International Journal of Biological Macromolecules 72 (2015) 680686

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Evaluation of biomaterial containing regenerated cellulose and

chitosan incorporated with silver nanoparticles
M.I. Niyas Ahamed a, , S. Sankar a , P.Mohammed Kashif b , S.K.Hayath Basha b , T.P. Sastry a
a
b

Bio-products Laboratory, Central Leather Research Institute, Adyar, Chennai 600020, India
Department of Biochemistry, Islamiah College (Autonomous), Vaniyambadi 635751, India

a r t i c l e

i n f o

Article history:
Received 21 July 2014
Received in revised form 13 August 2014
Accepted 21 August 2014
Available online 16 September 2014
Keywords:
Regenerated cellulose
Chitosan
Silver nanoparticles

a b s t r a c t
Biomaterials are used in regenerative medicine, implantable materials, controlled release carriers or scaffolds for tissue engineering. In the present study, the composites containing regenerated cellulose (RC)
and chitosan (Ch) impregnated with silver nanoparticles (AgNP) with and without antibiotic gentamicin
(G) were prepared. The composites prepared were characterized for their physico-chemical and mechanical properties and the results have shown the composite nature. RCChAg and RCChAgG composites
were used as wound dressing materials in experimental wounds of rats. The healing pattern of the wounds
was evaluated by planimetric studies, macroscopic observations, biochemical studies and mechanical
properties. The results have shown faster healing pattern in the wounds treated with RCChAg and
RCChAgG composites compared to untreated control. This study revealed that RCChAg composite
might be a potential, economical wound dressing material and may be tried on the clinical wounds of
animals before being applied on humans.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Wound healing is the bodys natural process of regenerating
dermal and epidermal tissue. It is the process whereby the body
restores the injured part to as near its normal condition as possible. Though wound healing takes place naturally on its own, some
of complications like sepsis, disruption of tissue and skin layer,
maggots formation, extension of infection to adjacent and interior organs occur in major cases. It is the primary response to
any tissue injury [1] and complex dynamic process that involves
many cascades of events like hemostasis, inammation, proliferation and remodeling of tissues in order to ll the damage area and
re-establish the skin barrier [2,3]. A good wound dressing should
maintain a moist environment upon absorption of the wound
exudates, protect the wound from secondary infection, provide
adequate gaseous exchange, regulate and/or mediate the release
of certain growth factors and cytokines, and also be elastic, biocompatible, non-toxic and non-antigenic [47]. Recently many
researchers are entailed to produce new and improved wound
dressing materials by synthesizing and modifying biomaterials that
are eco-friendly and sustainable.

Corresponding author.
E-mail address: iniyasahamed@gmail.com (M.I.N. Ahamed).
http://dx.doi.org/10.1016/j.ijbiomac.2014.08.055
0141-8130/ 2014 Elsevier B.V. All rights reserved.

Chitosan is nontoxic, biocompatible, biodegradable polymer

[812]. It is used in drug delivery, cell delivery systems, orthopedics, wound healing, ophthalmology and bone healing [13]; it
enhances the function of polymorphonuclear cells, macrophages
[14] and broblastic proliferation and migration [15]. Chitosan
exhibits antimicrobial activity against bacteria [16] fungi, and yeast.
It is hypoallergenic, has rapid blood clotting property, haemostatic and acts as fat attractor by binding to dietary lipids [17].
The presence of active groups in chitosan molecules allows for
easy chemical modication and therefore is used in many elds,
including medicine. Extensive investigations are being aimed at
the wider use of chitosan in wound dressings [18]. The increasing interest in the material is caused by its biological activity
resulting from its susceptibility to degradation under the inuence of enzymes present in body uids such as lysozyme and
N-acetylglucosoamidase. The degradation products, being chitooligomers, are able to stimulate macrophages and positively
inuence collagen sedimentation, thus accelerating the wound
healing process [19].
Silver metal and its compounds have been known for their
antimicrobial activities. Silver ions work against bacteria in a number of ways, they interact with the thiol groups of enzyme and
proteins that are important for bacterial respiration and transport of important substance across the cell membrane and within
the cell [20,21]. Silver ions also bound to the bacterial cell wall,
altering the function of the bacterial cell membrane [22]. Thus sil-

