White paper publications in 3 dimensional tissue engineering

Herbally Painted Biofunctional Scaffolds with Improved
Osteoinductivity for Bone Tissue Engineering
Shivaji Kashte1
, Gajanan Arbade2
, R.K. Sharma1,
and Sachin Kadam1,3
*
1
Center for Interdisciplinary Research, D. Y. Patil Education Society (Deemed to be University),
Kolhapur 416006, India
2
Defence Institute of Advanced Technology (DIAT, DU), Girinagar, Pune, India
3
Advancells, NOIDA, Uttar Pradesh 201301, India
Kadamsachin@gmail.com*
*
Corresponding Author
Keywords: graphene, graphene oxide, Cissus quadrangularis, Human umbilical cord Wharton's
jelly-derived Mesenchymal stem cells, Bone tissue engineering.
Abstract. In the bone tissue engineering composite scaffolds with osteogenic potential are emerging
as the new tool. Here, we investigated the graphene (GP), graphene oxide (GO) and Cissus
quadrangularis (CQ) callus extract for their spontaneous osteoinductive potential. Electrospun poly
ε-caprolactone (PCL) sheets were painted with varying combination GP, GO and CQ solutions as
ink. The prepared PCL-GO, PCL-GO-CQ, PCL-GP and PCL-GP-CQ scaffolds were characterized
for their physical, mechanical and biological properties. Addition of GO, GP, GO-CQ and GP-CQ to
PCL enhanced roughness, wettability, Yield strength and tensile strength, biocompatibility
.significantly. Presence of GO and CQ in PCL-GO-CQ scaffolds, while GP and CQ in PCL-GP-CQ
scaffolds showed a synergistic effect on the biocompatibility, cell attachment, cell proliferation of
human umbilical Wharton's jelly-derived mesenchymal stem cells (hUCMSCs) and their
differentiation into osteoblasts by 21st
day in culture without osteogenic differentiation media or any
growth factors. Similarly, confirmed by the Alizarin red S staining and Von Kossa staining. The
combination of PCL-GO-CQ scaffold prepared by novel paint method was found to be most potential
in bone tissue engineering.
1. Introduction
Bone healing is defined as a complex process of biology and biomechanics in order to restore the
healthy form and functioning of the bone [1]. Earlier, the process of bone regeneration was achieved
with the help of natural grafts such as autografts, allografts, and xenografts but with certain
limitations. These limitations drive the effort for development of bone graft substitutes, using the
principles of biomaterials and tissue engineering. A variety of biomaterials such as metals (stainless
steel, Cobalt-Chromium (Co-Cr) alloys and Titanium (Ti) alloys, etc.) [2], ceramics (Hydroxyapatite
(HA), tri-calcium phosphate (TCP), etc.) [3], natural polymers (collagen, chitosan, etc.) [4] and
synthetic polymers (poly (lactic acid) (PLA), poly (glycolic acid) (PGA), etc.) and their specific
combinations have been explored for the replacement and repair of damaged or traumatized bone
tissues.
The poly ε-caprolactone (PCL), an FDA approved polymer is used for fabrication of scaffold sheets
by electrospinning, which is the most common and versatile technique. PCL is biocompatible,
biodegradable and has been used largely in tissue engineering applications. In various studies, PCL
along with Hydroxyapatite (HA) [5], PCL-gelatin hybrid nano-fibrous membranes [6], PCL-(Poly-
1,4- butylene adipate-co-polycaprolactam (PBAPCL)-HA composite scaffold [7], PCL has shown its
potential in bone tissue engineering applications. Whereas, the electrospinnable synthetic polymers
such as PCL, PLA etc. are primarily hydrophobic in nature. Hence, are not sufficient in order to
perform the necessary functions in the process of bone tissue engineering.
The application of these materials in tissue engineering can be advanced through modification of
surfaces from hydrophobic to partially hydrophilic. Introduction of hydrophilic property to
Journal of Biomimetics, Biomaterials and Biomedical Engineering Submitted: 2019-02-27
ISSN: 2296-9845, Vol. 41, pp 49-68 Revised: 2019-04-10
doi:10.4028/www.scientific.net/JBBBE.41.49 Accepted: 2019-04-10
© 2019 Trans Tech Publications, Switzerland Online: 2019-04-22
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans
Tech Publications, www.scientific.net. (#114297820-22/04/19,08:15:30)

hydrophobic nanofibres has been attempted in various ways by surface modification technologies,
e.g. flame treatment, corona discharge treatment, plasma modification, and surface graft
polymerization [8]. Surface modification is an important aspect in the field of tissue engineering for
cell attachment and proliferation. Cell behaviour mainly depends on the porosity of rough surfaces.
Therefore, along with PCL, we used graphene oxide (GO), graphene(GP) and Cissus quadrangularis
(CQ) callus culture extract as physical, chemical, and biological property enhancers in the preparation
of scaffolds for bone tissue engineering.GP has gained attraction in bone tissue engineering because
of its large surface area, low biological toxicity and osteoinductive nature[9]. GP-chitosan films [10],
GP coated cobalt- chromium-molybdenum-based alloy [11], GP nanosheets coated titanium alloys
[12] showed biocompatibility and its potential in tissue engineering. GO has more hydrophilic groups
and easy dispersion abilities as compared to GP[13]. GO along with HA and chitosan functionalized
graphene nanoplatelets reinforced with polyvinyl alcohol [14], GO with Polylactic acid and HA [15],
GO doped poly (lactic-co-glycolic acid) (PLGA) scaffolds [13], GO-poly-L-lysine composites [16]
used for bone tissue engineering. GP and GO, are the most fascinating materials based on their
interaction with stem cells channelled cell attachment, cell proliferation and differentiation into
various lineages like osteoblasts, chondroblasts, and neurons [11,12,17].
Cissus quadrangularis (CQ) also known as Vajravalli, Hadjod, or Kandvel and used in Ayurveda as
Asthiyuk (bone strengtheners) [18]. The stem extract of this plant contains high calcium ions (4%
weight) and phosphorous [19] and ethanolic extract possess triterpenes, β-sitosterol, α and β- amyrins,
ketosteroids, phenols, tannins, carotene and vitamin C [18], that is proved beneficial for bone fracture
healing. The active constituents of CQ may promote proliferation and differentiation of MSCs into
Osteoblasts and bone formation via wnt-LRp5-β-catenin or MAPK dependent pathway. It has been
found that CQ increases the activity of the proliferation and differentiation of BM MSCs into
osteoblasts [20]. Isolated phytogenic steroid is believed to be the main constituent in CQ [21].
Methanolic extract of CQ [22], and its uses in PCL-CQ-HA nano-fibrous scaffolds has been found as
a potential biocompatible material for bone tissue engineering [23]. We have used CQ callus culture
extract obtained from callus culture, a plant tissue culture technique that is used to propagate the plant
in vitro. The obtained callus is brown in colour and contains high amount of phytosterols. Therefore,
callus culture extract contains pure and higher amount of metabolites as compared to crude stem
extract of the plant.
The purpose of this study was mainly to investigate osteoblastic differentiation potential of
hUCMSCs induced by GO, GP, and CQ callus culture extract. Thus, different combinations of PCL-
GO, PCL-GO-CQ, PCL-GP and PCL-GP-CQ scaffolds were evaluated for osteoinductive potential.
2. Materials and Methods
2.1 Source Materials
Graphene (GP), Graphene Oxide (GO) was a kind donation from Sachin Kochrekar, Department of
Chemistry, Defence Institute of Advanced Technology, Girinagar, Pune (GO and GP prepared by
Modified Hummer’s method), Cissus quadrangularis plant samples were collected from Kolhapur,
India. poly ε-caprolactone (PCL) (Mw= 80,000 g/mol; Sigma Aldrich)MS medium No.6, NAA (α-
Naphthalene acetic acid), BAP (6-Benzylaminopurine), sucrose, Agar, 70% ethanol, 0.01% mercuric
chloride (HgCl2) solution (Himedia), collagenase type IV, dispase, phosphate buffer saline (PBS),
Trypsin (0.05% and) EDTA (0.02%) (Sigma Aldrich), Dulbecco's Modified Eagles Medium
(DMEM), Ham's F12 (DMEM: HF12, 1:1), (Invitrogen), Penicillin, streptomycin, human umbilical
cord blood serum, (Sigma Aldrich).
2.2 Cissus quadrangularis Callus Culture and extraction
Cissus quadrangularis (CQ) is identified and confirmed in the surrounding region of Kolhapur, India.
Stem parts of the plant were isolated, cleaned and further used as explants for callus culture of CQ in
sterile conditions. MS medium No.6 (2.26 gm/L) along with NAA 2.5 mg/L, BAP 0.5 mg/L, sucrose
40 gm/L, Agar (10 gm/L) were used as callus induction medium. pH of the medium was adjusted to
50 Journal of Biomimetics, Biomaterials and Biomedical Engineering Vol. 41

