Collagen modified with epoxidized safrole for improving antibacterial activity

Article abstract of DOI:10.1039/c7ra08319j

An epoxidized safrole, 5-(oxiran-2-ylmethyl)-benzo[d][1,3]dioxole (OYBD), was synthesized and employed to modify collagen for improving its antibacterial activity. The interaction between collagen and OYBD, and the structure/properties of the modified collagen were investigated in detail. The results indicated that the OYBD-modified collagen showed a higher de-nature temperature (Td, 90.2 °C), improved hydrophobic properties (contact angle from 84.2° to 89.1°) and enhanced tensile strength (6.2-11.0%) without destroying its triple helix structure. From observation of scanning electron microscopy (SEM), a higher density of intertwining morphology and a more stable network structure were observed, which was consistent with improved tensile strength and reduced breaking extension stress. The antibacterial test and LIVE/DEAD Baclight bacterial viability assay illustrated that the modified collagen exhibited excellent antibacterial activity to both Gram-negative and Gram-positive bacteria. Furthermore, the OYBD-modified collagen still exhibited cytocompatibility, supporting human fibroblast proliferation, which holds a great potential for developing antibacterial collagen-based biomaterials.

Full text of DOI:10.1039/c7ra08319j

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PAPER
Collagen modified with epoxidized safrole for
improving antibacterial activity
Cite this: RSC Adv., 2017, 7, 50300
ab
a
a
a
a
Zhou Xu,
Jinming Chang, Peikun Zhang, Xiaoyu Guan, Yi Chen
a
and Haojun Fan*
An epoxidized safrole, 5-(oxiran-2-ylmethyl)-benzo[d][1,3]dioxole (OYBD), was synthesized and
employed to modify collagen for improving its antibacterial activity. The interaction between collagen
and OYBD, and the structure/properties of the modified collagen were investigated in detail. The
results indicated that the OYBD-modified collagen showed a higher de-nature temperature (Td,
ꢀ
ꢀ
ꢀ
9
0.2 C), improved hydrophobic properties (contact angle from 84.2 to 89.1 ) and enhanced tensile
strength (6.2–11.0%) without destroying its triple helix structure. From observation of scanning
electron microscopy (SEM), a higher density of intertwining morphology and a more stable network
structure were observed, which was consistent with improved tensile strength and reduced breaking
extension stress. The antibacterial test and LIVE/DEAD Baclight bacterial viability assay illustrated that
the modified collagen exhibited excellent antibacterial activity to both Gram-negative and Gram-
positive bacteria. Furthermore, the OYBD-modified collagen still exhibited cytocompatibility,
supporting human fibroblast proliferation, which holds a great potential for developing antibacterial
collagen-based biomaterials.
Received 27th July 2017
Accepted 21st October 2017
DOI: 10.1039/c7ra08319j
rsc.li/rsc-advances
identied as being of importance for its functional and bioac-
tive properties.
1
. Introduction
Collagen is the major structural and functional element of
Previous studies demonstrated that collagen cross-linked
connective tissues, and is also found in large quantities in the with epoxy compounds displayed comparable mechanical
5
interstitial tissues of virtually all parenchymal organs, where it properties to those cross-linked by glutaraldehyde. Addition-
maintains the structural integrity of tissues and contributes to ally, cross-linking with epoxy compound showed no signs of
1,2
9,10
various physiological functions.
Unfortunately, natural cytotoxic degradation products. To improve the antibacterial
collagen is particularly susceptible to microbial colonization. activity of collagen, we synthesized a new modier in this study,
Microbial contaminations compromise the quality of the named 5-(oxiran-2-ylmethyl)-benzo[d][1,3]dioxole (OYBD),
collagen product as they introduce variability and cause loss of derived from natural safrole (4-allyl-1,2-(methylenedioxy)benzene).
potency due to collagen degradation by microbial enzymes, This modier contained an epoxy group and a piperonyl group.
changes in impurity proles, and also an increase in the levels The former could react with amine groups of collagen, and the
of bacterial endotoxins. Generally, cross-linking is an efficient latter was known as antibacterial. Aer modication, the collagen
strategy to improve the durability of collagen towards bacteria would be expected to yield desirable antibacterial activity as well as
3
–5
degradation, as well as to optimize its mechanical properties.
enhanced thermal and mechanical properties.
