol kept as tissue culture plate with osteoblas-
tic differentiation media containing DMEM
medium supplemented with ascorbic acid
(50 µg/mL), β-glycerophosphate (5 mM), dexa-
methasone (1 × 10−7
M) and nonessential amino
acids (1%). After 14 and 21 days of culture,
mineralization was analysed by staining with
2% Alizarin Red S stain (pH 4.2). After washed
with PBS, the wash samples were observed for

6. 6 Journal of Bioactive and Compatible Polymers 00(0)
Ca++ minerals under an inverted phase-con-
trast microscope equipped with a digital cam-
era. Quantification of osteoblast formation was
performed using 10% acetic acid. After 30 min
incubation with acetic acid, the scaffolds were
placed into microcentrifuge tube, heated at
85°C for 10 min then cooled and centrifuged at
10,000 g for 15 min; 500 µL of the above solu-
tion was taken and neutralized with 10% ammo-
nium hydroxide. Also, 150 µL of this solution
was transferred to 96-well plate and quantity of
Alizarin Red S was determined by measuring
absorbance at 405 nm.36–38
Von Kossa staining for calcium. Scaffolds
were evaluated for mineralization using Von
Kossa staining. The samples were fixed by 10%
formalin and then 1 mL of 5% silver nitrate
(AgNO3) solution was used to stain samples at
room temperature for 60 min under UV light.
The stain was removed and samples were visu-
alized under an inverted phase-contrast micro-
scope and images were taken.
Statistical analysis. Statistical analysis of all
data was performed using Origin Pro 8.5 soft-
ware. Numerical data were presented as
mean ± standard deviation. Statistical signifi-
cance was evaluated using student’s t-test
(p ⩽ 0.05*; p ⩽ 0.005**).
Results and discussion
CQ callus culture and powder
extraction
Brown-coloured callus was observed after
4 weeks of culture (Figure 1(c)).39,40
The purified
form of CQ callus extract showed the presence of
phytosterol. It was then confirmed after the for-
mation of brown ring at the bottom of test tube as
indicated in Salkowski test (see Supplemental
Material). Similar results were mentioned by
Suganya et al.30
Phytosterols are osteoinductive
in nature.30
They stimulate increased expression
of osteopontin, and increased uptake of minerals,
such as calcium and sulphur, was shown by
osteoblasts in fracture healing.31
Phytosterols act
as a main component in bone regeneration.31
Preparation and characterization of
scaffolds
Morphological analysis. FESEM analysis of elec-
trospun PCL scaffolds and layer-by-layer modi-
fied scaffolds (Figure 1) showed electrospun
PCL scaffolds with smooth fibre structures.
Where layer-by-layer modified scaffolds coated
with GO and CQ had rough fibre structures.
Fibre diameter (Table 1) was increased in PCL-
GO scaffolds compared with PCL scaffolds.
Fibre diameter was significantly increased in
the PCL-GO-CQ scaffolds. GO layers on PCL-
GO and PCL-GO-CQ were randomly distrib-
uted throughout PCL scaffold. Fibre diameter
was significantly increased with deposition of
GO and CQ in the respective scaffolds. Higher
fibre diameter also migrate the cells and help in
cell penetration.41,42
The enhanced rough sur-
face increases potential of protein adhesion, cell
adhesion and cell proliferation.30
Surface properties of PCL and surface-
modified PCL scaffolds were analysed by
AFM using tapping mode (see Supplemental
Material). TheAFM images (Figure 3) showed
the rough surface of the scaffolds. Root mean
square roughness (RMS) values were PCL
(146 ± 10 nm), PCL-GO (222 ± 8 nm) and
PCL-GO-CQ (341 ± 9 nm). The modified
scaffolds PCL-GO and PCL-GO-CQ scaffolds
showed higher roughness as compared to PCL
scaffolds.
Similar results were analysed by other
researchers. The coating of polyethyleneimine–
GO on PLA films also showed rough, uneven,
mountain-like topography compared to
uncoated PLA films.17
GO films on Si/SiO2
showed nanoripples with high density.43
Rough
surface is beneficial in cell attachment and pro-
liferation. Surface roughness affects the adsorp-
tion of fibronectin and albumin in vitro.44
Surface roughness promotes cell attachment,
adhesion, osteoblast proliferation and differen-
tiation. It also promotes matrix synthesis and