5.6. Stem of the plant was washed for 10 min under running tap water and surface was then sterilized
by 70% ethanol (v/v) for 5 min. Stem explants immersed in 0.01% HgCl2 (mercuric chloride) solution
for 2 and 3 minutes respectively. Finally, stem explants were washed for 5 times using sterile distilled
water and cut into 10-13 mm pieces. These surface sterilized explants were inoculated into the centre
of sterile MS media containing culture tubes. Culture tubes were incubated at room temperature of
28o
C and incubated in dark. Followed by which, these culture tubes were observed daily for callus
formation for a span of 3-4 weeks.
A pure callus extract of CQ was then tested by Salkowski test [23] to determine the presence of
phytosterol, For this, a small quantity of extract was dissolved in 1mL of chloroform and few drops
of concentrated Sulphuric acid was added along the walls of the test tube. The formation of brown
ring at the bottom of the test tube indicated the presence of Phytosterol.
2.3 Preparation of Scaffolds
PCL solution (10% w/v) was prepared by dissolving PCL in THF (Tetrahydrofluran): Methanol (3:1)
and was kept on a magnetic stirrer for 30 h. These PCL scaffold sheets were fabricated by
electrospinning with parameters of the flow rate of 0.8 mL/h, voltage 12kV and 12.5 cm distance
between tip of the needle of a syringe and the collector. GP solutions (1 mg/mL), GO solutions
(1 mg/mL) and CQ solution (1 mg/mL) were prepared by dispersing/dissolving in distilled water and
sonication.
PCL scaffold sheets fabricated by electrospinning were used for further surface modification of
scaffolds. Preparation of PCL-GO, PCL-GO-CQ, PCL-GP, PCL-GP-CQ scaffolds was carried out by
a respective painting of the ink. The ink was simply painted using acrylic painting brush (Flat,
midsize) with hands. After painting, they dried at room temperature. Leaching study was carried out
for adhesion of GO and GP for respective scaffold as per ISO-10994-12 (Supplementary
information). The leaching study showed a small amount of GP leached from PCL-GP and PCL-GP-
CQ scaffolds. There was a small amount of GO leaching from PCL-GO and PCL-GO-CQ scaffolds.
2.4 Physico-chemical analysis
The morphologies of electrospun pure polymeric and composite scaffolds were examined by field
emission scanning electron microscope (FESEM, Carl Zeiss, Germany) at an accelerating voltage of
15 kV. For FESEM, the samples were cut into 5X5 mm squares, mounted on to sample stubs; sputter
coated with gold/palladium using an SC 7640 Sputter Coater (Quorum Technologies Ltd, UK). The
coated GO/GP were analysed on electrospun fibres of PCL. The fibre diameter ranges of scaffolds
were calculated from FESEM micrographs using image analysis software (ImageJ, National Institutes
of Health, Bethesda, USA).
Surface properties of the modified surface of PCL sheets were analysed by atomic force microscopy
(AFM, Asylum Research) with the help of tapping mode.
FTIR spectra were then recorded for all scaffolds using (FTIR, Brucker, Germany). The spectra were
obtained with 30 scans per sample ranging from 4000 to 500 cm-1
.
The thermal degradation behaviours of scaffolds were studied with a thermogravimetric analyser
(Universal V4.7A TA Instrument); instrument under nitrogen atmosphere between temperatures
ranging from 300
C to 8000
C at a heating rate of 100
C/min.
The water contact angle was determined by sessile drop method and Drop shape image analysis using
water as droplet with contact angle goniometer (KRUSS, Germany). Contact angle measurement of
liquid droplets on a solid substrate (n=3) was used to characterize surface wettability, surface
cleanliness and hydrophilic/hydrophobic nature of the surface.
Tensile properties were calculated at room temperature using a Universal tensile Machine. Scaffold
cut into cylinders (n=3) were tested. The loading was 100 N and crosshead speed was 5mm/min.
From the resulting stress-strain curves, Yield strength and tensile strength were calculated [23–25].
Journal of Biomimetics, Biomaterials and Biomedical Engineering Vol. 41 51