Collagen contains abundant amino and carboxyl side groups,
6
which provides convenience for its chemical modication.
Although some modiers, such as diisocyanates, glutaralde- 2. Experimental
hyde, and aziridin could connect the amine groups between two
2
.1 Materials
adjacent polypeptide chains of collagens, improving the resis-
Pigskins were obtained from a local tannery. Safrole ($97%)
was purchased from Sigma-Aldrich (St. Louis, MO) and used as
received. Pepsin (3000–3500 U g ), MTT (dimethyl thiazolyl
tetrazolium bromide), and 3-chloroperbenzoic acid (mCPBA)
were purchased from Sino Standards Biotechnology Co. Ltd.
7,8
tance against collagenase and bacteria, it is noteworthy that
these crosslinkers might cause cytotoxicity and further destroy
of the triple helical structure of collagen that has been
ꢁ
1
(
Chengdu, China). Dichloromethane, NaOH, MgSO , NaCl, and
4
a
HAc were obtained from Kelong Chemical Engineering Co. Ltd.
(Chengdu, China). All chemicals were of analytical grade or
biological reagent grade.
Key Laboratory of Leather Chemistry and Engineering of Ministry of Education,
Sichuan University, Chengdu 610065, China. E-mail: fanhaojun@scu.edu.cn
b
School of Life Science and Food Engineering, Yibin University, Yibin 644007, China
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2
.2 Synthesis of 5-(oxiran-2-ylmethyl)-benzo[d][1,3]dioxole
Differential scanning calorimetry (DSC) analysis was conducted
ꢀ
(
OYBD)
in the temperature range from 20 to 180 C with a heating rate
ꢀ
ꢁ1
of 4 C min
.
The synthesis of OYBD was conducted according to the
following procedure. m-chloroperbenzoic acid (mCPBA) (31.8 g,
0
.15 mol) was rst dissolved in 200 mL of dichloromethane
2.8 SEM observation
(DCM), followed by addition of safrole (16.2 g, 0.1 mol) at room
The morphology of the lyophilized collagen samples (1 cm ꢂ 1
cm) was observed with a scanning electron microscopy (SEM,
Nova Nano SEM450, FEI, USA). The collagen specimen was
sputter-coated with aurum and imaged at an accelerating
voltage of 5 kV.
temperature. Aer stirred for 24 h, the mixture was ltered, and
the precipitate was discarded. The supernatant was washed
with 5% NaOH solution three times and subsequently with
deionized water three times. The organic layer obtained was
dried over anhydrous magnesium sulfate (MgSO
4
). The solvent
ꢀ
was removed by distillation (110 C, 530 Pa), and OYBD was

nally obtained with a yield of 82%.
2
.9 Water contact angle (WCA) test
The hydrophilicity of the OYBD-modied collagen was
measured using goniometer (Dataphysics, OCAH200,
2.3 Preparation of native collagen
a
The pig skin was rst pulverized with a mill (Fristsch puluteri-
sette, ldar-Oberstein, Germany) and then suspended in
a mixture of 0.5 M HAc (3%, w/v) and pepsin (2%, w/w) at 4 C
for 48 h under magnetic stirring. Aer that, the supernatant was
collected through centrifugation at 10 000 rpm for 10 min (4 C)
and then salted using excessive NaCl. The precipitate was
subsequently dialyzed against 0.1 M HAc at 4 C for 3 days to
Germany) at room temperature. Briey, 5 mL per drop of
distilled water was drop-wise deposited on the surface of the
collagen, and the WCA was calculated using a shape DSA100
analysis system. Each sample was tested for ve different
surface areas to obtain an average value.
ꢀ
ꢀ
ꢀ
obtain native collagen. The collagen was lyophilized using 2.10 Mechanical measurement
ꢀ
a lyophilized at ꢁ50 C for 48 h for further use.