7. Kashte et al. 7
local factor production.45
Surface roughness has
a positive effect on bioactivity, water uptake
and cytocompatibility of composites.46
Physical analysis. FTIR spectra (Figure 2)
showed the presence of PCL, GO and CQ in
respective scaffolds. Distinctive absorption
Figure 1. FESEM images: (a) PCL, (b) PCL-GO and (c) PCL-GO-CQ.
Table 1. Properties of scaffolds: fibre diameter, contact angle and mechanical properties.
S.No. Type of
scaffolds
Fibre diameter
(nm), M ± SD
Contact angle,
(degree),
M ± SD
Nature of
scaffolds
Tensile
strength (MPa),
M ± SD
Yield strength
(MPa),
M ± SD
1 PCL 226.37 ± 16.92 126.5 ± 0.28 Hydrophobic 0.85 ± 0.11 0.46 ± 0.01
2 PCL-GO 1929.64 ± 694.94 58.9 ± 0.55 Hydrophilic 1.21 ± 0.01 0.51 ± 0.02
3 PCL-GO-CQ 2369.74 ± 681.45 55.7 ± 0.51 Hydrophilic 3.02 ± 0.04 1.51 ± 0.02
PCL: polycaprolactone; GO: graphene oxide; CQ: Cissus quadrangularis.

8. 8 Journal of Bioactive and Compatible Polymers 00(0)
peaks of asymmetric CH2 stretching at
2926 cm−1
and symmetric CH2 stretching at
2860 cm−1
, C=O/carbonyl stretching
at 1720 cm−1
, C–O and C–C stretching at
1293 cm−1
and asymmetric C–O–C stretching at
1240 cm−1
observed for PCL. Carboxylic C=O
bend at 1719 cm−1
, C=C bend at 1569 cm−1
and C–O bend at 1220 cm−1
observed for GO.
Alkane asymmetric C–H stretching at
2920 cm−1
, alkane symmetric C–H stretching at
2847 cm−1
, C=O stretching at 1708 and
1459 cm−1
, alkane C–H bending at 1377 and
1163 cm−1
, C–N stretching at 1150 and 1000
cm−1
and C=S stretching at 1032 cm−1
observed
for CQ. FTIR spectra (Figure 2) confirmed the
interaction of PCL with GO and CQ in respec-
tive scaffolds. In composite scaffolds, there
were many overlapping peaks observed
between PCL, GO and CQ; therefore, they were
not clearly differentiated. However, integrating
and/or broadening of peaks confirmed the pres-
ence of these multiple components. The promi-
nent peak of 1569 cm−1
and small peaks of GO
between 2000 and 2500 cm−1
were reduced in
PCL-GO and PCL-GO-CQ scaffolds. Similar
kind of results were obtained elsewhere.30,32
Figure 3. The FESEM images of cell attachment with scaffolds: (a) PCL, (b) PCL-GO and (c) PCL-GO-CQ.
Figure 2. Fourier-transform infrared (FTIR) spectra of the scaffolds.