2.5 Biological Analysis
Isolation and expansion of human umbilical cord Wharton's Jelly-derived mesenchymal stem cells
(hUCMSCs):
The human umbilical cords were collected from caesarean deliveries with proper consent from patient
and approval of Institutional Ethics committee (DMCK/57/2016). These umbilical cords were then
collected and transported to lab in L15 medium. Human umbilical cord Wharton's Jelly-derived
mesenchymal stem cells (hUCMSCs) were isolated as per protocol described by Kadam et.al[26]. In
brief, cords were washed with PBS to remove cord blood and blood clots. The surface of cords were
sterilized with Betadine® solution for 5 minutes and washed with sterile PBS. Cord tissues were
chopped into 1-2 mm length after removal of blood vessels. The chopped cord tissue was digested
with a sterile cocktail of enzymes Collagenase Type IV and Dispase (7:1 v/v) for 30 min at 400 rpm
and at temperature of 37°
C on magnetic orbital shaker. Similarly, tissue was incubated with Trypsin
(0.05%) - EDTA (0.02%) for 20 min. Homogenate was then centrifuged at 1500 rpm for 10 min. The
obtained cell pellet was washed and cultured in the Dulbecco's Modified Eagles Medium (DMEM),
Ham's F12 (DMEM: HF12, 1:1) medium supplemented with 10% serum and penicillin (100
units/mL) and streptomycin (100 µg/mL). The cells were incubated for 48 h at 37°
C, 5% CO2. The
medium was changed every 48 h and the first passage was carried out after 8 days followed by every
4 days. The isolated hUCMSCs of 3-5 passage were cryopreserved using 10% DMSO with standard
protocol [27].
Accordingly, the scaffolds were cut in order to fit the wells of 48 well plates. These scaffolds were
washed with PBS and sterilized by ethylene oxide (EtO). 1.0X104
cells/mL were seeded on to
scaffolds and cell-seeded plates were incubated at 37°C, 5% CO2 for 1st, 4th and 7th day for cell
attachment, cytotoxicity and proliferation studies.
MTT Cell cytotoxicity and Proliferation Assay: MTT [3-(4, 5-dimethylthiazol-2yl)-2, 5-
diphenyltetrazolium bromide] is prepared in DMEM at a final concentration of 5mg/mL (pH=7.4),
filter sterilized through 0.2 micron filter. 50 µL MTT solutions were added to each of the well-
containing cells seeded scaffolds. After incubation of 3 h, 450 mL of dimethyl sulfoxide (DMSO)
were added to each for dissolving formazan crystals. The quantity of purple coloured formazan,
formed by tetrazolium reduction is directly proportional to metabolically active viable cells. The
absorbance is measured at 570nm with reference of 650 nm using a plate reader spectrophotometer
(Hitachi) [28]. Same procedure is then followed for 4th and 7th day. Tissue culture plate is treated as
a negative control whereas; PCL is treated as a positive control.
Cell Attachment Study: Cell-seeded scaffolds were used for cell attachment study after 24 hours of
culture. At the end of incubation period, cells were fixed on scaffolds with 4% paraformaldehyde.
Plates were kept at 4 °C in the freezer and the samples were dehydrated with graded ethanol. After
completely drying, the samples were mounted on to sample stubs; sputter coated with palladium using
SC 7640 Sputter Coater (Quorum Technologies Ltd, UK) and observed under FESEM (Carl Zeiss,
Germany) at an accelerating voltage of 15 kV.
Confocal Microscopy Imaging: Cell-seeded scaffolds which were incubated for 1st, 4th and 7th day
werefixed with 4% paraformaldehyde and stained with DAPI for nuclear visualization. Slides were
mounted on mounting media and images were taken with the help of confocal microscope (Zeiss,
Germany). The images were then processed with Zen Software (Zeiss). Cell quantification was
performed with the help of ImageJ software.
Alizarin Red S staining for calcium: The calcium deposition was analysed by Alizarin Red S staining.
The scaffolds A: PCL; B: PCL GO; C: PCL GO CQ; D: PCL GP; E: PCL GP CQ; (A to E: without
Osteoblastic differentiation medium, A: PCL: Negative control); F: PCL with Osteoblastic
differentiation medium were seeded with hUCMSCs for 14th and 21st day of culture (F: positive
control).Osteoblastic differentiation medium consisted of DMEM medium which contain 50 µg/ml
ascorbic acid, 0.1 µM dexamethasone, and 10 mMβ-glycerophosphate. In case of alizarin staining,
the specimens were prepared by fixing the cells with 10% formalin. 2% Alizarin Red S stain (pH 4.2)
was added sufficient to cover cell scaffold and was later incubated in dark at room temperature for
45 min. Samples were observed using an Inverted Phase contrast microscope (Eclipse TS 100, Nikon)

equipped with a digital camera. Following, the scaffolds were washed with PBS and quantified using
a solution of 10% acetic acid. After 30 min of incubation with acetic acid, the scaffolds were placed
into a 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 solution was taken and neutralized with 10% ammonium hydroxide.
150 µL of this solution was transferred to 96 well plate and the quantity of Alizarin Red S was
determined by measuring absorbance at 405 nm [29–31].
Von Kossa staining for calcium: Similarly, these scaffolds were also evaluated for calcium deposition
by using Von Kossa staining. The samples were fixed by 10% formalin and then 1ml of 5% silver
nitrate (AgNO3) solution was used to stain the samples at room temperature for 60 min in UV light.
The stains were removed and samples were visualized under an Inverted phase contrast microscope
(Eclipse TS 100, Nikon) and images were taken.
2.6 Statistical analysis
Statistical analysis of all data was conducted by using the Origin Pro 8.5 software. Numerical data
were presented as Mean± standard deviation. Statistical analysis was performed by using a one-way
analysis of variance (ANOVA). A p values <0.05 was considered to be statistically different.
3. Results and Discussion
3.1 CQ callus culture and powder extraction
Figure 1: CQ callus culture. A: Callus formed from CQ stem explant; B: Control CQ stem
explant; C: Salwoski test. The brown ring formed at the bottom of the test tube confirms the
presence of phytosterols in the callus extract.
The brown coloured callus was observed after the 4 weeks of culture (Figure 1: A, B) as mentioned
elsewhere[32,33]. The fine powder was made using these 4-5 weeks of fully grown, dehydrated and
dried callus
Crude extract was prepared by using a Soxhlet apparatus with ethanol. 50 gm callus powder of CQ
yielded 3 gm of extract which was then dissolved in water, partitioned with petroleum ether yielding
0.5 gm of extract.
This CQ callus extract showed the presence of Phytosterol, confirmed by formation of brown ring at
the bottom of test tube as indicated in Salkowski test (Figure 1: C), as shown elsewhere [34]. CQ
callus extract was preferred over crude stem extract due to high content of phytosterols. These
Phytosterols are osteoinductive in nature [34]. They stimulate increase expression of osteopontin and
increase uptake of the minerals such as calcium, sulphur, by the osteoblasts in fracture healing [21],
acting as a main component in bone regeneration.

3.2 Physico-chemical analysis
FESEM analysis was done for morphological analysis of the electrospun PCL and painted scaffolds.
FESEM images (Figure 2) shows electrospun PCL scaffolds with smooth fibre structures while that
of painted scaffolds were decorated with the GO, GP and CQ that were rough in nature. The porosity
of painted scaffolds was intact. Fibre diameter (Table 1) was significantly increased with GO, GP and
further with CQ. Fibre diameter (Table 1) was increased in the PCL GO and PCL GP scaffolds as
compared to PCL scaffolds. Later the fibre diameter was significantly increased in the PCL-GP-CQ
and PCL-GO- CQ scaffolds.
GO layers on PCL-GO, PCL-GO -CQ and GP layers on PCL-GP, PCL-GP-CQ scaffolds were
randomly distributed all through the PCL sheet. The enhanced rough surface is potential for protein
adhesion, cell adhesion and cell proliferation [34].
Figure 2: Field emission scanning electron microscope (FESEM) images. A: PCL; B: PCL GO;
C: PCL GO CQ; D: PCL GP; E: PCL GP CQ.

Table 1: Properties of scaffolds. The contact angle and mechanical properties were mentioned
in the table.
Surface properties of PCL and surface modified PCL sheets were analysed by atomic force
microscopy (AFM) by tapping mode. AFM images (Figure 3) shows rough surface of scaffolds. The
modified scaffolds PCL-GO and PCL-GP were slightly rough as compared to PCL scaffold. PCL-
GP-CQ and PCL-GO-CQ scaffolds showed higher roughness as compared to other scaffolds. The
root mean square roughness (RMS) values were PCL (146 nm), PCL-GO (201 nm), PCL-GO-CQ
(231 nm), PCL-GP (330 nm) and PCL-GP-CQ (382 nm).
This rough surface is helpful in cell attachment and proliferation. Surface roughness also has a
positive effect on bioactivity, water uptake and cytocompatibility of composites [35]. Similar results
were analysed by other researchers. The coating of polyethyleneimine-GO on PLA films also showed
rough, uneven, mountain-like topography as compared to uncoated PLA films [36]. GO films on
Si/SiO2 showed nanoripples with high density [37]. The root mean square roughness of rGO coated
on Ti alloys and GO coated on Ti alloys was higher in comparison with Ti alloys alone [12].
Figure 3: Atomic force microscopy (AFM) images. A: PCL; B: PCL GO; C: PCL GO CQ; D:
PCL GP; E: PCL GP CQ.
FTIR spectra (Figure 4) shows distinctive absorption peaks of asymmetric CH2 stretching at
2926 cm-1
and symmetric CH2 stretching at 2860 cm-1
, C=O stretching at 1720 cm-1
, C-O and C-C
stretching at 1293 cm-1
, asymmetric C-O-C stretching at 1240 cm-1
for PCL. Carboxylic C=O bend
at 1719 cm-1
,C=C bend at 1569 cm-1,
C-O bend at 1220 cm-1
and 1029 cm-1
,for GO.C-C stretching
at 1576 cm-1
, C-OH stretching at 1365 cm-1
,alkoxy C-O stretching at 1150 cm-1
and 1069 cm-1
, for
GP. Alkane asymmetric C-H stretching at 2920 cm-1
, alkane symmetric C-H stretching at 2847 cm-1
,
C=O stretching at 1708 cm-1
and 1459 cm-1
, Alkane C-H bending at 1377 cm-1
and 1163 cm-1
, C-N
stretching at 1150 cm-1
-1000 cm-1
, C=S stretching at 1032 cm-1
for CQ.
Sr.No
Type of
Scaffolds
Fibre
Diameter
(nm) (Mean
±SD)
Contact
angle (Mean
±SD)
Nature of
Scaffolds
Tensile
strength
(MPa)
(Mean ±SD)
Yield
strength
(MPa)
(Mean ±SD)
1 PCL 226±16 126.5±0.28 Hydrophobic 0.85±0.11 0.46±0.01
2 PCL-GO 1029±183 55.6±0.72 Hydrophilic 1.72±0.02 0.53±0.01
3
PCL-GO-
CQ
1374±224 47.6±0.72 Hydrophilic 2.84±0.01 1.00±0.01
4 PCL-GP 823±124 68.1±0.95 Hydrophilic 1.63±0.03 0.81±0.01
5
PCL-GP-
CQ
1319±680 51.6±0.72 Hydrophilic 2.44±0.03 1.04±0.01

