Hilo quirúrgico

1. See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/272186517
Fabrication and feasibility study of an absorbable
diacetyl chitin surgical suture for wound healing
ARTICLE in JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B APPLIED BIOMATERIALS · FEBRUARY 2015
Impact Factor: 2.76 · DOI: 10.1002/jbm.b.33307 · Source: PubMed
READS
21
7 AUTHORS, INCLUDING:
Jinning Gao
Qingdao University
11 PUBLICATIONS 10 CITATIONS
SEE PROFILE
Weizhi Liu
Ocean University of China
27 PUBLICATIONS 275 CITATIONS
SEE PROFILE
Available from: Weizhi Liu
Retrieved on: 13 November 2015

2. Fabrication and feasibility study of an absorbable diacetyl chitin
surgical suture for wound healing
Kai Shao,1,2
Baoqin Han,2
* Jinning Gao,3
Zhiwen Jiang,2
Weizhi Liu,2
Wanshun Liu,2
Ye Liang4
1
Center of Laboratory Medicine, Qilu Hospital of Shandong University (Qingdao), Qingdao 266035, China
2
Laboratory of Biochemistry and Biomaterials, College of Marine Life Science, Ocean University of China, Qingdao 266003, China
3
Institute for Translational Medicine, The Medical College, Qingdao University, Qingdao 266021, China
4
Central Laboratory, Affiliated Hospital of Qingdao University, Qingdao 266003, China
Received 4 April 2014; revised 24 September 2014; accepted 1 October 2014
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33307
Abstract: Diacetyl chitin (DAC) is an acidylated chitin
obtained using acetic anhydride mixed perchloric acid sys-
tem. By wet spinning and weaving technique, DAC has been
successfully developed into a novel absorbable surgical
suture. Thanks to the unique properties of chitins, the poten-
tial application of this novel monocomponent multifilament
DAC suture may break the monopoly of synthetic polymer
sutures in wound closure area. In this study, DAC was syn-
thesized and characterized by multiple approaches including
elemental analysis, Fourier transform infrared spectrometry
(FTIR), and X-ray diffraction (XRD). In addition, we performed
the feasibility assessment of DAC suture (USP 2-0) as
absorbable suture for wound healing. Several lines of evi-
dences suggested that DAC suture had comparable mechani-
cal properties as synthetic polymer sutures. Moreover, DAC
suture retained approximately 63% of the original strength at
14 days and completely absorbed in 42 days with no remark-
able tissue reaction in vivo. Most important of all, DAC
suture significantly promoted skin regeneration with faster
tissue reconstruction and higher wound breaking strength on
a linear incisional wound model. All these results demon-
strated the potential use of DAC suture in short- or middle-
term wound healing, such as epithelial and connective tis-
sue. VC 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl
Biomater 00B:000–000, 2015.
Key Words: diacetyl chitin, absorbable suture, liner incisional
wound, wound healing, skin regeneration
How to cite this article: Shao K, Han B, Gao J, Jiang Z, Liu W, Liu W, Liang Y. 2015. Fabrication and feasibility study of an
absorbable diacetyl chitin surgical suture for wound healing. J Biomed Mater Res Part B 2015:00B:000–000.
INTRODUCTION
Skin wounds are common in surgery and trauma. Wound
healing process involves the cooperation of complex interac-
tions of extracellular matrix, cells, and signaling factors.1
Among various signaling compounds and growth factors,
transforming growth factor-b (TGF-b) is believed to act in
various functions as inhibitors or attractants of inflamma-
tory cells and keratinocyte fibroblast, in acceleration of
matrix turnover and collagen.2
In addition, TGF-b1 can
induce myofibroblast differentiation and collagen deposition,
marked by hydroxyproline (Hyp) content in wound tissues.3
Clinically, suture is the most effective method for wound
closure with a huge market exceeding $ 1.3 billion annu-
ally.4
Thus, a suitable suturing technique is essential for
wound healing and prevention of excessive scar. The devel-
opment of ideal absorbable suture for mechanical wound
closure with full recovery of its biological functions has long
been a major goal in surgery and trauma. In light of this
pivotal role of absorbable surgical suture for clinical use,
many different kinds of synthetic polymers have been devel-
oped, such as polyglycolide (PGA),5
polylactide,6
poly(lac-
tide-co-glycolide) (PLGA),7
polydioxanone (PDS),8
and other
copolymers. Currently, absorbable polymeric sutures have
got unprecedented commercial success, such as VicrylVR Plus
(Ethicon, USA), PDS IIVR (B. Braun, Germany), and PloysorbVR
(US Surgicals, USA).4
Compared with traditional nonabsorb-
able suture materials, these absorbable polymeric sutures
have advantages on reproducible degradability in biological
environment during the wound healing process.9
However,
most polymeric sutures are found to cause pronounced
inflammatory response during the degradation process, add-
ing severe side-effects for wound healing and much pain for
patients.10
Therefore, to solve this issue and discover new
absorbable and biocompatible sutures for clinical usage are
necessary.
