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Journal of Biomaterials Science, Polymer Edition

ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsp20

Development and evaluation of cross-linked


collagen-hydroxyapatite scaffolds for tissue
engineering
Niladri Nath Panda, Sriramakamal Jonnalagadda & Krishna Pramanik
To cite this article: Niladri Nath Panda, Sriramakamal Jonnalagadda & Krishna Pramanik
(2013) Development and evaluation of cross-linked collagen-hydroxyapatite scaffolds for
tissue engineering, Journal of Biomaterials Science, Polymer Edition, 24:18, 2031-2044, DOI:
10.1080/09205063.2013.822247
To link to this article: http://dx.doi.org/10.1080/09205063.2013.822247

Published online: 01 Aug 2013.

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Date: 13 September 2015, At: 22:44

Journal of Biomaterials Science, Polymer Edition, 2013


Vol. 24, No. 18, 20312044, http://dx.doi.org/10.1080/09205063.2013.822247

Development and evaluation of cross-linked collagen-hydroxyapatite


scaffolds for tissue engineering

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Niladri Nath Pandaa, Sriramakamal Jonnalagaddab and Krishna Pramanika*


a
Department of Biotechnology and Medical Engineering, National Institute of Technology,
Rourkela 769008, Odisha, India; bPhiladelphia College of Pharmacy, University of the Sciences,
600 S 43rd Street, Philadelphia PA 19104, USA

(Received 27 April 2013; accepted 2 July 2013)


This study examines the tissue engineering potential of type I collagen cross-linked
in the presence of hydroxyapatite (HAp). Scaffolds were prepared by controlled
freezing followed by lyophilization of composite mixtures of collagen and HAp in
acetic acid, followed by cross-linking with 0.3% glutaraldehyde. Scaffolds of three
ratios were prepared, corresponding to collagen/HAp ratios of 1:2, 1:4, and 1:6. The
scaffolds were evaluated for their microstructure, chemical and physical properties,
swelling behavior, mechanical strength, biodegradability hemocompatability, cytocompatibility, and histopathology following subcutaneous implantation in Sprague
Dawley rats. The collagen/HAp matrices showed a smaller pore size of 1040 m
compared to 50100 m for pure collagen scaffolds. Pure collagen showed a
mechanical strength of 0.25 MPa, and the value almost doubled for cross-linked
composites with collagen/HAp ratio 1:6. The improvement in mechanical strength
corresponded to a decrease in swelling and enzymatic degradation (measured by
resistance to collagenases). FTIR spectra results in conjunction with scanning electron micrographs showed that cross-linking in the presence of HAp did not signicantly alter the structure of collagen. MTT assay and calcein AM staining revealed
prominent and healthy growth of mesenchymal stem cells in both the pure collagen
as well as collagen:HAp composites of ratio 1:2. In vivo implantation in Sprague
Dawley rats showed an initial acute inammatory response during days 3 and 7, followed by a chronic, macrophage-mediated inammatory response on days 14 and
28. Overall, a cross-linked collagen/HAp composite scaffold of ratio 1:2 was identied as having potential for further development in tissue engineering.
Keywords: collagen; hydroxyapatite; scaffolds; tissue engineering

1. Introduction
Tissue engineering presents an alternative approach to repair or replace lost or damaged
human tissue resulting from injury or disease.[1] The discipline encompasses an understanding of the attachment and proliferation of progenitor cells [2,3] or growth factors
[4] onto scaffolds. Skeletal tissue is a highly organized structure comprising of mainly
type I collagen, nanometre-sized carbonate-substituted hydroxyapatite (HAp) crystals,
water, and various other noncollagenous proteins. Collagen type I presents natural binding sites such as Arg-Gly-Asp (RGD) and Asp-Gly-Glu-Ala (DGEA) peptide sequences

*Corresponding author. Email: kpr@nitrkl.ac.in


2013 Taylor & Francis

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N.N. Panda et al.