Figure 4: Fourier Transform Infra-Red spectroscopy (FTIR) spectra of the scaffolds.
FTIR spectra (Figure 4) confirm interaction of PCL with GP, GO and CQ in their respective scaffolds.
The peaks obtained here are similar to that of PCL [6,7,38], GP, GO [12,14,39] and CQ [18,23,24,40]
obtained elsewhere. In the composite scaffolds there are many overlapping peaks between PCL, GP,
GO and CQ, therefore could not be differentiated. But the integration and reduction or broadening of
peaks could be due to hydrogen bonds and/or van der Waals forces, which confirms the presence of
the multiple components and interaction between them [24,34].
Thermal degradation of scaffolds was studied by determining weight loss of sample upon linearly
increasing temperature. The samples were heated at 10℃/min up to 800℃ under nitrogen
atmosphere. There was no significant increase in the thermal stability of pure PCL scaffolds by
introduction of GO or GP into PCL sheets (Figure 5). In PCL TGA curve it mainly displays the
degradation at 250- 433 ℃ (88 % weight loss) and at 487 ℃ (10 % residue); PCL-GO: 213- 434 ℃
(88 % weight loss) and at 523 ℃ (9% residue); PCL-GO-CQ: 187- 433 ℃ (87 % weight loss) and at
580 ℃ (13 % residue); PCL-GP: 313- 451 ℃ (76 % weight loss) and at 578 ℃ (13 % weight loss);
PCL-GP-CQ: 235-498 ℃(85 % weight loss).
The complete degradation of composite scaffold occurred at more or less similar temperatures to pure
PCL scaffolds. There was no significant difference observed. It could be due to the GO, GP and
phytosterols from CQ extract which interacted with PCL via hydrogen bonds and/or Van der Waals
forces, but the mass loss for composite scaffolds may be due to pyrolysis of liable oxygen-containing
groups in GO, or phytosterols. Similar results were found in GO incorporated PLGA scaffolds [41].
The incorporation of polyethyleneimine -GO into PLA films were majorly degraded [36].
The water contact angle of PCL, modified PCL-GO, PCL-GO-CQ and PCL-GP, PCL-GP-CQ were
shown in Figure 6 and Table 1. The plain PCL scaffolds were found to be hydrophobic whereas the
painted scaffolds PCL-GP, PCL-GP-CQ, PCL-GO, and PCL-GO-CQ were hydrophilic in nature.
PCL-GO-CQ scaffolds showed lowest water contact angle and highest hydrophilicity. The
incorporation of GO, GP and CQ into respective scaffolds increased hydrophilicity of the scaffolds.
This is mostly due to the nature of GO and CQ possessing hydrophilic carboxylic and hydroxyl
containing functional groups [25]. The hydrophilic surface provides better cell attachment, spreading
and proliferation of cells compared to hydrophobic surfaces. Hydrophilic surface allows absorption
of fibronectin which is important in osteoblast adhesion in vitro [42]. A similar decrease in the
hydrophilicity was observed elsewhere. The contact angle of Poly (3-hydroxybutyrate-co-4-

hydroxybutyrate) also decreased on addition of GO making hydrophobic to hydrophilic. It also
showed that the increased concentration of GO decreases the contact angle [25]. The contact angle of
PLA decreased on addition of GO [36,43]. Hydrogen bond interactions between oxygen-containing
groups present in GO and water can help explain this behaviour. The contact angle of poly(lactic co
glycolic acid)(PLGA) was also decreased on addition of GO [41]. Hydrophobicity of PLLA [24] was
increased on addition of CQ crude extract and contact angle of PCL also decreased drastically on
addition of CQ [34]. The super-hydrophilic nature observed with rGO Ti and GO Ti alloys films,
water droplets on contact with surfaces, disappear and infiltrate inside samples completely [12].
Tensile strength and Yield strength of scaffolds have shown in Table 1. Both Tensile strength and
Yield strength of scaffolds have increased with the addition of GP and CQ into the PCL sheets (in
PCL-GP-CQ scaffold) and the addition of GO and CQ into the PCL sheets (in PCL-GO-CQ scaffold)
increased the mechanical strength of scaffolds. PCL-GO-CQ scaffolds showed highest tensile
strength and Yield strength as compared to other scaffolds.
Figure 5: The thermal degradation analysis (TGA) of scaffolds.
Figure 6: The water contact angle of scaffolds. A: PCL; B: PCL GO; C: PCL GO CQ; D: PCL
GP; E: PCL GP CQ.
The painted composite scaffolds PCL-GO, PCL-GO-CQ and PCL-GP, PCL-GP-CQ have shown
increased Tensile strength and Yield strength as compared to PCL alone (Table 1). Increase of GO,
GP and CQ concentration in the respective scaffolds increases the Tensile and Yield strength of the
scaffolds that has been previously documented. Also, Tensile strength and Young’s modulus of the
PLLA [24]. The tensile strength of PCL-PMMA increased from 101 MPa to 137 MPa on the addition
of GO [38]. The tensile strength of PCL nanofibers also improved from 0.79 MPa to 2.92 MPa with
the addition of CQ [34]. Mechanical properties of GP-chitosan were higher than chitosan alone [10].

The scaffolds with higher mechanical strength support cell-based bone regeneration via an
endochondral ossification [44]. The scaffolds must be mechanically stable so that they retain its
structure after in vivo implantation in load-bearing tissues such as bones [45,46]. Therefore, the
mechanical properties of implanted scaffolds should be comparable with native tissues [47,48].
3.3 Biological Analysis
Isolation and Culture of hUCMSCs: hUCMSCs (Figure 7) were successfully isolated from human
umbilical cord Wharton’s Jelly with a mixture of enzymes Collagenase Type IV and Dispase (7:1v/v)
and Trypsin-EDTA. The morphology of hUCMSCs exhibited spindle-shaped fibroblast-like
morphology. These cells were further used in vitro studies.
MTT Cell cytotoxicity and Proliferation Assay: The proliferation of hUCMSCs on different scaffolds
was evaluated by MTT assay at different time point 1st
, 4th
, and 7th
day. From (Figure 8) – it is evident
that proliferation of the cells, as determined from the absorbance, increases from day 1 to day 7 for
all substrates.
Figure 7: Isolated human umbilical cord Wharton's Jelly-derived mesenchymal stem cells
(hUCMSCs).