Chitin, a natural polysaccharide with antimicrobial prop-
erty, is one of the most promising biomaterials for effec-
tively accelerating wound healing and providing protection
from wound infection. In particular, chitins can promote
fibroblast proliferation and macrophage migration, and
accelerate vascularization and granulation during wound
healing process.3
Moreover, the non-toxic and biodegradable
*Present address: Room 220, Science Building, 5# Yushan Rd., Qingdao, China
Correspondence to: B. Han; e-mail: baoqinh@ouc.edu.cn
VC 2015 WILEY PERIODICALS, INC. 1

3. properties of chitin make it a promising biomaterial for
absorbable suture.11
Actually, due to the excellent fiber-
forming ability12
and biological activities of chitin, a few
commercial chitin-based dressings have been developed,
such as BeschitinVR (Unitika, Japan), Chitipack SVR (Eisai Co,
Japan), and TegasorbVR (3M, USA).13
Previous studies have
indicated that chitin has comparable properties to collagen
and lactide fibers,14
and chitin suture can be absorbed in
about four months in rat muscles.15
However, the current chi-
tin suture also has limitations in mechanical strength and
degradation time, and cannot meet the requirements of some
particular surgery. To improve the quality for chitin, various
acyl derivatives of chitin have been obtained by reaction of
acetylation, formylation, propionylation, and butyrylation,
using acid anhydrides mixed perchloric acid system.16,17
Dibutyryl chitin (DBC) is a recent entry in the list of acyl chi-
tins of interest in wound healing and radical scavenging func-
tion. Notably, DBC-based non-wovens mats made by
Muzzarelli et al.13
and J. Matthew et al.18
have been tested in
rats and obtained a good effect, demonstrating that DBC is a
good candidate for further evaluation as an effective wound
healing agent. Recently, Liu et al. have demonstrated that
chitosan-halloysite nanotubes composite sponges possess a
higher mechanical strength than pure chitosan sponges.19
Diacetyl chitin (DAC) is another multifunctional acylate of
chitin. Previous studies have already revealed that acetylated
chitin fibers, with greater tenacity and elongation than chitin
fibers,20
can be taken into consideration for potential applica-
tions of fiber-based medical devices. To investigate the poten-
tial for DAC as absorbable surgical suture material, a novel
monocomponent multifilament DAC suture was developed in
our lab. Our study also aimed at fundamental insights on the
feasibility of DAC suture as absorbable biomaterials for
wound healing. Thus, we investigated the mechanical proper-
ties and degradation behavior of DAC suture, and performed
functional study on a linear incisional wound model.
MATERIALS AND METHODS
Materials and reagents
Chitin with viscosity average molecular weight of 269 kDa
and intrinsic viscosity 10.72 dL/g (dissolved in
dimethylacetamide 1 5% LiCI solutions, 25
C) was supplied
by Biotemed Co., Ltd. VicrylVR Plus suture (USP 2-0) was
supplied by Ethicon, USA. Rat TGF-b1 ELISA Kit (96T) and
Rat Hyp ELISA Kit (96T) were from Colorfulgene Biotech-
nology Co. (Wuhan, china). Adult male Sprague-Dawley rats
(200 6 10 g body weight) were obtained from Qingdao
Institute for Drug Control. All chemicals were analytical
grade and received from Sinopharm Chemical Reagent Co.,
Ltd (China), unless otherwise indicated.
Preparation of DAC
DAC was prepared as described previously in heterogeneous
reaction with minor modifications, using acetic anhydride as
acylating agent and perchloric acid as a catalyst.21
The brief
process was as follows: Perchloric acid (4.0 mL) was added
dropwise in acetic anhydride (100.0 mL) at 0
C and stabilized
for 30 min. Then chitin (10.0 g) was dispersed into the acy-
lated conditions and stirred at 0
C for 24 h. The mixture was
then neutralized by NaOH powder and the final DAC product
was obtained after filtrated, purified (95% alcohol), and dried.
Characterization of DAC
Chemical structure of DAC product was characterized by
elemental analysis, Fourier transform infrared (FTIR), and
X-ray diffraction (XRD). To determine the degree of substitu-
tion (DS), CHN elemental analysis was performed on an Ele-
mental Analyzer-MOD 1106 (Carlo Erba Strumentazione,
Italy). FTIR spectra of chitin and DAC were recorded on a
Nicolet Nexus-470 Fourier Transform Infrared Spectrometer
(USA). KBr method was adopted and data analysis was car-
ried out on Jwstda-32. XRD was performed on X’Pert MPD
DY1291 (Koninklijke Philips Electronics NV, the Nether-
lands), using conventional Bragge-Brentano geometry in q-
2q configuration with Cu Ka radiation at 40 kV and 35
mA.22
The XRD patterns were collected over a range from
5
to 60
at a scanning rate of 2
/min and data analysis
was carried out on Jade 5.0 (Materials Data, Inc., USA).