9. Kashte et al. 9
Thermal degradation of scaffolds (Figure 5)
was studied by determining the weight loss of
the sample with increasing temperature. The
PCL scaffold displayed the main degradation at
433°C (88% weight loss) and 487°C (10%
weight loss) with 2% residue; PCL-GO: 431°C
(88% weight loss) and 526°C (12% weight
loss) with complete degradation; PCL-GO-CQ:
431°C (87% weight loss) and 586°C (13%
weight loss) with complete degradation. There
was a complete degradation of PCL-GO and
PCL-GO-CQ scaffolds. It could be due to
pyrolysis of liable oxygen-containing groups in
GO or phytosterols. The similar results were
found in GO incorporated PLGA scaffolds47
and PCL scaffolds.48
Also, polyethyleneimine-
GO-PLA films were majorly degraded.17
Fluctuations in the degradation temperature
could also occur due to different heating rates.49
Wetting properties. The water contact angles
of PCL, layer-by-layer modified PCL-GO and
PCL-GO-CQ are shown in Figure 6 and
Table 1. PCL scaffolds were hydrophobic,
while layer-by-layer modified scaffolds PCL-
GO and PCL-GO-CQ were hydrophilic in
nature. PCL-GO-CQ scaffolds showed the
lowest water contact angle and the highest
hydrophilicity. Incorporation of GO and CQ
into their respective scaffolds has increased
the hydrophilicity of scaffolds. This could be
due to the nature of GO and CQ having hydro-
philic carboxylic and hydroxyl functional
groups.33
The contact angle of poly(3-hydroxy-
butyrate-co-4-hydroxybutyrate) was decreased
with the addition of GO making it less hydro-
phobic and more hydrophilic. It also showed
that the increasing concentration of GO
decreases contact angle.33
Contact angle of
PLA was decreased with the addition of
GO.17,50
The hydrogen bond interactions
between oxygen-containing groups present in
GO and water explained this behaviour. The
contact angle of PLGA was also decreased
with the addition of GO.47
Hydrophilicity of
PLLA32
was increased with the addition of CQ
crude extract. Contact angle of PCL decreased
from 133 to 37 with the addition of CQ.30
Hydrophilic surface provides better cell
attachment, spreading and proliferation of cells
than hydrophobic surfaces. This hydrophilic
surface allows absorption of fibronectin which
is important in osteoblast adhesion in vitro.45
Mechanical properties. Tensile strength and
yield strength of scaffolds are shown in Table 1.
Both tensile strength and yield strength of scaf-
folds were increased with the addition of GO
and CQ into PCL scaffolds. PCL-GO-CQ scaf-
folds showed the highest tensile strength and
yield strength as compared to other scaffolds.
All the modified scaffolds PCL-GO and
PCL-GO-CQ showed increased tensile strength
and yield strength as compared to PCL alone
(Table 1). Tensile strength and Young’s modu-
lus of PLLA increased with the addition of
CQ.32
Tensile strength of PCL nanofibers also
improved from 0.79 MPa to 2.92 MPa with the
addition of CQ.30
The scaffolds with higher
mechanical strength support cell-based bone
regeneration via an endochondral ossification.51
These scaffolds should be mechanically stable
so that they retain the structure after in vivo
implantation in load-bearing tissues, such as
bones.14,52
Therefore, the mechanical properties
of implanted scaffolds should be comparable
with the native tissue.53,54
Isolation and culture of hUCMSCs. hUCMSCs
were successfully isolated from human umbili-
cal cord Wharton’s jelly and further passaged in
DMEM medium at 37°C, 5% CO2. These hUC-
MSCs were then used for further studies.
In vitro studies
Cell adhesion study. The cells were attached on
PCL, PCL-GO and PCL-GO-CQ scaffolds
after 24 h of culture. Morphology of these cells
was fibroidal in nature. The filopodia of T-cells
were attached to the surface of scaffold. The
rough surface and hydrophilic nature of scaf-
folds have contributed for better attachment
and spreading of cells on surfaces. hUCMSCs
were well spread and attached to PCL-GO-CQ
scaffolds (Figure 3). The hydrophilic and rough

10. 10 Journal of Bioactive and Compatible Polymers 00(0)
Figure 4. Confocal microscopy imaging from 1st to 7th day of culture: (a) PCL, (b) PCL-GO and (c) PCL-
GO-CQ.
surfaces of the scaffolds played an important
role in the cell attachment. The obtained results
were similar to those obtained by other
researchers. Human osteosarcoma cells (HOS)
adhered and spread showing flat morphologies
on PBAPCL blended with HA scaffolds.11
The
human foetal osteoblast cells (hFOB) showed
cuboidal osteoblast-like morphology with filo-
podia formation and bridging each other with
the help of extracellular matrix. Besides, for-
mation of mineral particles on cell surfaces was
observed after 10 and 15 days of culture.30
Confocal microscopy imaging. Cell-seeded scaf-
folds that were incubated for 1, 4 and 7 days
were also stained with DAPI for nuclear visu-
alization as shown in Figure 4. These cells were
in a progressive manner from 1st to 7th day of
culture. Z-stack images showed cell attachment
and cell movement deep into scaffold and not
just on the surface. Cell count obtained with
ImageJ software showed the number of cells on
1st, 4th and 7th day as 884, 2683 and 2773 on
PCL; 20, 578 and 2690 on PCL-GO and 94,
2164 and 4612 on PCL-GO-CQ, respectively.
All these modified scaffolds showed the highest
cell proliferation on 7th day as compared to
PCL scaffolds with PCL-GO-CQ scaffold
exhibiting the highest cell proliferation among
the lot.
Similar results were found in other
GO-containing scaffolds. GO films showed
progressive proliferation of MSCs from Day 1
to Day 7. There was a higher density of

11. Kashte et al. 11
blue-stained nuclei on GO films compared to
polydimethylsiloxane (PDMS) or Si/SiO2.55
HOS cells also showed adherence and high den-
sity on PBAPCL blended with HA scaffolds via
nuclei staining.11
It showed good proliferation
and penetration of cells on these scaffolds. It
again showed the resemblance in terms of cell
attachment on scaffolds. Rough surface and
hydrophilic nature of scaffolds have contrib-
uted to proliferation of cells. The higher
Figure 6. Alizarin Red S staining of layer-by-layer scaffolds after 14 and 21 days of differentiation of
hUCMSCs: (a) PCL, (b) PCL-GO, (c) PCL-GO-CQ (without osteoblastic differentiation medium) and
(d) tissue culture plate with osteoblastic differentiation medium.
Figure 5. The cell viability and proliferation of hUCMSCs on the scaffolds for 1, 4, and 7 days of the
culture studied with MTT assay (**p 0.01).