Figure 8: The cell viability and proliferation of hUCMSCs on the scaffolds for 1, 4, and 7 days
of the culture studied with MTT assay. Statistical significance compared with the TCP of the
respective group of days. No significance among groups of a number of days. (**p< 0.01, ***p<
0.001).
This shows the proficiency of all scaffolds to support proliferation of hUCMSCs. Cell proliferation
on all the scaffolds was found to be higher as compared to control. Cell proliferation on PCL-GO-
CQ was highest followed by PCL-GP-CQ as compared with other scaffolds. The improved
biocompatibilities of scaffolds are due to improved hydrophilic and rough surfaces. The presence of
GO on surface has improved hydrophilicity which is required for cell adhesion and protein adsorption.
The vitronectin and fibronectin protein adhesion are increased in hydrophilic surfaces. Also, similar
results were found in PLA/GO and PLA/GNP (graphene nano-platelets). There was a significantly
higher MG 63 cell proliferation on GO and GNP containing PLA scaffolds [43].
Similarly, human osteosarcoma cells (HOS) shows good biocompatibility on Poly(1,4-butylene
adipate-co-polycaprolactam) (PBA)-PCL blended with HA [7]. The MTT assay showed that there
was an equivalent growth of cells on GP coated substrate like that of a like glass slide or Si/SiO2 [37].
PCL-CQ scaffolds shows good growth and proliferation of human fetal osteoblast cells (hFOB) as
compared to PCL alone [34]. GP-Chitosan films showed good biocompatibility with L929 cell lines.
There was no visible reduction in viability after 24 and 48 hours of MTT study[10]. MG63 cells
seeded on PCL-PMMA-GO showed no cytotoxicity over a period of 1 to 7 days. There was an
improvement in cell viability as compared to PCL-PMMA films [38]. MSCs cultured on calcium
phosphate and biphasic calcium phosphate showed viability with MTT assay through day 3 and day
7 [49].
Figure 9: The FESEM images of cell attachment with Scaffolds. A: PCL; B: PCL GO; C:
PCL GO CQ; D: PCL GP; E: PCL GP CQ.

Cell Adhesion study: The cells on the scaffolds appeared after 24 hours of culture days (Figure 9)
were penetrated and attached well on PCL-GO, and PCL-GO-CQ, PCL-GP, PCL-GP-CQ scaffolds
as compared to PCL alone. hUCMSCs were well spread and attached on composite scaffolds mostly
on PCL-GO-CQ followed by PCL-GP-CQ scaffolds The morphology of cells was fibroidal in nature.
The filopodia of cells were attached to the surface of scaffold. Rough surface and hydrophilic nature
of scaffolds have contributed to good attachment and spreading of cells on surfaces.
Figure 10: Confocal Microscopy Imaging from 1st day to 7th day of culture. A: PCL; B: PCL
GO; C: PCL GO CQ; D: PCL GP; E: PCL GP CQ.
Table 2: Cell quantification of Confocal Microscopy Images through ImageJ
Type of scaffold PCL PCL GO PCL GO CQ PCL GP PCL GP CQ
Cell Count on Day 1 884 223 247 138 134
Cell Count on Day 4 2683 474 274 382 186
Cell Count on Day 7 2773 2933 3194 1793 2081
The results were then compared to other findings. hFOB cells cuboidal osteoblast-like morphology
with filopodia and bridging each other with the extracellular matrix, also there was a formation of
mineral particle on cell surfaces after 10 and 15 days of culture [34].HOS cells showed flat

morphologies as adherence and spread on (PBA)-PCL blended with HA scaffolds [7]. BMSCs
cultured on GP-CoCrMo, showed increase in cells with increasing time of culture. The appearance
of the BMSCs was flat and polygonal, ovoid or fusiform and displayed many cytoplasmic
lamellipodia. On days 3 and 5,the cells were colonized more densely [11]. The MG63 cells seeded
on PCL-PMMA-GO were seen well anchored and the cells spread after 7 days of culture. The quick
interaction due to nanoroughness has a favourable impact on cell attachment, cell survival and
proliferation [38].
Confocal Microscopy Imaging: Cell-seeded scaffolds incubated for 1, 4 and 7 days were also stained
with DAPI for nuclear visualization shown in (Figure 10, Table 2). The cells are seen in a progressive
manner from 1st
day to 7th
day of culture. Z stack images show the cell attachment and cell movement
deep into the scaffold and not just on the surface. It also shows good proliferation and penetration of
cells on these scaffolds. All the composite scaffolds showed the highest cell proliferation on 7th
day
when compared to PCL scaffolds. PCL-GO-CQ followed by PCL-GP-CQ scaffolds showed the
highest cell proliferation (Figure 10). It again shows the resemblance with the cell viability and
attachment of cells on these scaffolds. The rough surface and hydrophilic nature of scaffolds have
majorly contributed to proliferation of cells.
It allows absorption of fibronectin which is important in osteoblast adhesion in vitro [42,50] and
promotes cell attachment on the surface of composites and osteoblast proliferation, differentiation.
They also have a positive effect on bioactivity, water uptake and cytocompatibility of the composites
[35]. GP and GO films showed progressive proliferation of MSCs from day 1 to day 7. There was a
higher density of blue stained nuclei on GP and GO films as compared to PDMS
(polydimethylsiloxane) or Si/SiO2 [51]. MG63 cells seeded on PCL-PMMA-GO showed good
attachment and proliferation on DAPI staining of cell nuclei [38]. HOS cells also showed adherence
and high density on (PBA)-PCL blended with HA scaffolds via nuclei staining [7]. BMSCs culture
on rGOTi and GOTi films showed better cell adhesion and spreading after 24 hrs of DAPI staining
[12].
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 that
showed the presence of secreted mineralization. The differentiation of hUCMSCs into osteoblasts
was observed from 14th day onwards. There was mineralization on PCL-GO, PCL-GO-CQ, PCL-
GP, PCL-GP-CQ scaffolds after 21 days of culture. The maximum differentiation of hUCMSCs into
osteoblasts was confirmed on day 14 and day 21of culture on the modified scaffolds of PCL-GO-CQ.
These results indicated that the synergistic effect of GO and CQ extract could enhance the expression
of osteogenic differentiation markers and can stimulate calcium deposition (Figure 11 and Figure 12).
Spontaneous differentiation of hUCMSCs on the scaffolds (Figure 11.A to E) without use of
osteogenic medium was similar to that of control group where hUCMSCs were subjected to
osteogeneic differentiation media (Figure 11.F). The least mineralization was on PCL scaffolds
without an osteogenic medium. These results suggest that the scaffolds have great potential for
osteogenic differentiation of hUCMSCs.
Also, there was a higher amount of mineralization on PCL-GO-CQ and PCL-GP-CQ scaffold. There
was slight increase in the activity of PCL-GO-CQ scaffolds as compared to PCL-GP-CQ scaffolds.
Alizarin Red S staining confirmed calcium deposition due to the osteoblastic differentiation of
hUCMSCs after 21 days on the PCL GO CQ scaffolds (Figure 11,12).