The collagen membrane was cut into a dumbbell shape with
length and width of 50.0 mm ꢂ 10 mm using a dumbbell-
2.4 Modication of collagen by OYBD
shaped knife. The thickness of the samples was measured at
About 0.5 g native collagen was rst dissolved in 0.5 M HAc. The three different areas using a micrometer, and an average value
mixture was cast to form an aqueous membrane, which was was calculated. Using a universal tensile testing machine (GT-
ꢁ
1
ꢀ
then dried at 37 C until a constant weight. The resultant AI-7000S, Hi tech, Taiwan), a crosshead speed of 1 mm min
collagen membrane was immersed in 100 mL of an ethane- was applied until the collagen membrane ruptured. The tensile
sulfonic acid (MES, TRC, Canada) buffer solution (pH ¼ 8.0) strength, elongation at maximum force, and elongation at break
containing 5 wt% OYBD. The epoxidation reaction was con- of the collagen sample were calculated, and each sample was
measured three times.
ducted at room temperature for 36 h. Aer that, the collagen
membrane was rinsed in phosphate buffer saline for 24 h and
washed with deionized-water ve times. The resultant material
was denoted as collagen-OYBD.
2.11 Antibacterial measurement
Escherichia coli (E. coli, ATCC 25922) was selected as the indi-
cator in evaluating the antibacterial activity of the OYBD-
modied collagen. A small quantity of recovered cultures of
1
2
.5 H NMR detection
1
The H NMR spectrum of OYBD was recorded by a NMR spec-
trometer (AV11-600MH, Bruker, Swiss) with a test frequency of
8
E. coli was diluted with saline to a concentration of 1 ꢂ 10
colony-forming unit (cfu) per mL. Subsequently, 2 mL of E. coli
suspensions were transferred into a tube containing 18 mL of
sterile water, followed by addition of 0.15 g collagen membrane
that had been subject to irradiation sterilization. Furthermore,
the suspension was shook for 1 h. Aerwards, 1 mL of the
resulting mixture was incorporated into 9 mL of sterile water to
6
00 MHz. The triuoroacetic acid-D (99.5 atom% D) was used as
deuterium reagent, and tetramethylsilane (TMS) as internal
standard.
2
.6 FTIR spectra measurement
Fourier transform infrared (FTIR) spectra were collected using produce a series of dilution. For each diluted tube, 0.1 mL of the
a Nicolet iS10 FTIR spectrometer (Thermo Fisher Scientic, bacterial suspension was evenly distributed onto a sterilized
ꢁ1
United States) over a wavenumber range from 500 to 4000 cm
nutrient agar Petri dish and incubated for 48 h in an incubator
at 37 C. The number of viable micro-organisms colony and the
ꢁ
1
ꢀ
aer 64 scans at a resolution of 4 cm
.
antibacterial ratio were measured.
2
.7 DSC measurement
w
1
ꢁ w
2
Antibacterial rate ð%Þ ¼
ꢂ 100%
w
1
Briey, 3 mg collagen specimens were loaded in an aluminum
pan. The sample pan and the empty reference aluminum pan where, w
1
is the cfu of collagen and w
2
is the cfu of the OYBD-
2
were both placed in a Netzsch DSC 200 PC operating in N ow. modied collagen.
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.12 LIVE/DEAD Baclight bacterial viability test
Paper
2
The LIVE/DEAD Baclight bacterial viability kit was used to
visualize the bacterial cells on the modied collagen. The

uorescent dyes agents of green stain SYTO 9 (label both live
and dead cells) and red stain propidium iodide (only penetrate
cells with compromised or damaged membranes) could bind to
nucleic acids to assay cell viability. A suspension of Staphylo-
5
ꢁ1
coccus aureus (S. aureus, ATCC 25923) in PBS (10 CFU mL , 50
11
mL) was added onto the collagen samples, which was then
incubated for 24 h at 35 C. Subsequently, the collagen sample
ꢀ
was washed with PBS buffer and soaked in SYTO 9 (4 mL) and
propidium iodide (6 mL) dye solution in dark condition for
15 min. Finally, the stained bacterial cells were observed using
an IX-71 inversed uorescent microscope (Olympus America
Inc., Melville, NY).
2.13 Cytocompatibility analysis
The cell proliferation assay was carried out to evaluate the
cytocompatibility of the modied collagen according to ISO
1
0993-5. Normal human dermal broblasts (NHDFs) were
1
Fig. 1 H NMR spectra of (a) safrole and (b) 5-(oxiran-2-ylmethyl)
benzo[d][1,3]dioxole.
seeded in a culture dish and cultured to near conuence at
ꢀ
37
2
C under 5% CO . Collagen solution was diluted to
ꢁ
1
a concentration of 20 mg mL with 0.05 M acetic acid solution,
and then added into the wells of a 24-well polystyrene plate.
