Preparation and properties of tannic acid cross-linked collagen scaffold

and its application in wound healing

Venkatachalam Natarajan, Natarajan Krithica, Balaraman Madhan, Praveen Kumar Sehgal

Central Leather Research Institute (Council of Scientific and Industrial Research),
Sardar Patel Road, Adyar, Chennai 600 020, India

Received 11 January 2012; revised 30 August 2012; accepted 5 October 2012

Published online 19 December 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32856

Abstract: A biodurable porous scaffold of collagen with
good biocompatibility and enhanced wound healing poten-
tial is prepared through casting technique using tannic acid
(TA) as crosslinker. The morphological analysis of the tannic
acid cross-linked collagen scaffold (TCCs) distinctively shows
scaly interlinks with large pores. The enzymatic stability of
the scaffold is characterized in vitro to detail the role of TA
in stabilization of collagen matrix against collagenolytic deg-
has been established using 3T3 fibroblasts. Therapeutic and
wound healing potential of the TCCs has been studied in
vivo using excision wound model in rats. The results clearly
indicates that the TCCs has greater and significant effect in
wound closure and increased the wound healing rate com-
pared with native Cs. This biocompatible and biodurable
scaffold may find broad applications in the tissue engineer-
ing and drug delivery applications. VC 2012 Wiley Periodicals,

radation. TCCs shows more stability (>54%) against collage-
nase than that of the collagen scaffolds (Cs). The attenuated
total reflectance Fourier transform infrared analysis of the
TCCs confirms the noncovalent interaction between collagen
and TA. The biocompatibility of the scaffold (TCCs) in vitro
Inc. J Biomed Mater Res Part B: Appl Biomater 101B: 560–567, 2013.
Key Words: tannic acid, collagen scaffold, tissue engineering
and regeneration, wound healing, enzymatic stability

How to cite this article: Natarajan V, Krithica N, Madhan B, Sehgal PK. 2013. Preparation and properties of tannic acid cross-linked
collagen scaffold and its application in wound healing. J Biomed Mater Res Part B 2013:101B:560–567.

INTRODUCTION treatment,2 and variety of other chemical methods1,3 are the

In recent years, studies on engineering biopolymer to
three-dimensional (3D) architectures (i.e. scaffold) for tissue
engineering and regeneration have received a lot of atten-
tion. Collagen matrix, a successful resorbable biomaterial in
tissue engineering, exhibits very good hemostatic properties,
low antigenicity, low inflammatory, promotes the prolifera-
tion and migration of cells as well as angiogenesis.1–3 The
faster biodegradation of 3D collagen architectures compared
with synthetics in in vivo or in situ is a limitation in tissue
engineering applications. The enhanced degradation of colla-
gen scaffold (Cs) by the matrix metalloproteinase (MMPs)
from wound exudates and bacterial infections is one of the
major drawbacks for collagen-based wound dressing materi-
als.4–7 This leads to the breakdown of matrix faster, failure
to maintain the moisture conditions and therapeutic drug at
the wound site. This clearly establishes the requirement for
an advanced biodurable collagen biomaterial for wound
repair, which will substantially prevents bacterial infection,
keeps moisture and provides controlled release of the drug
or growth factors loaded. Synthetic peptides mimicking col-
lagen triple helices,8 cross-linking collagen with aldehydes,
such as formaldehyde and glutaraldehyde,1 dehydrothermal
approaches to develop biodurable scaffold for the applica-
tions in tissue engineering and drug delivery.9 The failure in
achieving the innate collagen properties using synthetics
and toxicity of the aldehyde crosslinkers has motivated
researchers to look out for the utilization of bioactive mole-
cules that can stabilize collagen.10,11
Plant polyphenols, which are widely used as therapeu-
tic agents in various inflammatory diseases, can be used
as potential cross-linking agents for collagen.12 Tannic acid
(TA) is a plant polyphenol found abundantly in the galls of
Rhus and Quercus species and they are categorized as an
astringent, antioxidant, antimicrobial, antiviral, and anti-
inflammatory agent. Structurally, TA has glucose moiety as
core and hydroxyl groups of glucose are esterified with
five digallic acid.13 In the early 20th century, it was used
in the treatment of burns and later it was discontinued
because of its inconclusive necrosis effect on liver.14
Recent studies on highly purified TA confirmed that toxic
effects observed in patients with burn were mainly due to
contaminants (particularly gallic acid) present in the prep-
arations used in clinical trials and toxicity studies
performed.15