Figure 11: Alizarin Red S staining of Layer by layer scaffolds after 14 days and 21 days of
differentiation of HUCMSCs. A: PCL; B: PCL GO; C: PCL GO CQ; D: PCL GP; E: PCL GP
CQ; (A to E: without Osteoblastic differentiation medium); F: Tissue culture plate.
Figure 12: Alizarin Red S staining quantification of Layer by layer scaffolds after 14 days and
21 days of differentiation of hUCMSCs. Statistical significance compared with the TCP of the
respective group of days. No significance among groups of a number of days. (*p< 0.05).
It may be due to the secretion of osteocalcin by differentiated osteoblasts. Osteocalcin plays an
important role in bone metabolic activities and bone building [34]. It shows PCL-GO-CQ scaffolds
as potential bone regenerative scaffold. The usage of Alizarin red S staining, and Von Kossa staining
for detection of mineralization was used by various researchers as differentiation for dental pulp stem
cell osteogenic differentiation on fluorapatite modified PCL fibres [52]. PLLA-CQ scaffolds also
showed mineralization with simulated body fluid /(SBF) after 14 days of incubation with Alizarin
Red S staining [24]. The human fetal osteoblast cells also showed good intensity of mineralization
on CQ containing scaffolds on the 15th
day of culture [34]. GP proved as an alternative to BMP-2
(Bone morphogenic growth factor-2) as GP 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 presence of osteogenic media [37]. Also, GP and GO showed differentiation of MSCs into

osteoblasts by mineralization on 12th
day in the presence of osteogenic media. It also reported higher
osteoblastic differentiation potential of GP due to its ability to preconcentrate dexamethasone and β-
glycerophosphate [51]. BMPs were assessed for an osteoinduction of MSCs for 21 days of culture.
There was significant mineralization and bone nodules formation when MSCs cultured with a
combination of BMP-2+BMP-6+ BMP-9, confirmed by ARS staining [53]. MC3T3-E1 cells seeded
on PCL-rGO-Cu showed mineralization on the 14th
day in the presence of osteogenic media[17].
Von Kossa staining for calcium: The Von Kossa staining was also used to evaluate secreted calcium
deposits in differentiated cells. (Figure 13) The appearance of black precipitates confirmed the
positive Von Kossa staining with secreted mineral deposition. The black precipitates were observed
from 14 days onwards and were maximum and broad after 21 days of culture on the modified
scaffolds of PCL-GO-CQ as compared to other scaffolds. The Von Kossa staining of hUCMSCs
differentiated on the scaffolds (Figure 13.A to E) without osteogenic medium and was comparable
with the control tissue culture plate (Figure 13.F) with osteogenic medium. The least mineralization
was on PCL scaffolds without an osteogenic medium.
Figure 13: Von Kossa staining after 14 days and 21 days of differentiation of HUCMSCs. A:
PCL; B: PCL GO; C: PCL GO CQ; D: PCL GP; E: PCL GP CQ; (A to E: without Osteoblastic
differentiation medium); F: Tissue culture plate with Osteoblastic differentiation
Similar results as that of Alizarin Red S staining confirmed the differentiation of hUCMSCs into
osteoblasts.
However, similar results were observed when MSCs cultured on biphasic calcium phosphate, calcium
phosphate in presence of conditioned medium containing significant growth factors, mineralization
and was observed with positive Von Kossa staining on 21 days of culture [49]. The Von Kossa
staining was used to evaluate the osteoblastic differentiation of MSCs through mineralization. Later
up to 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 [54]. BMPs were
assessed for an osteoinduction of MSCs for 21 days of culture. There was significant mineralization
and bone nodules formation when MSCs cultured with a combination of BMP-2+BMP-6+ BMP-9,
confirmed by Von Kossa staining [53]. Fetal rat calvariae (FRC) cells were cultured on osteoblastic
medium showed mineralization or bone nodules formation on day 14, as confirmed by Von Kossa
staining [55].
4. Conclusion
PCL, PCL-GO, PCL-GO-CQ and PCL-GP, PCL-GP-CQ scaffolds were prepared with the help of
paint method, of which PCL-GO-CQ and PCL-GP-CQ scaffolds showed promising results. PCL-GO-
CQ scaffolds showed higher biocompatibility, roughness, hydrophilic nature and mechanically stable
character as compared to PCL-GP-CQ and other scaffolds. These improved characteristics could be
due to synergistic effect of GO and CQ thathelped hUCMSCs to adhere, spread, proliferates and
differentiate into osteoblast-like cells. Mainly GO and CQ callus extract provided osteoinductive
properties to scaffold that helps hUCMSCs to spontaneously differentiate into osteoblast without any

osteogenic media or growth factors or added external stimuli. This property will help the scaffold for
speedy in vivo bone formation upon transplantation, thus saving in vitro differentiation time before
transplantation. Thus, the novel PCL-GO-CQ scaffolds which is prepared using the paint method
shows tremendous potential for in vivo bone tissue engineering and further studies to regenerate bone
tissues.
Acknowledgements
The author would like to acknowledge the University Grant Commission (UGC), Government of
India, New Delhi for a doctoral fellowship to Mr Shivaji Kashte. We thank Dr Manas Kumar Santra
and Mrs Neha Gupta, National Centre for Cell Sciences (NCCS), Pune for their assistance with
confocal imaging. We acknowledge Dr Anup Kale and Mrs Vedashree Sirdeshmukh, College of
Engineering, Pune (CoEP) for assistance with SEM. We acknowledge the assistance of Mr Chetan
Chavan, Defence Institute of Advanced Technology (DIAT, DU), Girinagar, Pune for
characterization of scaffolds.
Competing interests
The authors declare no competing interests.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
Supplementary information
Leaching Study:
The leaching of the additives (GO and GP) from the scaffolds was investigated as per ISO-10994-12.
Experiments were performed in batch mode as incubated with extractants. The 0.1 g of scaffolds per
1 ml of distilled water was incubated at 37o
C for 72 hours. Concentration of GO and GP in the
obtained extracts was determined by measuring the absorbance at 233 nm and 263 for GO and GP
respectively, using a UV-visible spectrophotometer. The standard calibration curve of GO and GP
were also plotted to calculate their respective concentrations. Ultraviolet-visible (UV-Vis) absorption
spectra were recorded using a 1cm path length quartz cuvette with distilled water as the reference on
a U-2001 UV/V is Spectrophotometer (Hitachi).
The leaching study showed a small amount of GP leached from PCL-GP and PCL-GP-CQ scaffolds.
There was a small amount of GO leaching from PCL-GO and PCL-GO-CQ scaffolds (Supplementary
Figure).

Supplementary Figure1: Leaching study of scaffolds
References
[1] R. Shrivats, P. Alvarez, L. Schutte, J.O. Hollinger, Bone Regeneration, Elsevier Inc., 2014.
doi:10.1016/B978-0-12-410396-2.00041-4.
[2] J.E.L. Buddy D. Ratner, Allan S. Hoffman, Frederick J. Schoen, Biomaterials Science : An,
Acad. Press. (2004) 2015.
[3] F.J. O’Brien, Biomaterials & scaffolds for tissue engineering, Mater. Today. 14 (2011) 88–95.
doi:10.1016/S1369-7021(11)70058-X.
[4] and C.-K.C. Yang, Shoufeng, Kah-Fai Leong, Zhaohui Du, The design of scaffolds for use in
tissue engineering. Part I. Traditional factors, Tissue Eng. 7 (2001) 679–689.
doi:10.1089/107632701753337645.Published.
[5] B. Chuenjitkuntaworn, T. Osathanon, N. Nowwarote, P. Supaphol, P. Pavasant, The efficacy of
polycaprolactone/hydroxyapatite scaffold in combination with mesenchymal stem cells for bone
tissue engineering, (2015) 264–271. doi:10.1002/jbm.a.35558.
[6] K. Ren, Y. Wang, T. Sun, W. Yue, H. Zhang, Electrospun PCL/gelatin composite nanofiber
structures for effective guided bone regeneration membranes, Mater. Sci. Eng. C. 78 (2017) 324–332.
doi:10.1016/j.msec.2017.04.084.
[7] V.Y. Chakrapani, T.S.S. Kumar, D.K. Raj, T. V Kumary, Electrospun Cytocompatible
Polycaprolactone Blend Composite with Enhanced Wettability for Bone Tissue Engineering, J.
Nanosci. Nanotechnol. 17 (2017) 2320–2328. doi:10.1166/jnn.2017.13713.
[8] Y.J. Son, H.S. Kim, H.S. Yoo, Layer-by-layer surface decoration of electrospun nanofibrous
meshes for air-liquid interface cultivation of epidermal cells, RSC Adv. 6 (2016) 114061–114068.
doi:10.1039/C6RA23287F.
[9] P. Yu, R.Y. Bao, X.J. Shi, W. Yang, M.B. Yang, Self-assembled high-strength
hydroxyapatite/graphene oxide/chitosan composite hydrogel for bone tissue engineering, Carbohydr.
Polym. 155 (2017) 507–515. doi:10.1016/j.carbpol.2016.09.001.
[10] H. Fan, L. Wang, K. Zhao, N. Li, Z. Shi, Z. Ge, et al., Fabrication , Mechanical Properties , and
Biocompatibility of Graphene-Reinforced Chitosan Composites, (2010) 2345–2351.
[11] Q. Zhang, K. Li, J. Yan, Z. Wang, Q. Wu, L. Bi, et al., Graphene coating on the surface of
CoCrMo alloy enhances the adhesion and proliferation of bone marrow mesenchymal stem cells,
Biochem. Biophys. Res. Commun. (2018). doi:10.1016/ j.bbrc.2018.02.152. This.