M.I.N. Ahamed et al. / International Journal of Biological Macromolecules 72 (2015) 680686

ver metal and its compounds are effective in preventing infection

of the wound [23].
Cellulose is a natural hydrogel whose properties are better
than the hydrogel produced from synthetic polymers; for example, it displays high water content (9899%), good adsorption of
liquids, high wet strength, and high chemical purity and can be
safely sterilized without any change to its structure and properties [24]. Being similar to human skin, bacterial cellulose can be
applied as skin substitute in treating extensive burns [25]. It is
an interesting material for using as a wound dressing since it can
control wound exudates and can provide moist environment to
a wound resulting in better wound healing. However, cellulose
itself has no antimicrobial activity to prevent wound infection. To
achieve an antimicrobial activity, in this work, silver nanoparticles
were impregnated into regenerated cellulose-chitosan composite (RCChAg). As per a study, documented that bacterial
cellulose-silver nanoparticle nanober had good antibacterial
activity and excellent healing effects in a second-degree rat wound
model [26,27].
These RCChAg composites were characterized for their various physico-chemical characterization studies like tensile strength,
thermo gravimetric analysis (TGA), differential scanning colorimetric analysis (DSC), scanning electron microscopy (SEM) and energy
dispersive X-ray analysis (EDX). It was used as wound dressing
material in experimental rats. The progress of the wound healing
in both experimental and control groups was evaluated by planimetric studies, macroscopic observations, biochemical studies and
mechanical properties.

681

2.5. Incorporation of silver nanoparticles on to the RCCh

biocomposites (RCChAg)
The prepared RC-Ch biocomposite was soaked in solution
containing 0.01% of silver nanoparticles for about 24 h. Silver
nanoparticles incorporated RC-Ch was taken out of the solution
and dried at 30 C. The dried material was denoted as RCChAg.
2.6. Coupling of gentamicin drug into RCChAg biocomposites
(RCChAgG)
The drug gentamicin (G) 0.04% was coupled by soaking
RCChAg composites into the gentamicin containing solution and
dried at 30 C. The dried material was denoted as RCChAgG and
sterilized by UV irradiation under sterile conditions.
2.7. Characterization
The mechanical properties of the composites were measured
using universal testing machine (Instron model 4501). Thermo
gravimetric analysis was carried out with a PerkinElmer TGA over a
temperature range of 30800 C at a heating rate of 20 C/min under
nitrogen atmosphere. Differential scanning calorimetry was carried
out using DSC Q200 V23.10 Build 79 at a heating rate of 5 C/min
under nitrogen atmosphere (ow rate is 40 ml/min). The morphological studies were carried out on a Leica stereo scan-440 Scanning
electron microscope equipped with phoenix EDX attachment.
2.8. Animal experiments

2. Materials and methods

2.1. Materials
Crab shells were collected from nearby sh market (Chennai,
India). Silver nitrate (Analytical grade) was purchased from
SigmaAldrich Chemical Co., USA. All other reagents used were of
analytical grade.

All experiments were performed according to the Institutional Animal Ethical Committee approval and guidelines
[1142/ab/07/CPCSEA]. Male Albino Wister rats, weighing
150200 g, were divided into four groups: control, standard
soframycin ointment, RCChAg and RCChAgG dressings.
Throughout the experiment, rats were maintained in an airconditioned room at 25 1 C with a lighting schedule of 12 h light
and 12 h dark and were fed with commercial balanced diet and
water ad libitum.