that modulate the adhesion of osteoblasts and broblasts.[5,6] A major limitation for
the use of collagen alone is its low mechanical strength, high swelling capacity, and
rapid degradation. HAp is a common excipient used to reinforce collagen in matrices
intended for tissue engineering.[710] HAp is a mineral (a form of calcium apatite),
and, therefore, lacks the natural binding sites presented in collagen. It also has certain
drawbacks such as limited biodegradability and osteointegration properties.[11]
Several additional excipients have, therefore, been explored to limit the use of high
ratios of HAp in collagen/HAp matrices. Examples include gelatin,[12] poly(-lactideco-verepsilon-caprolactone),[3] Poly L-lactic acid,[13] and Poly-benzyl-L-glutamate.[14]
This research proposes to strengthen a collagen/HAp matrix that is reinforced via crosslinking with glutaraldehyde as the cross-linking agent. Strengthening via cross-linking is
expected to enhance mechanical strength, decrease swelling and degradation rate at relatively low concentrations of HAp. Glutaraldehyde has previously been used to strengthen
collagen scaffolds.[15] The microstructural mechanism for reinforced strength has been
reported to be due to increased resistance to bril rotation.[16]
2.
2.1.

Materials and methods


Collagen isolation

The use of rat tail tendon for tissue engineering applications is well established in literature.[17] In this study, type I collagen was extracted from Sprague Dawley rat tail tendons in a manner similar to that described by Liu et al. [18] with modications.
Sprague Dawley rat tails were obtained from the animal house of Indian Institute of
Chemical Biology, India. HAp was prepared by microwave synthesis using the method
used by Liu et al. [19]
2.2.

Determination of collagen purity

The purity of isolated collagen was analyzed using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) according to the method described by lammelli.[20]
Briey, a 3% stacking gel was prepared from a stock solution of 30% w/w acrylamide and
0.8% w/w of N,N bis methylene acrylamide. The nal concentration of gel prepared for
separation was 0.375 M TrisHCl (pH 8.8) and 0.1% SDS. Tetramethylenediamine and
ammonium persulfate were used for gel polymerization, and the casted gel was cut to a
dimension of 15 cm  6 cm. The sample was immersed in boiling water for 1.5 min to
enable protein dissociation. The electrophoresis was performed with a current of 3 mA per
gel until the bromophenol blue marker reached the bottom of the gel (about 78 h). The
proteins were xed in the gel with 50% TCA overnight, stained for 1 h at 37 C with a
0.1% coomassie brilliant blue solution made freshly in 50% TCA. The gels were then
washed repeatedly in 7% acetic acid prior to taking the autoradiograph.
2.3.

Fabrication of scaffolds

A 0.5% w/v solution of collagen in acetic acid was used as the stock solution for scaffold
preparation. HAp was added to correspond to a collagen:HAp ratio of 1:2, 1:4, or 1:6.
The solution mixtures were gradually frozen and subsequently lyophilized to obtain
scaffolds. The HAp-containing scaffolds were cross-linked by immersion in a
glutaraldehyde bath of concentration 0.3% and pH 8.0 for 2 h at room temperature. The
scaffolds were then washed repeatedly with distilled water, and then lyophilized for an
additional 14 h at 40 C to eliminate water without loss of structural integrity (Table 1).

Journal of Biomaterials Science, Polymer Edition


Table 1.

Collagen-HAp composites compositions.

Specimen

Collagen:
hydroxyapatite (w/w)

Concentration of collagen
solution (w/v) in acetic acid, %

Collagen
CH1
CH2
CH3

1:2
1:4
1:6

0.5
0.5
0.5
0.5

2.4.