Additional Supporting Information may be found in the online version of this article.
Correspondence to: B. Madhan; e-mail: bmadhan76@yahoo.co.in
Contract grant sponsor: Council of Scientific and Industrial Research (CSIR) under Young Scientist Award (YSA) project scheme

560 C 2012 WILEY PERIODICALS, INC.

V

Recent reports on the stability enhancement of collagen
and elastin in the newly synthesized fibers,16–18 cardiovas-
cular constructs,13 and extra cellular matrix (ECM) using TA
moved its category to ‘active biomolecule’ in Medicinal field
from the category of ‘vegetable tanning agent’ in leather
industry. Such plant polyphenols are well established to
crosslink and stabilize collagen.19,20 Zhang et al.21 and Jack-
son et al.22 in their studies concluded that TA prevents col-
lagen matrix degradation via cross-linking fibrous collagen
and inhibiting MMPs. Very recently, Cass and Burg23 have
prepared collagen gels using TA as crosslinker and con-
cluded that 1 mg mL
1 is best for inducing apoptosis in
MCF-7 cancer cell lines and this concentration was not toxic
to mesenchymal stem cells (D1 cells).
In this study, we have presented the preparation of TA
cross-linked collagen scaffolds (TCCs) as an advanced
matrix and characterized them for enzymatic stability and
effectiveness as a wound dressing material.
MATERIALS AND METHODS
Materials
TA (of Chinese galls), collagenase type IA (2.8 units mg
1 solid
collagen), and 4,6-diamidino-2-phenylindole dihydrochloride
(DAPI) were purchased from Sigma–Aldrich (Bangalore,
India), type I collagen was isolated from bovine achilles ten-
dons using the previously reported procedure and purified.24
Other reagents and solvents used were of analytical grade.
Methods
Fabrication of TCCs. The collagen solution of 5 mg mL
1
and TA of 25 mg mL
1 were prepared as stock using 0.5 M
acetic acid and double distilled water, respectively, and
stored at 4 C. The TCCs was prepared by adding the
required volume of TA to collagen solution from TA stock to
make a final concentration of 1.5 mg mL
1. Previously, the
deaerated collagen solution of required volume to make a
final concentration of 3 mg mL
1 was taken with 300 lL of
Triton X-100 in a beaker. The final volume was made using
0.5 M acetic acid. This mixture was continuously stirred
under IKA T25 Homogenizer at 13,500 rpm to generate uni-
form foam. The foamed TA–collagen solution was poured
into the molds (7 cm  3 cm, with depth of 2.5 cm) and
frozen at
80 C for 24 h followed by lyophilization for 48 h
using Operon Co., Korea. The completely dried scaffolds
were stored in airtight containers. The Cs was prepared as
control as above except the addition of TA.
Characterization
Morphological studies. Morphology of the scaffold samples
were examined by a scanning electron microscope (SEM,
JEOL-JFC 6360 SEM, Japan). The scaffolds were kept on one
side of the adhesive stub. The stub was then coated with
gold using sputter coater (JEOL-JFC 1600 AUTO COATER)
and then the SEM micrographs were taken under 15 kV
emission voltage.
Water uptake capacity. Water uptake capacity of Cs and
TCCs was determined by gravimetric method. In this
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MAY 2013 VOL 101B, ISSUE 4
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ORIGINAL RESEARCH REPORT
method, 1 cm2 of scaffolds were weighed using electronic
balance (Sartorius electronic balance, model: BSA224S-CW,
Germany) and placed separately in a beaker containing 20
mL of millipore water for 60 min. The swollen scaffolds
were blotted with filter paper and immediately weighed in
the electronic balance. The percentage water uptake was
calculated using the following equations:
Wt
Wo
S¼ (1)
Wo
S
U¼ (2)
cm2
where S is the percentage water uptake ability, Wt denotes
the weight of the scaffolds at time t, and W0 is the initial
weight of the scaffolds, U ¼ water uptake in % per unit
area (cm2). Triplicates were analyzed for both Cs and TCCs.
Tensile strength measurements. Mechanical properties of
the prepared scaffolds (dry) were characterized by tensile
strength measurement using Instron Instruments-Series IX
automated materials testing system. The dumb bell-shaped
samples of size 1 cm  4 cm were cut out in the middle of
the prepared scaffold. The width of samples was 0.5 cm in
the middle. Triplicates were analyzed for both Cs and TCCs.
Enzymatic stability. The enzymatic stability of prepared
scaffolds were conducted in vitro by incubating (42 h) the
Cs and TCCs in 100 lg mL
1 collagenase solution prepared
using 0.05 M CaCl2 solution buffered at pH 7.4 with 0.05 M
of Tris–HCl. The samples were centrifuged at 1500 rpm for
10 min, aliquot of the clear supernatant was hydrolyzed
using conc. HCl (final conc. 6 M) at 120 C for 12 h.25 The
amount of collagen degraded was calculated from the
estimation of amount of hydroxyproline in the supernatant.
The hydroxyproline was estimated from standard curve
prepared using known concentration of hydroxyproline.25
Triplicates were analyzed for both Cs and TCCs.
ATR-FTIR analysis. Attenuated total reflectance (ATR) Fou-
rier transform infrared (FTIR) spectra were taken (using
instrument ABB MB3000, ABB Bomem, Canada) after desic-
cation to investigate the possible chemical interactions
between TA and collagen in the TCCs.
Bioevaluation
In vitro studies using fibroblasts. NIH 3T3 fibroblasts
were cultured in Dulbecco’s Modified Eagle’s Medium sup-
plemented with 10% fetal bovine serum at 37 C in 5% CO2
atmosphere. The cells were harvested by trypsin–ethylene-
diaminetetraacetic acid treatment and used for seeding. All
scaffolds were sterilized by exposing to ethylene oxide gas.
The scaffold was then placed in 24-well polystyrene plates
and seeded with 5  104 cells well
1. The culture medium
was changed every 2 days. Cell proliferation was monitored
at days 1, 3, 5, and 7 using 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay.
561