Aer incubation for 12 h, the solution was neutralized with
0
.05 M sodium hydroxide solution, and the wells were rinsed
5
with PBS three times. Subsequently, NHDFs (1 ꢂ 10 cells) in
2
mL Dulbecco's Modied Eagle's Medium (DMEM) with 10%
ꢀ
fetal bovine serum were seeded. Aer incubation at 37 C with
2
5% CO for 1, 3, and 7 d respectively, cell proliferation was
measured using the methyl thiazolyl tetrazolium (MTT) assay.
In addition, the morphologies of cells were observed by
employing an IX83 2-deck inverted microscope (Olympus).
2.14 Statistical analysis
The reported data are the means ꢃ standard deviation of trip-
licate samples for each measurement. Statistical analysis was
performed with Student's t-test. P < 0.05 was accepted as
statistically signicant.
Fig.
2 Schematic illustration of possible interactions between
collagen and OYBD.
3. Results and discussion
3.1 Cross-linking between collagen and OYBD
1
modied by monofunctional and multifunctional poly epoxy
The H NMR measurement was performed to conrm the
structure of 5-(oxiran-2-ylmethyl)benzo[d][1,3]dioxole (OYBD)
and the results were shown in Fig. 1. Safrole was oxidized by the
weak oxidant (mCPBA), forming a monofunctional epoxy group,
which could react with those amino groups originated from
14
compounds under alkaline condition. The synthesized OYBD
contained an epoxidized structure that could react with the
amine groups of lysine residues in collagen under alkaline
condition (pH 8.0). Thus, the antibacterial piperonyl group can
be covalently introduced into the collagen macromolecules.
1
collagen. The H NMR detection results of OYBD are as follows:
1
H NMR (600 MHz, DMSO-d
6
) d: 6.91–6.66 (m, 3H, ArCH), 5.98
O–), 3.06 (dp, J ¼ 5.9, 3.0 Hz, 1H, –OCH(CH )CH –),
.79–2.61 (m, 3H, –ArCH –CH and –OCH
CH), 2.53 (dd, J ¼ 5.1,
.6 Hz, 1H, –OCH CH). Such result is consistent with that as An ideal modication strategy should not destroy the special
(
s, 2H, –OCH
2
2
2
3.2 The triple-helical structure retention of collagen
2
2
2
2
2
12,13
previously reported.
triple-helical structure of tropocollagen molecules that is the
Possible interactions between collagen and OYBD are illus- basis of many important and specic biological functions of
15,16
ꢁ1
trated in Fig. 2. According to previous studies, collagen can be collagen.
In general, the FTIR absorption band at 1235 cm
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corresponds to the C–N bond in-plane vibration (amide III) and
the N–H stretch (amide I), which is associated with the triple-
helical structure of tropocollagen molecules. The absorption
ꢁ
1
band at 1450 cm can be ascribed to the pyrrolidine ring
vibration of proline and hydroxyproline, which is not affected by
changes in collagen secondary structure. Thus, it is widely
accepted that the intensity ratio of FTIR absorption at 1235 and
ꢁ
1
1
450 cm (I1235/1450 cmꢁ1) can be used as a sensitive indicator of
17
the integrity of triple-helical structure in collagen. Typically,
an I1235/1450 cmꢁ1 close to unity indicates intact triple-helical
structure, whereas an I1235/1450 cmꢁ1 signicantly lower than
18
unity suggests the triple helicity has been completely destroyed.
Based on the FTIR spectra in Fig. 3, the I1235/1450 cmꢁ1 of the
OYBD-modied collagen was 0.91 and is comparable to the value
of native collagen (1.0). This result revealed that the triple helix
conformation was not destroyed in the modication process
by OYBD.
Fig. 4 CD spectra of (a) native collagen and (b) OYBD-modified
collagen.
In addition, quantitative analysis of molar ellipticity changes
in CD spectra before and aer OYBD treatment could also
provide valuable information regarding the integrity of triple-
helical structure. CD spectroscopy is the most widespread
technique used for estimating the secondary structures of
proteins and polypeptides in solution. This technique can be
used to distinguish between unordered (random coil) and
ordered (triple helix) structures of collagen. CD detects
that of the native collagen (0.14), suggesting that modication
by OYBD did not destroy the triple helix structure of collagen.