2.2. Preparation of chitosan (Ch) solution

2.9. Surgical procedure and treatment
Chitosan was obtained by alkaline deacetylation of chitin from
crab shells. The chitosan solution was prepared by dissolving 2 g of
chitosan in 100 ml of 0.1 N HCl solutions.
2.3. Preparation of regenerated cellulosechitosan biocomposite
(RCCh)
In 90 ml distilled water 9.5 g of sodium hydroxide and 4.5 g
of Thiourea were dissolved and then the resultant solution was
pre-cooled to 5 C to prepare a new solvent of cellulose according to a modied method of Dong et al. [28]. Various amounts
of cellulose were dissolved separately in this solution; 2% chitosan solution was added to each of this solution (Table 1). Finally,
the pH of the solutions was brought to 7.0 and sieved to get the
regenerated cellulosechitosan (RCCh) composites. The resultant
materials were dried at 30 C. The stoichiometric ratio which gave
better tensile strength to the RCCh biocomposite was used for the
incorporation of AgNP solutions.
2.4. Preparation of silver nanoparticles (AgNP) solution
Silver nanoparticles were prepared using tea grains (Camellia
Sinensis) as a reducing as well as capping agent for AgNO3 as per
earlier method [29].

Each animal was given a dose of sodium pentobarbital 40 mg/kg

body weight intra peritoneal and the dorsal surface of the rat below
the cervical region was shaved on its back under aseptic conditions. An open excision wound of 2 cm 2 cm was created on the
shaved dorsal side of rats using sterile surgical blade. For the control animals (group I) sterile cotton gauze dipped with gentamicin
was applied on the wound. The group II was applied with standard
soframycin ointment, the group III was applied with wound dressing material of RCChAg and for the group IV; the RCChAgG
was applied. The dressings were periodically changed at an interval
of 5 days with the respective materials. Three rats were sacriced
periodically on 5th, 10th, 15th, 20th and 25th day of post wound
creation and the granulation tissues formed were removed and
stored at 70 C until analysis. The progress of wound healing in
the four groups was evaluated visually, planimetrically and biomechanically by periodical monitoring of wound surface.
2.10. Biochemical parameters
In the present study total protein, collagen, hexosamine and
uronic acid levels were estimated in the granulation tissue of control and experimental wounds on days 5, 10, 15, 20 and 25. The
granulation tissue was collected after sacricing the animals on the
respective days. Estimation of protein was determined by Lowrys

682

M.I.N. Ahamed et al. / International Journal of Biological Macromolecules 72 (2015) 680686

Table 1
Mechanical properties of the biocomposites.
Sample ID

Chitosan (%)

Regenerated cellulose (%)

Elongation at break (%)

Tensile strength (MPa)

1
2
3
4

2
2
2
2

0.5
1.0
1.5
2.0

37.23 1.73
25.13 0.96
30.18 0.88
18.34 1.45

1.05 0.03
2.45 0.21
1.55 0.11
1.95 0.18

Values are expressed as X S.D. for six animals in each individual experiment. Within a line, values without a common letter are signicantly different from the control group
at p < 0.05 as determined by ANOVA.

method [30]. Collagen and hexosamine were determined in defatted dried granulation tissue [31,32]. Extraction of uronic acid from
the tissue was carried out according to the method of Schiller et al.
[33] and estimated by the method of Bitter and Muir [34].

to the decomposition of cellulose. The second weight loss (7%) was

observed between 335 C and 422 C, this may be due to the decomposition of chitosan and a total of 52% weight loss was observed
in the case of RCChAg composite. A thermal stability of 7% was
exhibited by the composite compared to RC.

2.11. Tensile strength of the healed wound

3.2. Differential Scanning Calorimetry (DSC)
On the 30th day after creating the wound, the animals were
anesthetized. Healed tissue along with normal skin at the two ends
was excised for tensile strength (MPa) and percentage of elongation
at break (%) measurements.
2.12. Planimetric studies
Hair was clipped around the scar for proper visualization and the
individual contour of the wounds of both control and experimental animals was measured periodically, using a transparent graph
sheet, rate of healing was calculated and expressed as percentage
contraction [35].
2.13. Statistical analysis
All data were analyzed with SPSS/10 student software. Hypothesis testing methods included one-way analysis of variance (ANOVA)
followed by LSD. The values are expressed as mean S.D. and
results were considered signicantly different if p < 0.05.
3. Results and discussion
Recently many researchers have developed several wound
materials by synthesizing and modifying biomaterials. An ideal
wound dressing should have several key attributes [36] and only
those dressing material with good mechanical properties can be
applied on to wound properly.
In this study, the composites containing regenerated cellulose
and chitosan impregnated with silver nanoparticles (RCChAg)
were prepared. Among the various compositions studied, 1% of
regenerated cellulose and 2% of chitosan gave higher tensile
strength and good elongation properties (Table 1). Increase in the
amount of RC above 1% shown decrease in the tensile strength of
the samples. The same was used for further characterization studies
such as mechanical properties, SEM, TGA, DSC and in vivo studies.