FIGURE 1. Scanning electron micrographs (top view) of collagen scaffold (A) and tannic acid cross-linked collagen scaffold (B). 1300.
The attachment of NIH 3T3 fibroblasts cells seeded to
the scaffolds was observed after 7th day using confocal laser
scanning microscopy (CLSM), Carl Zeiss, Germany, LSM 710
laser scanning microscope and SEM. For CLSM analysis, the
scaffolds were incubated for 10–15 min with DAPI, which
was prepared using PBS to a final concentration of 2.5 lg
mL
1. The scaffolds were visualized by laser-diode 405
through the objective plan apochromate 40  0.95|corr M27.
For SEM analysis, the scaffolds were rinsed with PBS to
remove nonadherent cells and then fixed in 2.5% glutaralde-
hyde prepared using 0.1 M sodium cacodylate buffer (pH
7.4) for 24 h. Subsequently, the scaffolds were dehydrated
through a series of graded alcohol solutions and finally with
100% ethanol.26 After complete dehydration, samples were
freeze dried overnight and observed through SEM as per the
procedure given in Morphological studies section.
In vivo studies. The male wistar albino rats (n ¼ 45)
weighing 200–250 g were housed under the norms of the
Committee for the Purpose of Control and Supervision of
Experiments on Animals. The rats were divided into three
groups with 15 each and anesthetized by intraperitoneal
injection of thiopentone sodium at 40 mg kg
1 body weight.
Full thickness excision wounds measuring 1 cm  1 cm
were created using scalpel blade after shaving the dorsal
area of the skin. The actual wound area was traced in poly-
ethylene terephthalate (PET) transparent sheets using per-
manent marker and the wound was dressed with scaffold of
1 cm  1 cm size which was previously sterilized by expos-
ing to ethylene oxide gas. The scaffold on the wound was
further covered with the nonadherent gauze around the
body to protect the scaffold from rat and unpeel. The
animals are grouped as follows:
• Group 1 (control): undressed wounds covered with sterile
nonadherent gauze.
• Group 2 (reference): Cs dressed covered with sterile
nonadherent gauze.
• Group 3 (test): TCCs dressed covered with sterile nonad-
herent gauze.
The dressed rats were placed in individual cages and
effects of scaffolds in wound healing were observed on 4th,
8th, and 12th days from the day of wound dressed by
562 NATARAJAN ET AL.
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observing % wound closure and histological examination.
Five rats from each group were used for this study at every
sampling time.
The % wound closure8 was calculated using the follow-
ing equation:
ðAi
AtÞ
%Wound closure ¼  100 (3)
Ai
where Ai denotes initial wound area, At denotes the wound
area measured at the time of biopsy. Ai and At were traced
in PET transparent sheets using permanent marker. The cir-
cumferences in the traced sheets were measured using GGT
digitizer (size A0) and areas were measured using Gerber
AccuMark version 8.5.
The histology of excised wound skins were formalin
fixed, processed and embedded in paraffin. Sections of thick-
ness 3–5 lm were stained using hematoxylin and eosin
(H&E) and Masson’s trichrome (MT) stain as per procedures
reported earlier.8,24
Statistical analysis
The quantitative assays were analyzed statistically using
Student’s t-tests. The data were presented as mean 6 stand-
ard error mean (SEM) with p < 0.05 considered statistically
significant.
RESULTS AND DISCUSSION
Fabrication of TA–Cs
The collagen solution with the combination of TA showed
less foam formation during the final steps of fabrication
compared with the native collagen solution. This could be
due to the crosslinking of collagen by TA. The color of the
Cs was white, whereas TCCs was observed to be slightly
brownish. The handling stability of the Cs was lower than
that of TCCs (i.e. the thickness of TCCs was consistent
throughout the handling compared with the Cs, which fails
to maintain the thickness).
Scaffold characterization
Morphological studies. SEM morphology of Cs and TCCs
(Figure 1) showed the presence of porous mesh with inher-
ent interconnectivity. These factors are said to have a prom-
inent role in cell proliferation, function, and migration. The
TANNIC ACID CROSSLINKED COLLAGEN SCAFFOLDS
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ORIGINAL RESEARCH REPORT