[12] J. Qiu, J. Guo, H. Geng, W. Qian, X. Liu, Three-dimensional porous graphene nanosheets
synthesized on the titanium surface for osteogenic differentiation of rat bone mesenchymal stem cells,
Carbon N. Y. (2017). doi:10.1016/j.carbon.2017.09.064.
[13] Y. Luo, H. Shen, Y. Fang, Y. Cao, J. Huang, M. Zhang, et al., Enhanced proliferation and
osteogenic differentiation of mesenchymal stem cells on graphene oxide-incorporated electrospun
poly(lactic-co-glycolic acid) nanofibrous mats, ACS Appl. Mater. Interfaces. 7 (2015) 6331–6339.
doi:10.1021/acsami.5b00862.
[14] T. Kaur, A. Thirugnanam, K. Pramanik, Effect of carboxylated graphene nanoplatelets on
mechanical and in-vitro biological properties of polyvinyl alcohol nanocomposite scaffolds for bone
tissue engineering, Mater. Today Commun. 12 (2017) 34–42. doi:10.1016/j.mtcomm.2017.06.004.
[15] H. Ma, W. Su, Z. Tai, D. Sun, X. Yan, B. Liu, et al., Preparation and cytocompatibility of
polylactic acid/hydroxyapatite/graphene oxide nanocomposite fibrous membrane, Chinese Sci. Bull.
57 (2012) 3051–3058. doi:10.1007/s11434-012-5336-3.
[16] W. Qi, W. Yuan, J. Yan, H. Wang, Growth and accelerated differentiation of mesenchymal
stem cells on graphene oxide/poly-l-lysine composite films, J. Mater. Chem. B. 2 (2014) 5461–5467.
doi:10.1039/c4tb00856a.
[17] L.R. Jaidev, S. Kumar, K. Chatterjee, Colloids and Surfaces B : Biointerfaces Multi-
biofunctional polymer graphene composite for bone tissue regeneration that elutes copper ions to
impart angiogenic , osteogenic and bactericidal properties, Colloids Surfaces B Biointerfaces. 159
(2017) 293–302. doi:10.1016/j.colsurfb.2017.07.083.
[18] A. Siddiqua, S. Mittapally, Formulation and Evaluation of ethanolic extract of Cissus
quadrangularis herbal gel, 4 (2017) 9–29.
[19] M.S. Rao, P. Bhagath Kumar, V.B. Narayana Swamy, N. Gopalan Kutty, Cissus quadrangularis
plant extract enhances the development of cortical bone and trabeculae in the fetal femur,
Pharmacologyonline. 3 (2007) 190–202.
[20] B.K. Potu, M.S. Rao, N.G. Kutty, K.M.R. Bhat, M.R. Chamallamudi, S.R. Nayak, Petroleum
ether extract of Cissus quadrangularis (LINN) stimulates the growth of fetal bone during intra uterine
developmental period: a morphometric analysis., Clinics (Sao Paulo). 63 (2008) 815–820.
doi:10.1590/S1807-59322008000600018.
[21] N. Singh, V. Singh, R. Singh, A. Pant, U. Pal, L. Malkunje, et al., Osteogenic potential of cissus
qudrangularis assessed with osteopontin expression, Natl. J. Maxillofac. Surg. 4 (2013) 52.
doi:10.4103/0975-5950.117884.
[22] D.K. Deka, L.C. Lahon, a Saikia, Mukit, Effect of Cissus quadrangularis in accelerating healing
process of experimentally fractured radius-ulna of dog : a preliminary study., Indian J Pharmacol. 26
(1994) 44–45.
[23] S. Suganya, J. Venugopal, S. Ramakrishna, B.S. Lakshmi, V.R. Giri Dev, Herbally derived
polymeric nanofibrous scaffolds for bone tissue regeneration, J. Appl. Polym. Sci. 131 (2014) n/a-
n/a. doi:10.1002/app.39835.
[24] K. Parvathi, A.G. Krishnan, A. Anitha, R. Jayakumar, M.B. Nair, Poly(L-lactic acid) nanofibers
containing Cissus quadrangularis induced osteogenic differentiation in vitro, Int. J. Biol. Macromol.
110 (2018) 514–521. doi:10.1016/j.ijbiomac.2017.11.094.
[25] T. Zhou, G. Li, S. Lin, T. Tian, Q. Ma, Q. Zhang, et al., Electrospun Poly(3-hydroxybutyrate-
co-4-hydroxybutyrate)/Graphene Oxide Scaffold: Enhanced Properties and Promoted in Vivo Bone
Repair in Rats, ACS Appl. Mater. Interfaces. 9 (2017) 42589–42600. doi:10.1021/acsami.7b14267.
[26] S.S. Kadam, M. Sudhakar, P.D. Nair, R.R. Bhonde, Reversal of experimental diabetes in mice
by transplantation of neo-islets generated from human amnion-derived mesenchymal stromal cells
using immuno-isolatory macrocapsules, Cytotherapy. 12 (2010) 982–991.
doi:10.3109/14653249.2010.509546.