The DSC was carried out under continuous ow of dry nitrogen gas (10 ml/min) at a heating rate of 20 C/min. Fig. 2A and
B illustrates the thermograms of RC and RCChAg biocomposite
respectively. In Fig. 2A endothermic event appeared as a peak at
87.73 C corresponding to water evaporation. There was a broad
melting temperature (Tm ) with single step degradation found at
87.73 C. In Fig. 2B, RCChAg biocomposite exhibits decreased
melting temperature (Tm ) at 78.92 C when compared to RC, it is
due to the addition of chitosan into the RC and the difference is
around 8.8 C.
3.3. Scanning electron microscopy (SEM) and energy dispersive
X-ray analysis
Scanning electron microscope pictures exhibit the surface morphology of biomaterials, Fig. 3A and B shows the SEM micrographs
of the RCChAg biocomposite. The brous nature of cellulose is
clearly visible with the coating of chitosan along with incorporated
silver nanoparticles. The diameter of the cellulose ber is in the
range of 1020 m. The spherical shape particles formed are predominant, some of the silver nanoparticles are individual and some
of the silver nanoparticles are aggregated. The size of smallest silver nanoparticles is around 60 nm and the size of aggregated silver
nanoparticles is around 150 nm. The EDX spectrum of RCChAg
biocomposite has clearly shown the presence of silver with a corresponding signal (Fig. 4).
3.4. Animal studies
3.4.1. Photographic evaluation
Surface of the wounds were photographed periodically from a
constant distance for control (Fig. 5A) and experimental (Fig. 5BD)
groups. Faster rate of healing was observed in the experimental
wounds compared to that of control. The results are in agreement
with those of planimetric observations.

3.1. Thermo gravimetric analysis (TGA)

Normally, when a biomaterial is heated to higher temperature it
decomposes into CO2 , CO and H2 O. In the present investigation, the
biomaterials prepared were heated in a nitrogen atmosphere from
30 C to 800 C. A single step weight loss was observed in the case
of RC from 223 C to 396 C. The weight loss was found to be 59%,
this weight loss may be due to the decomposition of RC. There was
about 8% loss of water and bound water up to 223 C, a residue of
21% was observed at 800 C (Fig. 1A). In Fig. 1B, RCChAg biocomposite exhibits two step weight loss, rst weight loss of 45% was
observed from 204 C to 335 C. This weight loss may be attributed

3.4.2. Wound contraction and period of healing

Fig. 6A indicates the rate of wound contraction in control,
standard soframycin, RCChAg and RCChAgG treated groups.
Periodical observation of animals up to 25 days showed a significant increase in the rate of contraction in experimental groups,
when compared to control. On day 25, the wounds dressed with
RCChAgG contracted completely, while those treated with
RCChAg, standard soframycin data shown 96% contraction as
against the control groups, which showed only 74% of contraction. The observed increase in the contraction of wounds in the
animals treated with chitosan based dressings (experimental) is

M.I.N. Ahamed et al. / International Journal of Biological Macromolecules 72 (2015) 680686

683

Fig. 1. Thermogravimetric analysis of (A) RC and (B) RCChAg biocomposite.

attributed to the increased rate of healing. Among the experimental groups those treated with RCChAgG showed quick healing
when compared with those of RCChAg and standard soframycin.
The increase in wound contraction in the treated rats might be
due to the enhanced activity of broblasts which might resulted
in more collagen synthesis. There was no signicant difference in
the gross healing pattern between the dressing materials RCChAg
and RCChAgG.
3.4.3. Biochemical parameters
The biochemical parameters i.e. collagen, hexosamine and
uronic acid in the granulation tissues of the control and experimental rats on different days after wound creation were analyzed.