12. 12 Journal of Bioactive and Compatible Polymers 00(0)
proliferation is due to hydrophilic and rough
surfaces of scaffolds. They allow absorption of
fibronectin which is important in osteoblast
adhesion in vitro44,45
and promote cell attach-
ment on the surfaces of composites, osteoblast
proliferation and differentiation. They also have
a positive effect on bioactivity, water uptake
and cytocompatibility of composites.46
MTT cell proliferation assay. The proliferation of
hUCMSCs on different scaffolds was evaluated
by MTT assay at a point in time on 1st, 4th and
7th day. From Figure 5, it was evident that pro-
liferation of cells, as determined by the absorb-
ance, increases from Day 1 to Day 7 for all
scaffolds. This showed the proficiency of all
scaffolds to support the proliferation of hUCM-
SCs. Cell proliferation on all scaffolds was
found to be higher as compared to tissue culture
plate and PCL from 4th to 7th day. Cell prolif-
eration on PCL-GO-CQ is higher as compared
to other scaffolds on 7th day.
Also, similar results were found in the
PLA-GO and PLA-GNP. There was signifi-
cantly higher MG-63 cell proliferation on
GO and GNP containing PLA scaffolds.50
HOS
cells showed good biocompatibility on
PBAPCL blended with HA.11
The MTT assay
showed that there was an equivalent growth of
cells on graphene-coated substrates like that of
a glass slide or Si/SiO2.43
PCL-CQ scaffolds
showed good growth and proliferation of hFOB
compared to PCL alone.30
Cell culture experi-
ments exhibit improved biocompatibility of
PCL-GO, PCL-GO-CQ scaffolds as compared
to PCL alone (Figure 5). The improved biocom-
patibilities of scaffolds were due to improved
hydrophilic and rough surfaces. GO presence
on the surface of scaffold has improved hydro-
philicity which is then required for cell adhe-
sion and protein adsorption. Vitronectin and
fibronectin protein adhesion are increased in
hydrophilic surfaces.50
Osteoblastic differentiation
Alizarin Red S staining for calcium. Alizarin Red S
staining was used to evaluate calcium deposits in
differentiated cells. There was a red–orange
complex formed with Alizarin Red S staining
showed the presence of secreted mineralization.
Differentiation of hUCMSCs into osteoblasts
was observed from 14 days onwards. There was
mineralization on PCL-GO, PCL-GO-CQ scaf-
folds after 14 days of culture. There was higher
mineralization on PCL-GO-CQ scaffolds at the
21st day as compared to 14th day. The maximum
differentiation of hUCMSCs into osteoblasts
was confirmed after 21 days of culture on modi-
fied scaffolds of PCL-GO-CQ. These results
indicate that the synergistic effect of GO and CQ
extract could enhance the expression of osteo-
genic differentiation markers and can stimulate
calcium deposition (Figures 6 and 7). hUCMSCs
differentiated on scaffolds (Figure 6(a)–(c))
without an osteogenic medium is comparable
with the control tissue culture plate (Figure 6(d))
containing an osteogenic medium. Least miner-
alization was observed on PCL scaffolds without
an osteogenic medium. These results suggest
that these scaffolds have great potential for oste-
ogenic differentiation of hUCMSCs.
There was increased activity on PCL-
GO-CQ scaffolds as compared to PCL-GO and
PCL scaffolds on 21 days of differentiation. It
may be due to the secretion of osteocalcin by
differentiated osteoblasts. Osteocalcin plays an
important role in bone metabolic activities and
bone-building.30
It shows PCL-GO-CQ scaf-
folds as potential bone regenerative scaffold.
PLLA-CQ scaffolds also showed mineraliza-
tion with simulated body fluid (SBF) after
14 days of incubation with Alizarin Red S stain-
ing.32
The human foetal osteoblast cells also
showed good intensity of mineralization on CQ
containing scaffolds on the 15th day of cul-
ture.30
The graphene proved as alternative to
bone morphogenic growth factor-2 (BMP-2), as
graphene showed equivalent amount of hMSCs
differentiation into osteoblastic cells, with a
significant amount of osteocalcin secretion on
the 15th day as that of BMP-2 in the presence of
osteogenic media.43
Also, graphene and GO
showed differentiation of MSCs into osteo-
blasts by mineralization on 12th day in presence
of osteogenic media.