Preparation of DAC suture
Using 12% solution of DAC in formic acid, DAC fibers were
obtained by wet spun into coagulation baths composed of
acetone and ethanol.23
With application of modern weaving
technology, a novel monocomponent multifilament DAC
suture was obtained.
Mechanical properties of DAC suture
There must be a proper match between the suture strength
and the tissue strength.24
To investigate the breaking
strength, breaking elongation, knot strength, knot-pull
strength, and strength with needle of DAC suture (USP 2-0),
Universal Testing Instruments AGS-X (Shimadzu, Japan) was
performed at a testing speed of 5.0 mm/s. VicrylVR Plus
suture (USP 2-0), a multifilament braided PLGA suture
impregnated with triclosan to provide antimicrobial protec-
tion, was employed as control. The parameters above were
measured ten times and the suture materials 20 cm in
length were incubated in phosphate buffer saline (PBS, pH
7.2) for 30 min at 25
C before testing. For the mechanical
properties assay, breaking strength and breaking elongation
were the tensile and elongation at which suture failure
occurred separately; strength with needle was the tensile at
which suture with needle broke; knot strength was the
amount of tensile necessary to cause a knot to slip (the
middle of the suture were tied into a knot separately before
testing); while knot-pull strength was the breaking strength
of knotted suture in the middle. Moreover, the swelling
(sutures with 25 cm in length were incubated in PBS 7.2 at
25
C for 24 h), pliability and memory characteristics of DAC
suture were also evaluated.
In vivo breaking strength retention
The animal experiments in this study were carried out in
compliance with the National Institute of Health Guide for
the Care and Use of Laboratory Animals. Thirty-six adult
male Sprague-Dawley rats were housed singly in standard
2 SHAO ET AL. DIACETYL CHITIN SURGICAL SUTURE FOR WOUND HEALING

4. cages and kept under controlled temperature and humidity
with free access to food and water.25
The rats were anesthe-
tized with intraperitoneal administration of pentobarbital
sodium (3% in saline solution) at a dose of 1 mL/kg. The
dorsal side of the rats was shaved and disinfected with 75%
alcohol for surgery. Sterile multifilament DAC suture (USP
2-0), 10 cm in length, was implanted by sewing with a long
needle (8 cm) inserting through the left dorsal subcutis of
each rat.26
Treated with VicrylVR Plus suture (USP 2-0) in
the right dorsal subcutis was employed as control. At 7, 14,
21, 28, 35, and 42 days after operation, six rats were sacri-
ficed respectively and the breaking strength retention of the
implanted sutures was determined on Universal Testing
Instruments AGS-X (Shimadzu, Japan).
In vivo biocompatibility and biodegradability
Thirty adult male Sprague-Dawley rats were housed and
anesthetized as described above before operation. The glu-
teal side of the rats was shaved and disinfected with iodine
solution before surgery. Sterile multifilament DAC suture
(USP 2-0), each 3 cm long, was implanted into the left
side of the gluteal muscle. To avoid secession of the suture
construction, both ends of the DAC suture were tied into a
knot with a little space. Treated with VicrylVR Plus suture
(2-0) in the right gluteal muscle was employed as control.
Six rats were sacrificed at 7, 14, 21, 28, and 42 days after
operation, respectively. Macroscopic examination was per-
formed to investigate the biocompatibility and biodegrad-
ability of DAC suture. On the other hand, hematoxylin–eosin
(HE) staining was employed to investigate suture degrada-
tion and inflammatory reaction caused by implantation.
Creation of linear incisional wound on rats and
suturing
Adult male Sprague-Dawley rats were housed and anesthe-
tized as described above before operation. The back hair of
the rats were shaved and disinfected with iodine solution.
Three centimeters long, a linear paravertebral incision was
made with a sterile surgical blade through the full thickness
of the skin.27,28
The wound closure was performed with
sterile multifilament DAC sutures (USP 2-0) of 0.5 cm apart,
and treated with VicrylVR Plus suture (USP 2-0) was
employed as control. The rats were housed individually for
investigation of wound healing process at each time point.
Macroscopic evaluation and wound breaking strength
The wound area of 12 rats was macroscopically evaluated
for 21 days continuously after treatment. The following
parameters were determined: wound healing rate, wound
breaking strength, suture retention, presence of ulcers, and
scars. Rats with complete skin reconstruction and wound
healing at 14 days was considered to be successful case for
wound healing rate. Wound breaking strength was the ten-
sile strength of a healing wound at which separation of the
wound edges occurs. Thus, it was determined 7 days and
14 days after operation using a tensiometer (Meixun,
Wuhan, China). Skin ulcers and scars were examined and
scored as absent and present.