TABLE I. Results of the Enzymatic stability, Water Uptake, presented in Table I. The amount of collagen degraded was
and Tensile Strength of Cs and TCCs quantified based on the release of hydroxyproline and was
Scaffold Enzymatic Water Uptake Tensile observed to be only 9.9% for TCCs and 64% in the case of
Type Degradation (%) Capacity (%) Strength (MPa) Cs control. This result clearly emphasizes higher stability of
the TCC scaffolds when compared with that of the Cs
Cs 64.09 6 1.22 669 6 98 3.57 6 0.197
TCCs 9.88 6 2.81 242 6 10 2.94 6 0.229
against collagenase. TA containing galloyl groups with
multiple hydroxyl groups has the ability to form hydrogen
Values are presented as mean 6 SEM of triplicates (n ¼ 3). bonding with backbone peptide groups and side chain func-
tional groups of collagen. TA also has the potential to form
hydrophobic association with the hydrophobic sites of colla-
Cs (Fig. 1(A)) show very narrow pore size and a large num- gen.20 The stability enhancement could be due to crosslink-
ber of thin fibril-like networks compared with TCCs ing of TA with collagen.19,20,25,28–30 Recently, Jackson et al.22
(Fig. 1(B)) which showed increased pore size with very low studied the role of TA in stabilizing collagen against collage-
number of fibril-like networks. In TCCs, instead of fibrils, nases and revealed that the galloyl containing polyphenols
scaly appearance was seen which might be due to the such as TA may inhibit collagenase activity by binding with
merged appearance or loss of fibril-like network forming it. Therefore, the increase in the stability of Cs against
property. TA might have barred the collagen to form multi- collagenase after the introduction of TA is important for the
ple fibril-like network thereby establishing interconnectivity development of collagen biomaterial with enhanced biodur-
through cross-linking collagen with both inter and intra ability, particularly in tissue engineering and bioprosthesis
molecular collagen–TA hydrogen bond formation. application.