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Characterization of scaffolds

(1) Scanning electron microscopy: The surface microstructures of the scaffolds


before and after cross-linking with glutaraldehyde were observed under scanning
electron microscope (SEM, JeolJsm 5200) operated at an accelerating voltage of
215 kV. The mean pore size of the scaffolds was determined from SEM images
by importing data to image-J software. Ten pores were measured for each measurement, in a perpendicular direction through the long axis and the minor axis.
Average values were reported as the mean pore size. The tensile strength of the
scaffolds was evaluated on an Instron Material tensile testing machine 4204
(UK) using pneumatic grips with load cell 500 N at room temperature. The
cross-head speed was set at1 mm min1. Four samples were tested for each composition. The specimens were of rectangular shape and the dimensions were
measured with dial calipers. Three readings were taken at different positions,
and mean values were used to calculate the cross-section. Infrared spectra were
obtained in absorption mode using a FTIR spectrometer (NicoletTM Thermo
380) from collagen-HAp scaffolds.
(2) Swelling behavior: The water absorption capacity of collagen-HAp scaffolds was
determined by equilibrium swelling studies.[21] Four scaffolds were taken from
each specimen, and their dry weights recorded. The scaffolds were then dipped
in 30 mL PBS solution in Petri dishes, and weights recorded at 10 min intervals
for up to 2 h. At each immersion interval, the samples were removed, dabbed
with a lter paper to remove excess water, and weighed on a microbalance with
a sensitivity of 0.01 mg. The sample weight before and after immersion was
denoted as W0 and We, respectively. The percent swelling at equilibrium, Esw,
was calculated from the Flory-Huggins swelling equation shown below:

Esw



We  W0
100
W0

(3) Water permeation: Water permeation studies were performed following protocol
described by Sato et al. (2010).[22] Briey, a water reservoir was attached with
a polyethylene tube. The collagen and its composite scaffold of about 4 cm in
length were connected to the other end of polyethylene tube. The water reservoir
was set to a height of about 165 cm. A pressure transducer was set to verify a
120 mm Hg pressure constantly at its distal end. The permeated water through

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N.N. Panda et al.

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the scaffold structure was collected with respect to time(s) in a graduated cylinder in order to calculate in mL/min/cm2.
(4) Biodegradability: In vitro degradation tests of cross-linked collagen-HAp
scaffolds were performed using bacterial collagenase obtained from Clostridium
histolyticum (EC 3.4.24.3, Sigma Chemical Co., St. Louis, MO, USA) with a
collagenase activity of 300 units/mg. The in vitro degradation studies for
collagen and its composite scaffolds were performed at 37 C in 10 mL phosphate buffered solution (pH 7.4) containing collagenase in CO2 incubator
(Lishen HF151UV). The dynamic degradation of scaffolds was monitored for
four compositions with known dry weights by incubated at varying collagenase
concentrations (0.1, 0.5, 1, and 2 mg) for 24 h.

2.5.

Preliminary biocompatibility testing

(1) Hemo-compatibility testing: Human peripheral blood was collected in a 15 mL


tube containing 3.8% sodium citrate to avoid coagulation.[23] Blood diluted
with PBS (pH 7.4) at a ratio of 1:20 was used as the negative control, while
blood mixed with triton X was the positive control. Punched scaffolds of 5 mm
diameter were sterilized by exposure to UV light in a sterile culture hood, followed by rinsing with 70% ethanol. The sterile scaffolds were immersed in
100 l of blood diluted with PBS solution followed by incubation at 37 C for
60 min. The samples were then centrifuged at 3000 rpm for 10 min. The absorbance of the supernatant was measured at 545 nm.
(2) Calcein AM staining: Pure and composite scaffolds of 6 mm diameter were
added individual wells of a 12 well-tissue culture plate. MSCs in DMEM media
were added to these wells at the concentration of 105 cells/mL. The cells were
placed in a 5% CO2 incubator for 1012 h to enable attachment to scaffolds, following which the media was discarded, and calcein green AM added at a concentration of 10 M. The stain was prepared by dissolving in DMSO (less than
0.01%), and adding to fresh DMEM media.
(3) Inside the cells, calcein-AM is hydrolyzed by endogenous esterases into the
negatively charged green uorescent calcein, which is retained inside the cytoplasm. After incubation in the stain for an hour, the media was discarded and
the cells thoroughly washed with PBS (pH = 7.2). Around 810 beads were
then taken on a clean microscopic slide and mounted with glycerol: PBS (1:1
ratio), and observed under an upright uorescence microscope (Carl Zeiss
E600, Wavelength = 450 nm) using a green lter (wavelength = 490 nm) at
50 magnication. The cell-scaffold constructs were sectioned and the cell
adhesion and viability were also observed in the inner regions of the scaffolds.
The images were captured using a Carl Zeiss Axiocam camera attached to the
microscope.
(4) Cell culture studies: Mesenchymal stem cells (MSCs) were isolated from umbilical cord blood (obtained from Ispat General Hospital, Rourkela, India) using
DMEM F-12 medium (Gibco BRL, USA) supplemented with 10% fetal bovine
serum (Gibco BRL, USA) and 1% U/mL streptomycinpenicillin and incubated
at 37 C and 5% CO2. The MSCs were sub-cultured every three days by rst
adding 0.25% trypsin and 0.02% EDTA for detachment.