13. Kashte et al. 13
Von Kossa staining for calcium. Von Kossa stain-
ing was used to evaluate secreted mineraliza-
tion in differentiated cells (Figure 8). The
appearance of black precipitates confirmed
positive Von Kossa staining with secreted min-
eral deposition. The black precipitates were
observed from 14 days onwards and were maxi-
mum and broad after 21 days of culture on mod-
ified scaffolds of PCL-GO-CQ as compared to
other scaffolds. hUCMSCs differentiated on the
scaffolds (Figure 8(a)–(c)) without osteogenic
medium was comparable with the control tissue
culture plate (Figure 8(d)) containing osteo-
genic medium. Least mineralization was seen
on PCL scaffolds without an osteogenic
medium. Similar results as that of Alizarin Red
S staining confirmed differentiation of hUCM-
SCs into osteoblasts. The osteogenic differenti-
ation of MSCs was shown in the presence of
serum and human plasma after 28 days of cul-
ture in osteogenic media.56
MSCs cultured on
biphasic calcium phosphate and calcium phos-
phate in the presence of conditioned medium
containing significant growth factors. Minerali-
zation was observed with positive Von Kossa
staining on the 21st day of culture.57
Von kossa staining was used to evaluate
osteoblastic differentiation of MSCs through
mineralization. After 24 days of culture of
MSCs into osteoblastic induction medium,
there was mineralization from 14 days onwards
in an increasing manner, as confirmed by Von
kossa staining.58
Foetal rat calvariae (FRC)
cells were cultured on osteoblastic medium
showed mineralization or bone nodules forma-
tion on Day 14, as confirmed by Von kossa
staining.59
BMPs were assessed for osteoinduc-
tion of MSCs for 21 days of culture. There was
significant mineralization and bone nodules
formation when MSCs cultured with a combi-
nation of BMP-2 + BMP-6 + BMP-9, con-
firmed by Von kossa staining.60
Figure 7. Alizarin Red S staining quantification of layer-by-layer scaffolds after 14 and 21 days of
differentiation of hUCMSCs (*p 0.05).

14. 14 Journal of Bioactive and Compatible Polymers 00(0)
Conclusion
The prepared PCL-GO-CQ scaffolds are novel,
herbal and cell compatible with an osteoinduc-
tive nature. Their porous, rough and hydrophilic
nature, along with mechanically stable charac-
ter helped hUCMSCs to adhere, spread, prolif-
erate and spontaneously differentiate into
osteoblast-like cells. The synergistic effect of
GO and CQ in PCL-GO-CQ scaffold enhanced
the roughness, mechanical properties and wet-
tability of scaffolds. Mainly GO and CQ callus
extract provided osteoinductive properties to
scaffold that helps hUCMSCs to spontaneously
differentiate into osteoblast without any osteo-
genic media or growth factors or added external
stimuli. This property will help the scaffold for
speedy in vivo bone formation upon transplan-
tation, thus saving in vitro differentiation time
before transplantation. Thus, the novel PCL-
GO-CQ scaffolds which is prepared using
layer-by-layer method shows tremendous
potential for in vivo bone tissue engineering and
further studies to regenerate bone tissues.
Acknowledgements
The authors would like to thank the University Grant
Commission (UGC), Government of India, New
Delhi, for a doctoral fellowship to Mr Shivaji Kashte.
They also thank Dr Manas Kumar Santra and Mrs
Neha Gupta from National Centre for Cell Sciences
(NCCS), Pune, for their assistance with confocal
imaging; Dr Anup Kale and Mrs Vedashree
Sirdeshmukh from College of Engineering, Pune, for
their helpful assistance with SEM; Mr Gajanan
Arbade and Mr Chetan Chavan from Defence
Institute of Advanced Technology (DIAT, DU),
Girinagar, Pune, for their assistance in characteriza-
tion of scaffolds.
Declaration of conflicting interests
The author(s) declared no potential conflicts of inter-
est with respect to the research, authorship, and/or
publication of this article.
Funding
The author(s) received no financial support for the
research, authorship, and/or publication of this article.
ORCID iD
Shivaji Kashte https://orcid.org/0000-0002-9937
-4736
Supplemental material
Supplemental material for this article is available
online.
Figure 8. Von Kossa staining after 14 and 21 days of differentiation of hUCMSCs: (a) PCL, (b) PCL-
GO, (c) PCL-GO-CQ (without osteoblastic differentiation medium) and (d) tissue culture plate with
osteoblastic differentiation medium.

15. Kashte et al. 15
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