Hyp and TGF-b1
Myofibroblast differentiation and collagen deposition were
assayed with Hyp, which was mainly secreted by the fibro-
blasts and gave rise to collagen secretion, using a Rat Hyp
ELISA Kit (96 T). In addition, another relevant cytokine TGF-
b1, which was critical to wound contraction and marked by
synthesis of collagen and fibronectin, was determined in
wound tissue with a Rat TGF-b1 ELISA Kit (96 T). At weeks 1
and 2 after operation on the linear incisional wound model,
wound skin tissue (1.0 g) of each linear incision was collected
with 5.0 mL PBS (pH 7.4), completely homogenized for 30 s
with a Tissue Tearor (IKA, Germany), and centrifuged with
Sigma 3K 30 (USA) at 3000 rpm for 20 min. The supernatant
was collected to measure the levels of Hyp and TGF-b1 by
ELISA, using commercially available assay systems according
to the manufacturer’s instructions. Rats treated with VicrylVR
Plus suture (USP 2-0) were employed as control.
Histological examination
At weeks 1, 2, and 3 after operation on the linear incisional
wound model, seven rats were sacrificed separately and the
trauma samples were cut into 5 lm for histopathological
examination. HE-staining was carried out for investigation
of inflammatory reaction, collagen arrangement, suture
retention, re-epithelialization, and re-growth of skin appen-
dages in wounding area.
Statistics analysis
Statistical analysis of data was performed by one-way
ANOVA on SPSS (version 20.0).
RESULTS
Characterization of DAC product
Figure 1 displays the synthetic route of DAC from chitin. The
basic physical and chemical properties are shown in Table I. As
FIGURE 1. Synthetic route of DAC from chitin.
TABLE I. Basic Physical and Chemical Properties of the DAC Product
Sample
Elemental Analysis Results
DS Free Amino (%) Water Solubility Organic SolubilityN (%) C (%) H (%) C/N
DAC 4.15 42.26 6.57 10.18 1.94 2.12 Water-insoluble Formic acid-soluble
ORIGINAL RESEARCH REPORT
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2014 VOL 00B, ISSUE 00 3

5. shown above, the DAC product was water-insoluble and the
intrinsic viscosity in formic acid at 25
C was 2.06 dL/g. The
results of CNH elemental analysis was also given, suggesting the
degree of esterification was very close to 2.
In the FTIR spectrum of DAC (Figure 2), the characteris-
tic absorption peak of hydroxyl group at 3500 cm21
almost
disappeared, indicating they were esterified and the DS was
approximately to 2. New strong characteristic peaks at
1747 cm21
and 1232 cm21
, which attributed to the esters
of fatty acids, revealed the acetylation of chitin at 3 and 6
positions. Furthermore, the intensification of bands at
790 cm21
and 740 cm21
corresponding to methyl groups
confirmed the chemical structures of the DAC product.21
XRD (Figure 3), another independent approach, was
employed to further characterize DAC. The spectroscopy
showed the characteristic structure for the pure chitin pow-
der at peak 20.10
. The spectrum of DAC resembled that of
chitin. However, two stronger peaks of DAC spectrum at
9.36
and 19.32
attributed to acetylation,29
revealed the
chemical structures of the DAC product.
Mechanical characterization of DAC suture
The monocomponent DAC suture was manufactured by
modern multifilament braiding technique with a diameter
range 0.30–0.35 mm, with no coating materials or dyes, as
shown in Figure 4(A). There are several characteristics
which are essential for all sutures, such as physical and
mechanical properties, handling characteristics, biological
and degradation behaviors. In light of the significance of
mechanical properties to a suture, relevant mechanical
parameters were investigated and the results are given in
Table II. Figure 4(B) shows the load–displacement profile of
DAC suture (USP 2-0) and the control (VicrylVR Plus, USP 2-
0) at testing speed of 5 mm/s. The results indicated that
VicrylVR Plus suture had advantage in mechanical strength
over DAC suture, about 20% higher. Both the two sutures
could meet the strength and percentage elongation require-
ments for conventional suturing. It should be noted that,
due to the modest swelling property and excellent pliability,
DAC suture showed excellent handling characteristic for
knotting.
Breaking strength retention in vivo
The basic parameters of the DAC suture and the control
suture are given in Table III. The duly degradability inside
a biological environment is pivotal for an ideal absorbable
suture. Here, the breaking strength retention rate of DAC
suture was further assessed in subcutis of rats. As shown
in Figure 5, during a 6-week period observation for DAC
suture group, a strength loss of 23.1% was detected dur-
ing the initial 7 days, which had a significant difference
with control (32.6% at 7 days). After one week of implan-
tation, a relative slow strength loss occurred and DAC
suture remained approximately 63% of the original
strength at 14 days. DAC suture was completely absorbed
at 42 days with excellent biocompatibility and no broken
or liquefaction. On the other hand, strength of the control
showed a burst loss after 7 days, and this kind of syn-
thetic absorbable suture provided strength retention of
20% at 14 days. Notably, the suture material liquefied
significantly at 21 days observation time, leading to
obvious tissue reactions and entirely loss of suture
strength. All of these results indicated that DAC suture had
higher breaking strength retention rate than control in
vivo. Therefore, it is believed that in the critical post-
operative period (traditionally within 3 weeks), DAC suture
was able to maintain its physical and biochemical functions
for longer time.