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Journal of Biomaterials Science, Polymer Edition

2035

The prepared scaffolds were cut into cylindrical disks with a 6 mm dermal biopsy
punch (Miltex) and kept in a 96-well plate. They were sterilized with 70% ethyl alcohol
(v/v) followed by UV treatment, followed by hydration in sterile PBS (pH 7.4). The
scaffolds were then conditioned in DMEM medium for 4 h before cell seeding. Cells
were seeded at densities of 1  105 cells/well on these scaffolds. The seeded constructs
were incubated for 3 or 5 days at 37 C and 5% CO2 to examine for growth and proliferation. The numbers of living cells inside the scaffold were determined using the MTT
assay. After discarding the culture media, the cells were further washed with PBS to
remove the non-adhered cells. One ml/well of fresh medium and 100 l/well of MTT
was added to the wells, and the cells were cultured for an additional 4 h. The same procedure was repeated for 3 identical samples, except that these were cultured for an additional 12 h. The medium was then removed, and 150 l of dimethyl sulfoxide (DMSO)
was added to each well. The optical density (OD) was measured at wavelength 595 nm
using a microplate reader (Model PerkinElmer 2030 explorer). The percentages of
attached cells were calculated using following formula as:
The OD595 of cells on matrix after washing
 100%
The OD595 of cells in culture wells

2.6. Animal implantation and histology analysis


Sprague Dawley rats were implanted subcutaneously with 1 cm2 ECM scaffold materials
with 1 mm thickness. The rats were sacriced at days 3, 7, 14, and 28 post-implantation. The tissues along with the scaffold were collected in a neutral buffered formalin
xative. After xation, the tissues were processed in an automated tissue processor and
tissue sections of 4 m thickness were cut using Leica microtome (Leica RM2255,
Germany). The tissue sections were then stained with routine hematology and eosin
stain and examined under inverted Olympus microscope.
2.7. Statistical analysis
The data are expressed as the mean SEM for three independent experiments and its
statistical signicance was evaluated by one way ANOVA followed by Bonferronis
post hoc test (GraphPad Prism). p < 0.05 was considered statistically signicant.
3.

Results

3.1. Characterization of composite scaffolds


3.1.1. Microstructure evaluation
Figure 1 shows SEM photographs of the surface morphology and pore structure of single collagen and its composite, i.e. collagen: HAp scaffolds. All scaffolds showed relatively similar macroporous morphology with a high degree of interconnectivity.
Collagen-HAp scaffolds appeared less regular compared to pure collagen scaffolds.
Scaffolds prepared from pure collagen had a larger pore size of approximately
50100 m compared to the composite scaffolds that were considerably smaller at
1040 m. The 3-dimensional interconnectivity that could be seen in all scaffolds is
considered necessary to enable host cell inltration to guide the differentiation and proliferation of cells toward the targeted functional tissue or organ.[24,25]

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N.N. Panda et al.

Figure 1. Scanning electron micrographs of: (a) collagen scaffold; (b) collagen:HAp (1:2), (c)
collagen:HAp (1:4); and (d) collagen:HAp (1:6).