[27] S. Sachin, S. Sachin, Islet neogenesis from the constituvely nestin expressing human umbilical
cord matrix derived mesenchymal stem cell, 2 (2010) 112–120. doi:10.4161/isl.2.2.11280.
[28] A.K. Jaiswal, S.S. Kadam, V.P. Soni, J.R. Bellare, Improved functionalization of electrospun
PLLA/gelatin scaffold by alternate soaking method for bone tissue engineering, Appl. Surf. Sci. 268
(2013) 477–488. doi:10.1016/j.apsusc.2012.12.152.
[29] N. Thadavirul, P. Pavasant, P. Supaphol, Fabrication and Evaluation of Polycaprolactone–
Poly(hydroxybutyrate) or Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Dual-Leached Porous
Scaffolds for Bone Tissue Engineering Applications, Macromol. Mater. Eng. 302 (2017) 1–17.
doi:10.1002/mame.201600289.
[30] K.-Y. Tsai, H.-Y. Lin, Y.-W. Chen, C.-Y. Lin, T.-T. Hsu, C.-T. Kao, Laser Sintered
Magnesium-Calcium Silicate/Poly-ε-Caprolactone Scaffold for Bone Tissue Engineering, Materials
(Basel). 10 (2017) 65. doi:10.3390/ma10010065.
[31] H. Chhabra, J. Kumbhar, J. Rajwade, S. Jadhav, Three-dimensional scaffold of gelatin – poly (
methyl vinyl for regenerative medicine : Proliferation and differentiation of mesenchymal stem cells,
(2016). doi:10.1177/0883911515617491.
[32] P. Garg, C.P. Malik, Multiple shoot formation and efficient root induction in Cissus
quadrangularis, Int. J. Pharm. Clin. Res. 4 (2012) 4–10.
[33] P.S. R Mehta, K Teware, International Journal Of Ayurvedic And Herbal Medicine 2 : 4 ( 2012
) 661 : 678, Int. J. Ayurvedic Herb. Med. 2 (2012) 229–233.
[34] S. Suganya, J. Venugopal, S. Ramakrishna, B.S. Lakshmi, V.R. Giri Dev, Herbally derived
polymeric nanofibrous scaffolds for bone tissue regeneration, J. Appl. Polym. Sci. 131 (2014) 1–11.
doi:10.1002/app.39835.
[35] S.K. Misra, T. Ansari, D. Mohn, S.P. Valappil, T.J. Brunner, W.J. Stark, et al., Effect of
nanoparticulate bioactive glass particles on bioactivity and cytocompatibility of poly(3-
hydroxybutyrate) composites., J. R. Soc. Interface. 7 (2010) 453–465. doi:10.1098/rsif.2009.0255.
[36] X. He, L.L. Wu, J.J. Wang, T. Zhang, H. Sun, N. Shuai, Layer-by-layer assembly deposition of
graphene oxide on poly(lactic acid) films to improve the barrier properties, High Perform. Polym. 27
(2015) 318–325. doi:10.1177/0954008314545978.
[37] T.R. Nayak, H. Andersen, V.S. Makam, C. Khaw, S. Bae, X. Xu, et al., Graphene for Controlled
and Accelerated Osteogenic Differentiation of Human Mesenchymal Stem Cells, (2011) 34.
doi:10.1021/nn200500h.
[38] F. Pahlevanzadeh, E. Hamzah, In-vitro biocompatibility, bioactivity, and mechanical strength
of PMMA-PCL polymer containing fluorapatite and graphene oxide bone cements, J. Mech. Behav.
Biomed. Mater. (2018). doi:10.1016/j.jmbbm.2018.03.016.
[39] A. Oyefusi, O. Olanipekun, G.M. Neelgund, D. Peterson, J.M. Stone, E. Williams, et al.,
Graphene nanoparticles as osteoinductive and osteoconductive platform for stem cell and bone
regeneration, Biochem. Biophys. Res. Commun. 132 (2017) 410–416.
doi:10.1016/j.carbon.2017.09.064.
[40] S. Chanda, Y. Baravalia, K. Nagani, Spectral analysis of methanol extract of Cissus
quadrangularis L . stem and its fractions, 2 (2013) 149–157.
[41] E.J. Lee, J.H. Lee, Y.C. Shin, D. Hwang, J.S. Kim, O.S. Jin, et al., Graphene Oxide-decorated
PLGA/Collagen Hybrid Fiber Sheets for Application to Tissue Engineering Scaffolds, Biomater. Res.
18 (2014) 18–24.
[42] M. Yang, S. Zhu, Y. Chen, Z. Chang, G. Chen, Y. Gong, et al., Studies on bone marrow stromal
cells affinity of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate), Biomaterials. 25 (2004) 1365–
1373. doi:10.1016/J.BIOMATERIALS.2003.08.018.
[43] A.M. Pinto, S. Moreira, I.C. Gonçalves, F.M. Gama, A.M. Mendes, F.D. Magalhães,
Biocompatibility of poly(lactic acid) with incorporated graphene-based materials, Colloids Surfaces
B Biointerfaces. 104 (2013) 229–238. doi:10.1016/j.colsurfb.2012.12.006.

[44] H. Sun, F. Zhu, Q. Hu, P.H. Krebsbach, Controlling stem cell-mediated bone regeneration
through tailored mechanical properties of collagen scaffolds., Biomaterials. 35 (2014) 1176–84.
doi:10.1016/j.biomaterials.2013.10.054.
[45] S. Yang, K.F. Leong, Z. Du, C.K. Chua, The design of scaffolds for use in tissue engineering.
Part I. Traditional factors., Tissue Eng. 7 (2001) 679–689. doi:10.1089/107632701753337645.
[46] C.Y. Lin, N. Kikuchi, S.J. Hollister, A novel method for biomaterial scaffold internal
architecture design to match bone elastic properties with desired porosity, J. Biomech. 37 (2004) 623–
636. doi:10.1016/J.JBIOMECH.2003.09.029.
[47] M. Tarik Arafat, I. Gibson, X. Li, State of the art and future direction of additive manufactured
scaffolds-based bone tissue engineering, Rapid Prototyp. J. 20 (2014) 13–26. doi:10.1108/RPJ-03-
2012-0023.
[48] J. Wang, X. Yu, Preparation, characterization and in vitro analysis of novel structured
nanofibrous scaffolds for bone tissue engineering, Acta Biomater. 6 (2010) 3004–3012.
doi:10.1016/j.actbio.2010.01.045.
[49] J. Wang, D. Liu, B. Guo, X. Yang, X. Chen, X. Zhu, et al., Role of biphasic calcium phosphate
ceramic-mediated secretion of signaling molecules by macrophages in migration and osteoblastic
differentiation of MSCs, Acta Biomater. 51 (2017) 447–460. doi:10.1016/j.actbio.2017.01.059.
[50] D.. Deligianni, N. Katsala, S. Ladas, D. Sotiropoulou, J. Amedee, Y.. Missirlis, Effect of surface
roughness of the titanium alloy Ti–6Al–4V on human bone marrow cell response and on protein
adsorption, Biomaterials. 22 (2001) 1241–1251. doi:10.1016/S0142-9612(00)00274-X.
[51] W.C. Lee, C.H.Y.X. Lim, H. Shi, L.A.L. Tang, Y. Wang, C.T. Lim, et al., Origin of Enhanced
Stem Cell Growth and Differentiation on Graphene and Graphene Oxide, ACS Nano. 5 (2011) 7334–
7341. doi:10.1021/nn202190c.
[52] T. Guo, G. Cao, Y. Li, Z. Zhang, J.E. Nör, B.H. Clarkson, et al., Signals in Stem Cell
Differentiation on Fluorapatite-Modified Scaffolds, J. Dent. Res. (2018).
doi:10.1177/0022034518788037.
[53] Y. Açil, A.A. Ghoniem, J. Wiltfang, M. Gierloff, Optimizing the osteogenic differentiation of
human mesenchymal stromal cells by the synergistic action of growth factors, J. Cranio-Maxillofacial
Surg. 42 (2014) 2002–2009. doi:10.1016/j.jcms.2014.09.006.
[54] P.S. Hung, Y.C. Kuo, H.G. Chen, H.H.K. Chiang, O.K.S. Lee, Detection of Osteogenic
Differentiation by Differential Mineralized Matrix Production in Mesenchymal Stromal Cells by
Raman Spectroscopy, PLoS One. 8 (2013) 1–7. doi:10.1371/journal.pone.0065438.
[55] N. Yamamoto, K. Furuya, K. Hanada, Progressive development of the osteoblast phenotype
during differentiation of osteoprogenitor cells derived from fetal rat calvaria: model for in vitro bone
formation., Biol. Pharm. Bull. 25 (2002) 509–515. doi:10.1248/bpb.25.509.

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