Water uptake capacity. Uniform surface area of Cs and

TCCs were taken and their water uptake capacity was meas- ATR-FTIR analysis. In the ATR-FTIR spectrum of Cs shows
ured and presented in Table I. The water uptake of TCCs was (Fig. 2) the characteristic collagen functionality peaks at
observed to be 242% which is significantly lower from that frequencies of 3317 cm
1 (amide A), 3080 cm
1 (amide
of 669% of Cs. The swelling behavior of Cs may be due to the B), 1647 cm
1 (amide I C¼ ¼O stretching), 1552 cm
1 (am-
large number of narrow pores which may entrap and hold ide II NH bending and CN stretching), and 1243 cm
1
more water through capillary action, where TCCs failed to (amide III CN stretching and NH bending). The broad
entrap and hold water due to scaly pores and could also be band between 3650 and 3150 cm
1 represents the ali-
due to change in polarity of the matrix. TA may also prevent phatic –OH stretching. The peak at the frequency of 2880
the collagen matrix distension in TCCs, through its native sta- cm
1 was due to the aliphatic side chains present in the
bilization effect. Reduction in the water uptake of TCCs paves collagen. TCCs shows the absorption peaks at the frequen-
way for localizing the scaffold in the wound for a longer time. cies of 3307, 1649, 1550, 1340, 1242, 1203, 1085, and
1036 cm
1. The little shift and broadening of peak at 3307
Tensile strength of scaffolds. The tensile strength of the indicate the stretching of both phenolic hydroxy of TA and
prepared scaffolds (Table I) and its comparison shows that aliphatic hydroxy of collagen. This may also be due to the
the Cs is having slightly higher strength than the TCCs. The
tensile strength of TCCs was not statistically different from
that of Cs. It is well known that tensile strength (r) ¼ F/A
(where F ¼ force and A ¼ area). This equation shows that
the area calculated from width and thickness of the mate-
rial is the key factor in determining the tensile strength. In
the porous matrix, the tensile strength of the scaffolds
depends on the microstructure (i.e. pore size arrangement
and wall thickness) to pass the stress and bear the load.
The decline in tensile strength of TCCs may be due to the
larger pores and its irregular occurrence. This agrees with
previous findings on the mechanical properties of chemi-
cally cross-linked collagen constructs reported by Madha-
van et al.27 In this case, both the sponges resulted in two
different microstructures and thickness after freeze-drying.
However, the TA inclusion in the scaffold did not impart
any improvement in mechanical strength, which may be
due to the difference in microstructures and its arrange-
ment compared with Cs.
FIGURE 2. ATR-FTIR spectra of collagen scaffold (Cs) and tannic acid
Enzymatic stability. Enzymatic stability of Cs and TCCs cross-linked collagen scaffold (TCCs). [Color figure can be viewed in
against collagenase was studied and the results are the online issue, which is available at wileyonlinelibrary.com.]