Biocompatibility and biodegradability in muscle
To investigate the degradation behavior and tissue reaction
of the monocomponent multifilament DAC suture, micro-
scopic observation was performed. DAC suture (USP 2-0)
and the control suture were completely absorbed at 35–42
days and 42–49 days, respectively (Figure 6). At 7 days
post-implantation, both the two sutures were surrounded
by a large amount of inflammatory sells [Figure 6(A,F)]. A
thick collagen-like capsule with macrophages and fibro-
blasts was observed and the suture kept its original shape
in control group [Figure 6(F)]. Besides, due to the capillar-
ity and lack of coating material, DAC suture showed com-
pact interaction with the inflammatory zone [Figure 6(A)].
FIGURE 2. FTIR spectrum of chitin and DAC. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
FIGURE 3. X-ray diffraction spectra of chitin and DAC. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
4 SHAO ET AL. DIACETYL CHITIN SURGICAL SUTURE FOR WOUND HEALING

6. At 14 days observation, only a small amount of inflamma-
tory cells at the contact region were observed surrounding
both the suture strands. In the DAC group [Figure 6(B)],
the suture kept its original shape, yet limited fissures were
observed. And the diameter of the DAC fiber reduced to
some extent, indicating a gradual degradation process has
occurred. In contrast, the collagen-like capsule still
remained in control group, and the arrangement of the
multifilament fibers inside was broken and some fibers
were partly absorbed [Figure 6(G)]. As the degradation
proceeded, no remarkable tissue reaction was observed for
DAC suture at 21 days and the diameter of DAC fibers fur-
ther reduced [Figure 6(C)]. In the control group, the area
of visible fibrous region decreased to approximately half of
the original region and some fissures were observed [Fig-
ure 6(H)]. At 42 days, both the two sutures were com-
pletely degraded and absorbed, and no obvious
inflammatory reactions or other tissue reactions were
observed [Figure 6(E,J)].
Macroscopic observation and wound breaking strength
A linear full thickness wound model was surgically created
and the feasibility of the DAC suture for wound closure and
further healing was evaluated by comparison with VicrylVR
Plus. The operation process of suturing is shown in Figure
7(A). DAC suture could efficiently close the wound with no
suture breaking or knot slipping. During a 3-week period
observation, the wound healing process after treated with
DAC suture [Figure 7(B)] and the control suture [Figure
7(C)] was separately recorded daily. It should be stated that
DAC suture broke approximately at 6 days and disappeared
macroscopically at 11 days. On the other hand, tensile
strength of the wound skin at 6 days was enough to main-
tain its own shape and the healing process was fully com-
pleted at about 13 days. In the control group, the situation
was similar to that of the DAC suture at the initial stage.
Suture broke at approximately 5 days. But the suture reten-
tion time of the control was longer (about 20 days) and the
complete skin regeneration was accomplished at about 20
days. Generally, both the two sutures could break after los-
ing their functions to avoid the risk of complications.
The present results indicated that the wound breaking
strength of the DAC group and the control group at 7 days
and 14 days (Figure 8) were significantly higher than that
of the untreated group (p 0.05), indicating both the two
FIGURE 4. A: Macroscopic image of DAC suture (USP 2-0) and (B) the load–displacement profile of DAC suture (USP 2-0) and the control (VicrylVR
Plus, USP 2-0). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
TABLE II. Mechanical Parameters of DAC Suture (USP 2-0)
and the Control (VicrylVR Plus, USP 2-0)
Parameter
Suture
DAC
VicrylVR Plus
(Edicon, USA)
U.S.P. size 2-0 2-0
Breaking
strength (MPa)
189.95 237.65
Breaking
elongation (%)
16.77 25.13
Knot-pull
strength (MPa)
144.37 194.06
strength with
needle (MPa)
135.88 169.57
Knot strength
(MPa)
97.52 104.88
Swelling (%) 134.65 18.63
Pliability Soft (in PBS, 7.2) Stiff (in PBS, 7.2)
Memory Scarce Modest
TABLE III. Basic Parameters of DAC Suture (USP 2-0) and the
Control (VicrylVR Plus, USP 2-0)
Parameter
Suture
DAC
VicrylVR Plus
(Edicon, USA)
Composition Diacetyl chitin (DAC) Poly (LL to GL) (PLGA)
U.S.P. size 2-0 2-0
Diameter 0.30–0.35 mm 0.30–0.35 mm
Length 30 mm 90 mm
Formation Multifilament braided Multifilament braided
Coatings Not coated Coated
Dyed Not dyed Dyed (purple)
ORIGINAL RESEARCH REPORT
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2014 VOL 00B, ISSUE 00 5

7. sutures could enhance wound healing acceleration. In par-
ticular, there was also significant difference between the
two sutures (p 0.05) that DAC group showed higher
wound breaking strength.