The collagen content in group IV was signicantly higher when

compared with the corresponding values of groups I, II and III
throughout the experimental period. On the 10th day, the collagen
level was found to elevate to about 1.82, 1.65 and 1.68 fold in groups
II, III and IV respectively, when compared to control. The signicant
increase in the collagen content of granulation tissue isolated from
experimental groups, may be due to an increased synthesis of collagen and could be correlated with the effective healing of wounds
[37,38]. A decreasing trend in the collagen content after day 10 in all
the four groups was observed. The marked increase in collagen content of granulation tissue isolated from experimental groups may
be due to increased synthesis of collagen and could be correlated
with the enhanced migration of broblasts and epithelial cells to

Fig. 2. DSC curve of (A) RC and (B) RCChAg biocomposite.

684

M.I.N. Ahamed et al. / International Journal of Biological Macromolecules 72 (2015) 680686

Fig. 3. SEM images of RCChAg biocomposite

the wound site for effective healing [37,38]. The decreased content
of collagen in control group is due to the prolonged inammatory
phase [39].
Hexosamine content of granulation tissue is shown in Fig. 6C.
There is a gradual decrease of hexosamine content during the
course of wound healing in all the groups. However, remarkable difference between the control and experimental groups was noted.
A signicant increase in the concentration of uronic acid content was observed upto day 15 in experimental groups, whereas
in the control group, the peak was seen only on day 15. A gradual
decrease of uronic acid content was observed after the 15th day in
the experimental and control rats respectively. Decrease in uronic
acid content was observed in the treated group. The decrease in
uronic acid is attributed to an increase in collagen synthesis. This
was further supported by Cohen and Haynes, who found that an
increase in uronic acid will lead to a decrease in collagen synthesis
[40,41].
Hexosamine and uronic acid are matrix molecules, which act as
ground substratum for the synthesis of new extra cellular matrix.
It is reported that there is an increase in the levels of these components during the early stages of wound healing, following which
normal levels are restored. A similar trend was observed in chitosan based biomaterials treated wounds where in the levels of
hexosamine and uronic acid increased up to day 15 post wounding
and decreased thereafter. Hexosamine and uronic acid are matrix
molecules, which act as ground substratum for the synthesis of

Fig. 5. Photographs showing the wound healing pattern of A Control, B standard

soframycin, C RCChAg and D RCChAgG treated groups taken on day 20
after wound creation.

Fig. 4. EDX Spectrum of RCChAg biocomposite.

M.I.N. Ahamed et al. / International Journal of Biological Macromolecules 72 (2015) 680686

685

Fig. 6. (A) Percentage of contraction, (B) Total protein content, (C) collagen content, (D) hexosamine content and (E) the uronic acid content in granulation tissue of control,
standard soframycin, RCChAg and RCChAgG treated groups on various days of healing respectively. Values are expressed as X S.D. for six animals in each individual
experiment. Within a line, values without a common letter are signicantly different from the control group at p < 0.05 as determined by ANOVA.

new extra cellular matrix. It is reported that there is an increase

in the levels of these components during the early stages of wound
healing, following which normal levels are restored [42].
Increase in the levels of this biochemical increase of total protein
content in the initial phase of healing is due to the active synthesis
and deposition of matrix proteins in the granulation tissues, this
resulted in the increased synthesis of collagen. Hexosamine acts as
ground substratum for the synthesis of extracellular matrix; signicant decrease in the hexosamine content was associated with
associated increase in the extracellular parameters i.e. collagen,

total protein, uronic acid and hexosamine in the experimental

groups gave an indication of the faster rate of wound healing compared to control. Similar results were observed in the earlier studies
[1,43].
3.4.4. Tensile strength
The tensile strength values exhibited by healed excision wounds
of experimental groups are greater than control group (Table 2).
Increased tensile strength indicates increase in collagen matrix.
There is rapid biosynthetic activity in experimental groups during