This conclusion is well consistent with FTIR analysis.
3.3 De-nature temperature of collagen
wavelength-dependent differences in the absorption of right Differential scanning calorimetry (DSC) is used to analyze the
and le circularly polarized light by optically active molecules endothermic shrinkage transition of collagen. As shown in
such as collagen. The CD spectrum of unordered collagen is Fig. 5, the OYBD-modied collagen showed higher de-nature
ꢀ
ꢀ
usually characterized by a single band below 200 nm, whereas temperature (90.2 C) than native collagen (69.9 C). This
triple-helical structure usually presents one negative band at phenomenon occurred because OYBD reacted with –NH from
2
19,20
223 nm, along with one positive band at 197 nm.
It has been lysine or hydroxylysine side groups of collagen and, forming
well established that the absolute ratio value of molar ellipticity a more stable structure, which enhanced the thermal stability of
2
1
at 220 nm over molar ellipticity around 195–197 nm (Rpn) can collagen.
be used to characterize the integrity of triple-helical confor-
mation of collagen in solution. As shown in Fig. 4, the OYBD
3
.4 Microstructure appearance of collagen
modied collagen exhibited a positive peak at 223 nm and
a negative peak at 197 nm, respectively. Additionally, the Rpn
value of the OYBD-modied collagen (0.13) was comparable to
The morphology and porosity of porous materials are very
important for the scaffolds. The morphology of collagen
Fig. 3 FTIR spectra of (a) native collagen and (b) OYBD-modified Fig. 5 Differential scanning calorimetry curves of (a) native collagen
collagen.
and (b) OYBD-modified collagen.
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membrane was observed by SEM. As shown in Fig. 6, the bers 3.6 Mechanical properties of collagen
of the OYBD-modied collagen exhibited an intertwining
Cross-linking can efficiently increase the mechanical properties
morphology and more dense, intensive ber structure under
ꢂ100, ꢂ1200, and ꢂ5000 magnication compared with native
collagen, indicating efficient crosslinking between collagen and
OYBD.
of the collagen-based materials, especially, the tensile strength
23
and strain modulus. As illustrated in Fig. 8, collagen cross-
linked with OYBD at pH 8.0 had exhibited increased tensile
strength (42.9 MPa) and elongation at break compared with
native collagen. The enhanced tensile strength of the modied
collagen may be related to the nature of inter-microbrillar
3.5 Hydrophobic–hydrophilic properties of collagen
14
The hydrophobic/hydrophilic properties of collagen can be cross-links. As shown in the SEM images in Fig. 6, the modi-
evaluated by the value of water contact angle (WCA). In general, ed collagen displays a more dense microbrillar structure
a low value of WCA means higher hydrophilicity, whilst a high which suggests that the OYBD-induced cross-links hinder the
22
value of WCA indicates better hydrophobicity. As shown in slippage of collagen brils and consequently creates internal
ꢀ
Fig. 7, the value of WCA is 89.1 ꢃ 1.4 for OYBD-modied stresses in the bers. Thus, the modication with OYBD
ꢀ
collagen and 84.2 ꢃ 0.8 for native collagen, suggesting that increases the mechanical properties of the collagen.
OYBD modication increased the hydrophobicity of collagen.
The underlying mechanism is that a certain amount of the
hydrophobic phenoxy groups is introduced into the collagen
3.7 Antibacterial activity of collagen
side chains in the modication process, which leads stronger As shown in Fig. 9, the colony-forming unit (cfu) of all dilution
water repellence of the surface, and thus hydrophobicity. An from the modied collagen is less than those from the native
increased hydrophobic property of the OYBD-modied collagen collagen. The antibacterial ratio of the OYBD-modied collagen
is believed to be favorable in preventing water and bacteria is 51.6% when the suspension was diluted to 1 : 100, while the
intrusion for collagen-based biomaterials.
antibacterial ratio reached up to 100% at the dilution of
1
: 1000. The antibacterial activity against E. coli is the result of
the unique piperonyl structures from OYBD. This structure is
suggested to be very important in many compounds with
24
physiological activity. For E. coli, a type of typical Gram-
negative bacteria, the inhibitory activity of the OYBD-modied
collagen is attributed to the destruction of the peptidoglycan
layer and lipo-polysaccharide. More interestingly, the antibac-
terial activity of the OYBD-modied collagen for Gram-negative
bacteria has
biomaterials.