To quantify the performance of the sutures for full thick-
ness wound, wound healing rate, skin ulcers, and scars
were investigated by macroscopic evaluation at 14 days
post-operation. Skin ulcers were observed in DAC group (2/
12) and control (3/12), and hypertrophic or keloid scars
were evaluated in DAC group (1/12) and control group (2/
12). Rats with complete skin reconstruction and wound
healing at 14 days was considered to be successful case for
wound healing rate. The wound healing rate was 100%
(12/12, DAC) and 92% (11/12, control), respectively. Gener-
ally, all data obtained by macroscopic evaluation indicated
that this monocomponent multifilament DAC suture rapidly
restored the structural and functional properties of
wounded skin and had advantage over the control.
Inflammation and collagen arrangement
Histological examinations were performed at weeks 1, 2,
and 3 post-procedures to investigate the healing process
and the structure, in terms of inflammatory reaction, cell
proliferation, collagen arrangement, suture retention, and
re-epithelialization. The results showed that the suture
material and the wound site were surrounded by a large
amount of macrophages and neutrophile granulocytes in
both the two groups at 7 days [Figure 9(A,D)]. As the skin
reconstruction proceeded, DAC fiber were hydrolyzed gradu-
ally by glycosidases and became oligomers and monomers
and the fiber fragment could still be observed around the
wound site at 14 days [Figure 9(B)]. Beside this, the inflam-
mation reaction in regenerated tissue almost disappeared
and numerous types of cell differentiation were observed
possibly induced by the products of DAC, leading to acceler-
ation of the wound healing process. While for control group
at 14 days [Figure 9(E)], there were still a massive quantity
of inflammation cells and fibroblasts surrounding the
wound site, and extensive suture strands could also be
observed. At 21 days histological examination, there was no
remarkable tissue reaction or fiber fragment observed in
both the DAC group [Figure 9(C)] and the control group
[Figure 9(F)]. The fibroblasts differentiation and collagen
deposition are the key points of skin regeneration.30
There-
fore, the collagen content and arrangement were also inves-
tigated by histological examination. For DAC treated group
at 21 days [Figure 9(C)], more activated fibroblasts in the
wound site were observed than the control group. In partic-
ular, the collagen fibers in the wound site tended to run
parallel to the epidermal layer as the normal skin in DAC
group, while those in control group [Figure 9(F)] ran per-
pendicular to the epidermal layer. Nevertheless, DAC exhib-
ited loose collagen fiber arrangement and this phenomenon
might explain the higher wound breaking strength for DAC
group.
Re-epithelialization and re-growth of skin appendages
Generally, keratinocytes at the wound edge migrate over the
epidermis to differentiate the new outer layer in a process
termed re-epithelialization. At 21 days in the DAC group
[Figure 9(C)], the linear wound was epithelized completely
FIGURE 5. Breaking strength retention of DAC suture (USP 2-0) and
the control (VicrylVR Plus, USP 2-0) in vivo.
FIGURE 6. HE staining for inflammatory reaction and biodegradation of DAC suture (A–E) and the control (VicrylVR Plus, F-J) at 7 days, 14 days,
21 days, 28 days, and 42 days after implantation in rats, respectively. Bars represent 100 lm. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
6 SHAO ET AL. DIACETYL CHITIN SURGICAL SUTURE FOR WOUND HEALING

8. with the epidermic cells fully differentiated and basal cells
closely arranged. Also, the superficial crust disappeared and
a flat horny layer could be observed. However, in the control
group at 21 days [Figure 9(F)], the structure of new epider-
mis was uneven and incomplete. On the other hand, the for-
mation of skin appendages in DAC group was faster than
that in control group. At week 2 after treatment, a small
amount of sebaceous gland cells and hair follicle cells were
observed in DAC group [Figure 9(B)] while only inflamma-
tory cells and fibroblasts were observed in control group
[Figure 9(E)]. At week 3 post-operation, numerous mature
hair and sebum were found in DAC group [Figure 9(C)],
while the growth of skin appendages in control group [Fig-
ure 9(F)] was slower. All these results indicated that the
DAC suture stimulated the re-epithelialization and the
growth of skin appendages.
Hyp and TGF-b1 determination
To investigate myofibroblast differentiation and collagen
deposition in the wound site, a Rat Hyp ELISA Kit (96 T)
was employed. It was demonstrated that the contents of
Hyp in DAC group were significantly higher than that in
FIGURE 7. The operation process of suturing with DAC suture (A). Pretreatment (A1); linear incisional wound (A2); suturing (A3–A6). The repre-
sentative images of the wound healing process after treated with DAC suture (B) and the control (C) during a 3 week-period observation. Bars
represent 5 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
ORIGINAL RESEARCH REPORT
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2014 VOL 00B, ISSUE 00 7

9. control group at weeks 1 and 2 separately (p 0.05), indi-
cating more deposition of collagen [Figure 10(A)].