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MAY 2013 VOL 101B, ISSUE 4 563

FIGURE 3. The viability analysis of fibroblast cells in Cs and TCCs by
MTT assay. There was no significant difference between Cs and TCCs.
The values are mean 6 SEM (n ¼ 3).
formation of hydrogen bond between free NH group of col-
lagen and OH of the TA. The new medium intensity peak at
1340 cm
1 and intensified peak than Cs at frequencies of
1242, 1203, 1085, and 1036 cm
1 may be due to the CN
stretching coupled with NH bending. This study clearly rep-
resents that the presence of several carboxyl and hydroxyl
groups in the TA molecule influencing the possibility of
noncovalent multipoint interaction with collagen and
agrees with the previous findings.25
FIGURE 4. CLSM images of fibroblast-seeded DAPI counterstained (A) collagen scaffold (Cs) and (B) tannic acid cross-linked collagen scaffold
(TCCs) after day 7. Scanning electron micrographs of fibroblast-seeded (C) collagen scaffold (Cs) and (D) Tannic acid cross-linked collagen scaf-
fold (TCCs) after day 7 h by glutaraldehyde fixation. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
564 NATARAJAN ET AL.
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Biostudies
In vitro studies using fibroblasts. The viability (propor-
tional to number of cells) of fibroblasts cells were assayed
by MTT assay and the results are shown in Figure 3. The
statistical analysis shows that there was no significant
difference between Cs and TCCs in cell viability. Initially in
TCCs (up to the 3rd day), % viability of fibroblasts was less
compared with that of Cs. Further culture on scaffolds for
up to day 7 showed an increase in the % viability of fibro-
blasts which may be due to the presence of scaly supports
for the attachment and proliferation of fibroblasts. This
clearly demonstrates that TCCs promotes cell adhesion and
growth, as well as reiterate the evidence of TCCs compati-
bility as much as that of the Cs. This shows that the TA
concentration used in the scaffolds did not affect the bio-
compatibility of fibroblasts.
The observation of DAPI counterstained nuclei of fibro-
blasts in Cs and TCCs shown in Figure 4(A,B), respectively.
Although the figures show low amount of cells stained, it
clearly indicates that TCCs showed dense proliferation of
3T3 fibroblasts compared with that of Cs. These results
corroborate MTT results.
The scaffolds fixed with glutaraldehyde were observed
through SEM and the images are shown in Figure 4(C,D).
The cell growth was clearly visible in the Cs (Fig. 4(C)) and
TCCs (Fig. 4(D)). Figure 4(D) shows better proliferation and
TANNIC ACID CROSSLINKED COLLAGEN SCAFFOLDS