a potential application in collagen based
The LIVE/DEAD Baclight bacterial viability test is another
analytical method to detect the antibacterial activity of both
Gram-negative and Gram-positive bacterial. The green stain
SYTO 9 is particularly useful as a nuclear counter stain for
Fig. 6 SEM images of (a
1 2 3 1 2
), (a ), and (a ) native collagen and (b ), (b ),
and (b ) OYBD-modified collagen under different magnifications.
3
Fig. 8 Typical uniaxial stress–strain curves of (a) native collagen and
ꢀ
Fig. 7 Water contact angle of native collagen and OYBD-modified (b) OYBD-modified collagen (20 C, distance between clamps: 1 cm,
ꢁ1
collagen. Statistical significance: *p < 0.05. The reported data are pull speed: 1 mm min , N ¼ 3). The reported data are mean ꢃ S.D.
mean ꢃ S.D. (N ¼ 3). (N ¼ 3).
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Fig. 9 The cfu of (a) native collagen and (b) OYBD-modified collagen.
ꢁ
1
ꢁ2
ꢁ3
a
1 1
(b ), a
(b
2 2
) and a
3
(b
3
) represent the dilution of 10 , 10 and 10 .
Fig. 11 (1) The morphology of fibroblasts on the culture plates at day
3
; (2) cytotoxicity tests from MTT assays of cell viability over a period of
bacterial assays since it can stain both live and dead Gram-
25
7 days ((a) collagen, (b) OYBD-modified collagen, (c) gelatin). Statistical
significance: *p < 0.05. The reported data are mean ꢃ S.D. (N ¼ 3).
positive bacteria. The red stain propidium iodide (PI) can
only penetrate cells with compromised or damaged
membranes. As shown in Fig. 10, the predominance of green

uorescence (live bacteria) on native collagen aer dyed by
similar morphologies to those on native collagen, exhibiting
typical broblast morphologies, a spindle shape. This obser-
vation demonstrated that the modied collagen supported
attachment and differentiation of human broblasts as effi-
ciently as native collagen. In addition, the broblast prolifera-
tion rate was also quantitatively evaluated by using the MTT
assay. As illustrated in Fig. 11(2), human broblast not only
remained viable, but also proliferated on the OYBD-modied
collagen, signicantly faster than those on gelatin-coated
wells for the same seeding time. This was because gelatin has
SYTO 9 and PI indicated most of the seeded S. aureus bacteria
remained viable, which suggested the collagen and/or poly-
peptide, amino acid are benecial for bacterial growth. Never-
theless, little green uorescence could be found on collagen-
OYBD aer stained by SYTO 9 and PI, which suggested that
the collagen-OYBD remained antibacterial activity for Gram-
positive bacteria.
3.8 Cytocompatibility assay
26
no triple-helical conrmation, and thus did not act as an
efficient support for cell attachment and proliferation.
With intact triple-helical conrmation, the biological function
of collagen modied with OYBD remained uncompromised.
The morphologies of human broblasts cultured in collagen-
coated PS wells for three days are illustrated in Fig. 11(1).
Clearly, the cells seeded on OYBD-modied collagen showed 4. Conclusions
Modication of collagen with epoxidized safrole, 5-(oxiran-2-
ylmethyl)-benzo[d][1,3]dioxole (OYBD) under alkaline condition
resulted in a modied collagen with increased thermal stability,
enhanced mechanical properties, improved hydrophobic
properties together with more density intermicro-brillar
structure. Especially, the introduction of OYBD did not
destroy the triple helix conformation of native collagen
according to FTIR and CD analysis. Additionally, antibacterial
tests revealed that the modied collagen inhibited proliferation
of both Gram-negative bacterial E. coli and Gram-positive
bacteria S. aureus efficiently. Simultaneously, the collagen-
OYBD exhibited excellent cytocompatibility. According to
these results, OYBD holds a great potential as a novel modier
for producing antibacterial collagen-based biomaterials.
Fig. 10 Fluorescent images of S. aureus seeded on native collagen
and OYBD-modified collagen (green, live bacteria; red, dead bacteria). Conflicts of interest
The merged images represent the superposition of live and dead
bacteria.