Another relevant cytokine TGF-b1, which was critical to
wound contraction and marked by synthesis of collagen and
fibronectin, was also studied to quantify the acceleration of
DAC suture to wound healing [Figure 10(B)]. At week 1
after operation, the level of TGF-b1 in DAC group was sig-
nificantly higher than control (p 0.05). The data obtained
at week 3 indicated there was no significant difference
between the two groups (p 0.05). According to all these
results, it was out of doubt that DAC suture enhanced colla-
gen deposition and relevant cytokines secretion, leading to
acceleration of the wound healing process.
DISCUSSION
To meet different surgical needs, the development of new
absorbable and biocompatible sutures with proper mechani-
cal strength, modest inflammatory response, and significant
wound healing promotion is necessary and urgent. In light
of the excellent fiber-forming ability,31
antimicrobial proper-
ties,32
wound healing promotion, and other outstanding bio-
chemical activities, chitin and its derivatives have been
influential in the development of interest in wound healing
devices. Actually, one of the most promising biomaterials
that can accelerate wound healing process is chitin, which is
a natural polysaccharide containing N-acetyl glucosamine
moiety.33
The latter is also found in certain human glyco-
proteins in connective tissues like keratin sulfate. It has
been reported that several chitin sutures have remarkable
properties over other fibers for biomedical applications.34
An ideal suture should have proper mechanical charac-
teristics, especially enough tensile strength, pliability for
ease of handling, and knot security. In this study, we found
that DAC suture met the strength requirement for conven-
tional suturing. Investigation into the breaking strength
retention of the DAC suture in vivo revealed that DAC suture
remained approximately 63% of the original strength at 14
days, which was higher than chitin suture reported (45% at
14 days).35
Therefore, it is believed that in the critical post-
operative period (traditionally within 3 weeks), DAC suture
is able to maintain its physical and biochemical functions,
showing the potential of replacing traditional short- or
middle-term absorbable sutures. It should be noted that
suitable percentage elongation is necessary for an ideal
suture. Since high percentage elongation can give an indica-
tion of the potential gap formation that may occur under a
particular load, while low percentage elongation may cause
difficulty for knotting.36
The result indicated that the break-
ing elongation of DAC suture was approximately 17%, which
was flexible to tie in a stable knot. Previous studies have
showed that DAC fiber has greater tenacity and elongation
than chitin fiber, and it is hydrophobic because the hydroxyl
FIGURE 8. Wound breaking strength of the DAC group and the control
group (VicrylVR Plus) at 7 days and 14 days. *p 0.05 vs. untreated
wound.
FIGURE 9. Histological examinations of the wound healing process of the DAC group (A–C) and the control group (D–F) 7 days, 14 days, and 21
days after operation. Arrows indicate suture fiber, and bars represent 100 lm. [Color figure can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
8 SHAO ET AL. DIACETYL CHITIN SURGICAL SUTURE FOR WOUND HEALING

10. groups are totally esterified.23
However, the multifilament
structure gives rise to the obvious capillarity, modest swel-
ling property, and excellent pliability of the DAC suture.
The results demonstrated that DAC suture (USP 2-0)
was completely absorbed in 42 days in rat muscle, which
was similar to VicrylVR Plus suture and 80 days faster than
chitin suture reported previously.15
The fast absorption
behavior of DAC suture may be attributed to the acetylation
and/or the fabrication of the suture. Particularly, the mono-
component multifilament DAC suture has relatively larger
specific surface area and the capillary action makes it possi-
ble to uptake more body fluids during the degradation pro-
cess. As a result, chitins are hydrolyzed gradually by
glycosidases such as chitinase and lysozyme,37
leading to
the fast absorption behavior. In addition, DAC suture
retained approximately 63% of the original strength at 14
days. All these results revealed the potential of DAC suture
used in short- or middle-term wound support. Certainly, no
type of suture is suitable for every situation, and various
sutures are being designed all the time. A previous study
has developed a self-reinforced poly-lactide (SR-PLLA)
suture.36
With prolonged strength retention and low elonga-
tion, it can be applied to close wounds that need prolonged
support, such as bone. Another synthetic absorbable bicom-
ponent monofilament suture (MonoFlex), composed of
poly(p-dioxanone) and its copolymer, has been prepared for
extended wound support.26
In this study, it should be noted that VicrylVR Plus suture
showed pronounced inflammatory reaction compared with
DAC suture, which elicited unremarkable tissue response.
Previous studies have also reported that traditional absorb-
able polymeric sutures may cause unavoidable inflammatory
reaction.38,39
DBC, another acylated derivative, also shows
high biocompatibility in the form of films and non-wovens
in previous studies.13,40
Considering the mechanical properties and biocompati-
bility, this monocomponent multifilament DAC suture was
found to be potential applied as an ideal absorbable suture.
Particularly, present evidence pointed out that DAC suture
could significantly accelerate the wound healing process
with higher wound breaking strength and less wound heal-
ing time. Histological examinations also indicated that the
collagen fibers in the wound site tended to run parallel to
the epidermal layer as the normal skin in DAC group. As a
result, DAC suture showed higher wound breaking strength.