FIGURE 5. The percentage wound closure on 4th, 8th, and 12th days
of control, Cs treated, and TCCs treated with statistical analysis (data
presents as mean 6 SEM (n ¼ 5), p < 0.05, * significant with respect
to control, # significant with respect to Cs).
infiltration of fibroblasts into the TCCs matrix. The MTT
assay results on day 7 further confirms infiltration and
proliferation of fibroblasts in TCCs is better than Cs. This
result strengthens and augments the applicability of TCCs in
tissue engineering and regeneration.
In vivo studies using rats. The study protocol was
approved by our Institutional Animal Ethics Committee.
Before dressing the rats with scaffolds (Cs and TCCs) on
0th day, the wound areas were traced in PET transparent
sheets using permanent marker. Similarly, the wound areas
were traced on 4th, 8th, and 12th days after removing the
gauze. The traced sheets were used for calculating percent-
age wound closure using GGT digitizer (size A0) and
FIGURE 6. Histological analysis of skin regeneration after 4th, 8th, and 12th days of treatment. The letter ‘H’ denotes hematoxylin/eosin stained
sections of skin, ‘M’ denotes Masson’s trichrome stained sections of skin, ‘a’ denotes the control, ‘b’ denotes the Cs-treated sections, and ‘c’
denotes the TCCs-treated sections. Supplementary data
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ORIGINAL RESEARCH REPORT
Gerber AccuMark software. Figure 5 shows plot of time
versus percentage wound closure. The wound started to
close very rapidly in the first 8 days and then showed sta-
bility (images given in Supporting Information Fig. 7). Cs
particularly showed a rapid and significant wound closure
when compared with control (p < 0.001) and TCCs (p <
0.001) on 4th day, whereas on 8th and 12th day, the TCCs
showed greater and significant effect in percentage wound
closure compared with control (p < 0.001) and Cs (p <
0.001). This shows that re-epithelialization began early in
Cs compared with TCCs, and later slowed down. This may
be due to the infections caused by bacteria present in the
wound or loss of matrix structure as ideal matrix to sup-
port cell attachment and growth by action of collagenase
secreted by bacteria. Sections sliced from the harvested
tissue were stained with H&E and MT, analyzed for the
formation of granulation tissue and re-epithelialization. On
4th day (Fig. 6), the granulation tissue (i.e. collagen) began
to replace the excised area of skin in both the Cs and
TCCs treated when compared with control wound. The MT
staining clearly shows the varied amount of granulation
tissue formed, which was particularly high and dense in
TCCs-treated wounds. The re-epithelialization in margins of
the wounds in both treatment cases (Cs and TCCs) was
evident in Figure 6. On the 8th day , histological analysis
(Fig. 6) revealed that, epidermal cells in the edge of TCCs-
treated wound started to migrate faster toward the wound
(i.e. above the newly formed granulation tissue as support)
and established half of the epidermal layer over the wound
which was higher than that of the Cs and control. The
results of % wound closure on this day supported higher
epithelialization in TCCs-treated wounds. It also shows the
maximized synthesis and remodeling of the granulation
565

tissue as scar. The MT staining pictures showed less colla-
gen deposition in control compared with that of Cs- and
TCCs-treated groups in all the sampling days. On 12th day
MT stain did not show any apparent difference in collagen
deposition between Cs and TCCs. The Eschar is observed
for both control and TCCs treated but not in the case of
Cs treated, which may be due to the faster degradation of
Cs. On day 12 (Fig. 6), the TCCs treated one was almost
re-epithelialized (98.72 6 0.52%) and there was distinctive
difference between the epidermis and the dermis. In con-
trol and Cs-treated wounds, the re-epithelialization were
55.84 6 11.38% and 88.15 6 2.23%, respectively. How-
ever, compared with Cs, epithelialization was better in
TCCs. The results clearly indicate that TCCs promotes bet-
ter and faster healing compared with Cs. Presence of TA in
TCCs provides stability against enzymatic degradation and
promotes formation of granulation tissue where epitheliali-
zation is faster when compared with that of Cs. These
results clearly demonstrate that TCCs promotes the tissue
construction through various mechanisms including enzy-
matic stability against collagenase, antimicrobial properties
and antioxidant and anti-inflammatory property of TA
released from the scaffold.
CONCLUSIONS
The introduction of TA into Cs imparts enzymatic stability
as well as promotes the functional property of the scaf-
folds in tissue engineering applications. TCCs had shown
good biocompatibility with 3T3 fibroblasts compared with
Cs. In addition, TA has established its role in hastening the
closure process of the wound that may be a result of its
enzymatic stability of Cs. The scaffold has wider applica-
tions in the tissue engineering and drug delivery where
the prolonged presence of scaffold and sustained release
of drug or drug-loaded delivery systems, such as micro-
spheres or nanoparticles, from scaffold is essential.
ACKNOWLEDGMENTS
The authors also wish to thank Mr. G. Sathiamoorthy, CLAD,
Department of CSIR-CLRI for assisting in carrying out the
wound area measurements. We wish to thank National Imag-
ing Facility, Department of Biotechnology, Indian Institute of
Technology Madras (IITM) for the utilization of CLSM. Authors
also thank Mrs. Neeraja Sridharan of CLRI for proof reading
the manuscript.
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