3.1.2. Mechanical analysis


Pure collage showed a tensile strength of 0.251 0.007 MPa. The tensile strength
increased signicantly (p < 0.005) with the increasing ratio of HAp in the composite
scaffold as shown in Figure 2. The mechanical strength almost doubled for scaffolds
prepared with a collagen:HAp ratio of 1:6.
3.1.3. Swelling and water permeability studies
The results of both these studies demonstrated that the inclusion of HAp lowers the
hydrophilic characteristics of the scaffold. Figure 3 shows that equilibrium swelling
decreased signicantly upon incorporation of HAp. The water permeability of the scaffold to water was also consistently lowered upon the inclusion of water. Water ux
through the scaffolds was measured at 57 5 mL/min/cm2 for pure collagen scaffolds,
and 39 4.3, 28 6, and 15 3.5 mL/min/cm2 for collagen composite scaffolds of
composition 1:2, 1:4, and 1:6 ratios, respectively.

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Journal of Biomaterials Science, Polymer Edition

Figure 2.

Effect of HAp ratio on the scaffold strength.

Figure 3.

The effect of HAp ratio on water uptake measured as present swelling.

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N.N. Panda et al.

Figure 4.

3.1.4.

A representative FTIR spectra of collagen-HAp scaffolds (collagen:HAp ratio 1:2).

Infrared analysis

These studies were performed to ensure that the cross-linking with HAp does not affect
the chemistry of collagen. This is important considering that cells are more prone to
attach surfaces containing natural binding sites.[26]
Infrared spectrum of a composite scaffold is shown in Figure 4. The FTIR spectra
showed peaks at 3485.6 cm1 and 1529.57 cm1. These were in the range of -NH
stretching and N-H bending of secondary amide on collagen type I, indicating that these
functional groups may still be available to promote cell attachment to collagen. HAp is
characterized by an anti-symmetric phosphate band at around 12001000 cm1.[27] The
peak observed at 1203.5 cm1 in Figure 4 corresponds to phosphate stretching, and
conrms the presence of HAp in the scaffold.
3.1.5. Biodegradability testing
Figure 5 shows that the cross-linked collagen-HAp scaffolds showed greater
resistance to degradation than the pure collagen scaffolds in all collagenase
solutions.
3.1.6. Hemo-compatibility testing
Blood compatibility is a critical requirement for the blood interfacing biomaterials. The
implants should not damage proteins, enzymes, or elements of blood (red blood cells,
white blood cells, and platelets). Figure 6 shows that the % hemolysis increased two to
three times for the cross-linked collagen/HAp scaffolds compared to pure collagen
scaffolds.

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Journal of Biomaterials Science, Polymer Edition

Figure 5.

2039

The effect of HAp ratio on scaffold degradation.

Figure 6. The effect of HAp ratio on hemo-compatibility, measured as percent% hemolysis in


human peripheral blood.

3.1.7. Attachment and proliferation of mesenchymal stem cells onto the scaffolds
The results for MTT and cell adhesion assay are shown in Figure 7. The results were
plotted by measuring the OD and reported as percentage of cells adhered relative to
seeded MSCs. The attachment ratios were found to be more than 50% in all cases when

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Figure 7.

N.N. Panda et al.

The effect of HAp ratio on the adhesion of MSCs.

Figure 8. (a) and (b) Two representative uorescent microscopy photographs of calcein AM
staining in collagen/HAp scaffolds of ratio 1:2. (c) The scaffold after 72 h of incubation.

MSCs were cultivated for 12 h in the media and seeded over the scaffold. Cell proliferations were performed for 3 and 5 days, and signicant increases were observed.

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Journal of Biomaterials Science, Polymer Edition

2041

Figure 9. Tissue reaction to cross-linked scaffolds of ratio 1:2 at 3 days (a), 7 days (b), 14 days
(c), and at 28 days (d) after subcutaneous implantation in Sprague Dawley rats. The representative
scaffold shown here represents a ratio of collagen/HAp of 1:2.

Morphological analysis of cells was performed through uorescence microscopy


after Calcein AM staining. Figure 8(a) and (b) shows viable green colored cells almost
evenly distributed on surface as well as within the scaffold as shown. Figure 8(c) shows
that after 72 h of incubation, the cells undergo a morphological change from rounded to
spreading type morphological features conrming a healthy microenvironment of the
scaffold for cellular differentiation and proliferation.
3.1.8.