686

M.I.N. Ahamed et al. / International Journal of Biological Macromolecules 72 (2015) 680686

Table 2
Tensile strength of healed wounds.
Groups/parameters
Control
Standard soframycin ointment
RCChAg
RCChAgG

Tensile strength (MPa)

Group I
Group II
Group III
Group IV

3.22 0.06
3.47 0.02
4.63 0.06
4.81 0.04

Values are expressed as X S.D. for six animals in each individual experiment.
Within a line, values without a common letter are signicantly different from the
control group at p < 0.05 as determined by ANOVA.

initial phase of granulation. In the remodelling phase, maturation

of collagen takes place by the formation of inter and intramolecular
cross links, hence, increased wound strength was observed [43]. But
in the case of control, the decreased tensile strength and prolonged
wound healing further support the slow rate of wound healing
due to dry wound-healing conditions offered by cotton gauze.
Among the experimental groups, those treated with RCChAg
and RCChAgG show higher tensile strength values when compared with the control and this is mainly attributed to its increased
hydrophilic capacity thereby maintaining moist environment at the
wound site, which is reected in its faster wound-healing nature.
Moreover, the tensile strength is directly related to the amount of
collagen synthesized at the wound site. If we observe the results
shown in Fig. 5A, the collagen content values in the RCChAg and
RCChAgG treated wounds were higher when compared to those
of control group at all intervals.
4. Conclusion
Evaluation of the RCChAg and RCChAgG composites as
a temporary biological wound dressing materials on the experimental wounds of rats has revealed that the experimental wounds
healed faster than the control wounds. The gross observations
have revealed that the complete closings of wounds were observed
on the 25th day for the animals treated with RCChAg and
RCChAgG and more than 30 days for the control wounds. There
was no signicant difference in the gross healing pattern between
the dressing materials RCChAg and RCChAgG and the presence of antibiotic has only reduced the inammatory cells. These
observations gave an indication that RCChAg and RCChAgG
composites might be tried as a wound dressing materials in the
clinical wounds of smaller and larger animals before applying on
to humans.
References
[1] M. Sumitra, P. Manikandan, L. Suguna, Int. J. Biochem. Cell Biol. 37 (2005)
566573.