There are no conicts to declare.
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1
2 L.-Y. Wang, X.-H. Wang and J.-L. Tan, Bull. Korean Chem.
Soc., 2012, 33, 3571–3575.
Acknowledgements
The authors wish to acknowledge the nancial support from 13 B. X. Zhao, D. W. Wang, H. Zuo, G. T. Qian and J. Y. Miao,
National Natural Science Foundation of China (No. 51273128,
Chin. J. Org. Chem., 2003, 23, 1026–1028.
1206096) and National Key R&D Program of China (No. 14 R. Zeeman, P. J. Dijkstra and P. B. V. Wachem, Biomaterials,
2
2
017YFB 0308600).
1999, 20, 921–931.
15 H. Zhou, R. P. Xun, Z. H. Zhou, Q. Q. Liu, P. Wu and K. J. Wu,
Fibers Polym., 2014, 15, 519–524.
References
1
6 M. E. Andrews, J. Murali, C. Muralidharan, W. Madhulata
and R. Jayakumar, Colloid Polym. Sci., 2003, 281, 766–770.
1
2
3
4
R. Zeeman, P. J. Dijkstra and P. B. Van Wachem, J. Biomed.
Mater. Res., 1999, 46, 424–433.
D. M. Simmons and J. N. Kearney, Biotechnol. Appl. Biochem.,
17 L. He, C. Mu, J. Shi, Q. Zhang, B. Shi and W. Lin, Int. J. Biol.
Macromol., 2011, 48, 354–359.
18 B. Madhan, V. Subramanian and J. R. Rao, Int. J. Biol.
Macromol., 2005, 37, 47–53.
19 D. I. Zeugolis, S. T. Khew, E. S. Yew, et al., Biomaterials, 2008,
29, 2293.
20 J. Chang, Y. Chen, S. Zhao, et al., Polym. Chem., 2015, 6,
8150–8160.
1
993, 17, 23–29.
R. Tu, S. H. Shen and D. Lin, J. Biomed. Mater. Res., 1994, 28,
77–684.
6
J. M. Lee, C. A. Pereira and L. W. K. Kan, J. Biomed. Mater.
Res., 1994, 28, 981–992.
5
6
T. Xi and F. Liu, J. Biomater. Appl., 1992, 7, 61–75.
R. Sripriya, R. Kumar, S. Balaji, M. S. Kumar and P. K. Sehgal, 21 B. Balakrishnan, M. Mohanty, P. R. Umashankar and
React. Funct. Polym., 2011, 71, 62–69.
A. Jayakrishnan, Biomaterials, 2005, 26, 6335–6342.
L. H. H. Olde Damink, P. J. Dijkstra, M. J. A. van Luyn, 22 P. Fratzl, Collagen: Structure and Mechanics, Springer, New
7
P. B. van Wachem, P. Nieuwenhuis and J. Feijen, J. Mater.
Sci.: Mater. Med., 1995, 6, 429–434.
York, NY, USA, 2008.
23 P. F. Gratzer and J. M. Lee, J. Biomed. Mater. Res., 1997, 37,
8
9
R. Tu, R. C. Quijano, C. L. Lu, et al., Int. J. Artif. Organs, 1993,
497–507.
16, 537–544.
24 D. Ge, Q. Jing, W. Zhao, H. Yue, et al., PLoS One, 2014, 9,
J. M. Lee, C. A. Pereira and L. W. K. Kan, J. Biomed. Mater.
Res., 1994, 28, 981–992.
e99378.
25 C. Yang, X. Ding, R. J. Ono, et al., Adv. Mater., 2014, 26, 7346–
7351.
26 G. Y. Li, S. Fukunaga, K. Takenouchi and F. Nakamura, Int. J.
Cosmet. Sci., 2005, 27, 101.
10 J. M. Lohre, L. Baclig, J. Sagartz, S. Guida, K. Thyagarajan
and R. Tu, Artif. Organs, 1992, 16, 630–633.
1
1 R. Garc ´ı apumarino, G. Pascual, M. Rodr ´ı guez, et al., J.
Biomed. Mater. Res., Part B, 2013, 102, 366–375.
50306 | RSC Adv., 2017, 7, 50300–50306
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