Animal implantation and histology analysis

The tissue reaction was assessed by histopathology (Figure 9). Tissue response at days
3 and 7 was that of an acute inammatory reaction mediated by neutrophilic inltration.
The reaction then shifted to chronic inammation predominated by macrophages by
14 days. The chronic inammation persisted at 28 days, but the reaction was less intense
compared to response at day 14. There was evidence of healing by brosis from day
seven, progressing until the 28th day.
4.

Discussion

Pore size is an important characteristic of membrane-based scaffolds as it determines


the overall surface area available for cell-attachment. The composite scaffolds reported
in this study have pore diameters in the range of about 10 to 40 m and, therefore,

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N.N. Panda et al.

possess signicantly greater surface area compared to pure collagen scaffolds. The pore
sizes obtained in our study were similar to that of Chen et al. [28] Such dissimilar morphologies consisting of a highly porous structure and high mechanical strength may
have potential for the design of dermal implants. The porous surface can facilitate the
recruitment and proliferation of dermal broblasts to promote wound healing, while
composite structure can contribute to mechanical strength and barrier efcacy to prevent
infection and dehydration.[29] The roughly spherical shape of the pores as well as their
presence on the surface suggests that pore diameters may be inuenced by the size of
the evaporating solvent droplets as well as particle size of HAp.[30] Pores over the surface of the scaffold are most common sites of stress concentrator and crack initiator.
But incorporation of HAp increases the density of the composite material which retards
crack propagation. Moreover, the decrease in pore size for the composite improves the
circularity index of the pores and hence decreases crack propagation.[31] This property
has been veried with measurement of ultimate strength (Figure 2) where increase in
addition of HAp improves compressive strength of the scaffold. These observations are
consistent with previous literature.[32] The possible reason may be explained by either
an increase in total binding area of collagen with HAp or reduction of defects in case
of composite scaffold, both of which may lead to an increase in mechanical strength.
This conclusion is further supported by our decrease in swelling ratio for composite
scaffold. Okoshi et al. proposed that porous grafts with water permeability of about 10
40 mL/min/cm2 induced endothelization and kept patency.[33] The porosity of both collagen and composite scaffolds was in a comparable range.
A decrease in availability of interactive surface area in the composite scaffold
results lowers swelling, water permeation, and therefore exposure to enzymes responsible for degradation. The collagen/HAp scaffolds may therefore be expected to have a
lower degradation rate and a greater residence time following implantation.
While the mechanical and degradation properties of the collagen/HAp scaffolds
seem favorable, a drawback of the glutaraldehyde cross-linking is the lower hemocompatibility compared to pure collagen scaffolds. As both collagen and HAp were considered biocompatible, the decrease may be a consequence of residual glutaraldehyde
entrapped in the collagen-HAp composites. It is possible that the amount of residual
glutaraldehyde is higher in scaffolds containing greater amount of HAp. This outcome
is further supported by the fact that the collagen:HAp (1:2) composite showed nearly
same proliferation rate as that of pure collagen, while the composite with higher ratio
showed decreased cell proliferation and attachment.
5.

Conclusion

This study shows that cross-linked collagen-HAp scaffolds may present a valuable bone
tissue engineering platform. Advantages include a relatively slow degradation rate, high
mechanical strength, and a collagenous macroporous structure with potential to trigger
cell attachment and proliferation. The biocompatibility of composite scaffold though
found to be less than those of pure collagen scaffold, the collagen:HAp with 1:2 composite has comparable degree of cell adhesion and viability with those of pure collagen.
However, there is decrease in haemocompatibility with the increasing concentration of
HAp which may be due to increased amount of glutaraldehyde retention in the
composite.

Journal of Biomaterials Science, Polymer Edition

2043

Acknowledgment
The Indian Institute of Chemical Biology, Kolkata for providing the facilities for conducting the
various tests, especially Dr T Chakraborty.

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