[2] C.M. Deng, L.Z. He, M. Zhao, D. Yang, Carbohydr. Polym. 69 (2007) 583589.
[3] S. Enoch, D.J. Leaper, Surgery (Oxford) 26 (2005) 3137.
[4] J.S. Boateng, K.H. Matthews, H.N. Stevens, G.M. Eccleston, J. Pharm. Sci. 97
(2008) 28922923.
[5] M. Kokabi, M. Sirousazar, Z.M. Hassan, Eur. Polym. J. 43 (2007) 773781.
[6] S.Y. Lin, K.O. Chen, L. Run-Chu, Biomaterials 22 (2001) 29993004.
[7] A.C. Varshney, K. Amresh, S. Harpal, Ind. Vet. J. 65 (1988) 436439.
[8] E. Daniela, E.O. Camelia, Chem. Eng. Commun. 195 (2008) 12691291.
[9] R. Shelma, P. Willi, C.F. Sharma, Trends Biomater. Artif. Organs 22 (2008)
107111.
[10] K. In-Yong, S.J. Seo, H.S. Moon, M.K. Yoo, P. In-Young, B.C. Kim, C.S. Cho, Biotechnol. Adv. 26 (2008) 121.
[11] B.D. Emir, M.O. Raphael, J. Bioact. Compat. Polym. 21 (2006) 351368.
[12] G. Saraswathy, S. Pal, C. Rose, T.P. Sastry, Bull. Mater. Sci. 24 (2001) 415420.
[13] S. Senel, S.J. Clure Mc, Adv. Drug Deliv. Rev. 5 (2004) 14671480.
[14] H. Ueno, H. Yamada, I. Tanaka, N. Kaba, M. Matsuura, M. Okumara, T. Kadosawa,
T. Fuginaga, Biomaterials 20 (1999) 14071414.
[15] C.H. Su, C.S. Sun, S.W. Juan, C.H. Hu, W.T. Ke, M.T. Sheu, Biomaterials 18 (1997)
11691174.
[16] M.S. Mohy Eldin, E.A. Soliman, A.I. Hashem, T.M. Tamer, Trends Biomater. Artif.
Organs 22 (2008) 154164.
[17] S.S. Koide, Nutr. Res. 18 (1998) 10911101.
[18] M.I.N. Ahamed, T.P. Sastry, Int. J. Res. Ayurveda Pharm. 2 (4) (2011) 12031209.
[19] R.A.A. Muzzarelli, Carbohydr. Polym. 20 (1993) 716.
[20] K.H. Cho, J.E. Park, T. Osaka, S.G. Park, Electrochim. Acta 51 (2005) 956960.
[21] S. Ivan, S.S. Branka, J. Colloid Interface Sci. 275 (2004) 177182.
[22] S.L. Percival, P.G. Bowler, D. Russell, J. Hosp. Infect. 60 (2005) 17.
[23] J.B. Wright, K. Lam, D. Hansen, R.E. Burrell, Am. J. Infect. Control 27 (4) (1999)
344350.
[24] D. Klemm, D. Schumann, U. Udhardt, S. Marsch, Prog. Polym. Sci. 26 (9) (2001)
15611603.
[25] W. Czaja, A. Krystynowicza, S. Bielecki, R. Malcolm Brown Jr., Biomaterials 27
(2006) 145151.
[26] J. Wu, Y. Zheng, W. Song, J. Luan, X. Wen, Z. Wu, X. Chen, Q. Wang, S. Guo,
Carbohydr. Polym. 102 (2014) 762771.
[27] J. Wu, Y. Zheng, X. Wen, Q. Lin, X. Chen, Z. Wu, Biomed. Mater. 9 (2014) 035005.
[28] Dong Ruan, Ang Lue, Lina Zhang, Polymer 49 (2008) 10271036.
[29] S.S.K. Kamal, P.K. Sahoo, J. Vimala, M.P. kumar, S.R.L. Durai, Acta Chim. Slov. 57
(2010) 808812.
[30] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.T. Randall, J. Biol. Chem. 193 (1951)
265276.
[31] J.R. Woessner, Arch. Biochem. Biophys. 93 (1961) 440447.
[32] L.A. Elson, W.T.J. Morgan, Biochem. J. 27 (1933) 18241828.
[33] S. Schiller, A. Slover, A. Dorfman, J. Biol. Chem. 236 (1961) 983987.
[34] T. Bitter, H.M. Muir, Anal. Biochem. 4 (1962) 330334.
[35] P.W. Morgan, A.G. Binnington, C.W. Miller, D.A. Smith, A. Valliant, J.F. Prescott,
Vet. Surg. 23 (1994) 494502.
[36] R.E. Horch, J. Kopp, U. Kneser, J. Beier, J. Bach, J. Cell. Mol. Med. 9 (3) (2005)
592608.
[37] R. Bernaebei, F. Landi, S. Bonini, G. Onder, A. Lambiase, R. Pola, L. Aloe, Lancet
354 (1999) 307.
[38] B.K. Pilcher, B.D. Sudbeck, J.A. Dumin, H.G. Welgus, W.C. Parks, Arch. Dermatol.
Res. 290 (1998) 37.
[39] M. Senthil Kumar, S. Kirubanandan, R. Sripriya, P.K. Sehgal, J. Surg. Res. 144
(2008) 94101.
[40] I.K. Cohen, J.H. Haynes, in: P. Altmeyer (Ed.), Wound Healing and Skin Physiology, Springer, Berlin, 1995, p. 27.
[41] R. Ahmed, Gopinath, Gomathi, Sehgal, Jayakumar, J. Biomed. Mater. Res. Part
B: Appl. Biomater. 69 B (2004) 241248.
[42] J.E. Dunphy, K.N. Udupa, Wound healing., Ann. Surg. 144 (1956) 304316.
[43] S.E. Noorjahan, T.P. Sastry, J. Biomed. Mater. Res. Part B: Appl. Biomater. 71B
(2004) 305312.