The mechanism of the promotion of skin regeneration by
DAC suture may be attributed to the outstanding biochemi-
cal significance of chitins. As the acetylated chitin fibers are
gradually degraded by glycosidases such as chitinase and
lysozyme,37
they turn into oligomers and monomers in the
wound area. It has been reported that chitins and its biode-
gradable substances could accelerate fibroblast proliferation
and macrophage migration, and they could also promote
granulation, cytokine production, and vascularization, lead-
ing to better organization and smooth wound healing.3
The
results in contents of Hyp and TGF-b1 in the wound site
were consistent with previous studies. Specifically, com-
pared with the control group, DAC suture tended to enhance
much smoother re-epithelialization. Recent evidence points
to DG42 protein and hyaluronan.41,42
The former can pro-
duce chitooligomers acting as templates for hyaluronan syn-
thesis. As a result, the high concentration of hyaluronan
devotes to the correct tissue reconstitution of the wounds
in the fetus heal.
CONCLUSIONS
A novel monocomponent multifilament absorbable suture,
DAC suture, was fabricated and characterized by multiple
approaches including elemental analysis, FTIR, and XRD.
Investigations into the mechanical properties revealed the
enough tensile strength and pliability for ease of conven-
tional suturing. Moreover, DAC suture (USP 2-0) retained
63% of the original strength at 14 days and completely
absorbed in 42 days with no remarkable tissue reaction in
vivo. Particularly, it was demonstrated that DAC suture sig-
nificantly accelerated the wound healing process on a lin-
ear full thickness wound model, with higher wound
breaking strength and less wound healing time. Thus, the
data obtained above indicated the potential application of
this novel DAC suture for short- or middle-term wound
support, such as epithelial and connective tissue. However,
the mechanism of the promotion action of DAC suture for
tissue regeneration needs to be verified and more time is
needed.
FIGURE 10. The level of Hyp (A) and TGF-b1 (B) in skin wounds 7 days and 14 days after operation. *p 0.05 vs. normal skin. **p 0.01 vs. nor-
mal skin.
ORIGINAL RESEARCH REPORT
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2014 VOL 00B, ISSUE 00 9

11. ACKNOWLEDGMENT
The authors are grateful to Biotemed Co., Ltd. for providing the
chitin.
REFERENCES
1. Yang Y, Xia T, Zhi W, Wei L, Weng J, Zhang C, Li X. Promotion of
skin regeneration in diabetic rats by electrospun core-sheath
fibers loaded with basic fibroblast growth factor. Biomaterials
2011;32:4243–4254.
2. Witte MB, Thornton FJ, Kiyama T, Efron DT, Schulz GS,
Moldawer LL, Barbul A. Metalloproteinase inhibitors and wound
healing: A novel enhancer of wound strength. Surgery 1998;124:
464–470.
3. Muzzarelli RA. Chitins and chitosans for the repair of wounded
skin, nerve, cartilage and bone. Carbohydr Polym 2009;76:167–
182.
4. Pillai CKS, Sharma CP. Review paper: Absorbable polymeric sur-
gical sutures: Chemistry, production, properties, biodegradability,
and performance. J Biomater Appl 2010;25:291–366.
5. Devi KS, Vasudevan P. Absorbable surgical sutures. J Macromol
Sci Rev Macromol Chem Phys 1985;25:315–324.
6. Albertsson A-C, Varma IK. Recent developments in ring opening
polymerization of lactones for biomedical applications. Biomacro-
molecules 2003;4:1466–1486.
7. Gunatillake P, Mayadunne R, Adhikari R. Recent developments in
biodegradable synthetic polymers. Biotechnol Annu Rev 2006;12:
301–347.
8. Yang K-K, Wang X-L, Wang Y-Z. Poly (p-dioxanone) and its
copolymers. J Macromol Sci Part C Polym Rev 2002;42:373–398.
9. Chu C-C, Von Fraunhofer JA, Greisler HP. Wound Closere Bioma-
terials and Devices. Boca Raton, Florida: CRC Press; 1997.
10. Sharp KW, Ross CB, Tillman VN, Dunn JF. Common bile duct
healing: Do different absorbable sutures affect stricture formation
and tensile strength? Arch Surg 1989;124:408.
11. Rinaudo M. Main properties and current applications of some
polysaccharides as biomaterials. Polym Int 2008;57:397–430.
12. Ravi Kumar MN. A review of chitin and chitosan applications.
React Funct Polym 2000;46:1–27.
13. Muzzarelli RA, Guerrieri M, Goteri G, Muzzarelli C, Armeni T,
Ghiselli R, Cornelissen M. The biocompatibility of dibutyryl chitin
in the context of wound dressings. Biomaterials 2005;26:5844–
5854.
14. Desai A. Biomedical implantable materials sutures. Asian Text J
2005;14:54–56.
15. Singh DK, Ray AR. Biomedical applications of chitin, chitosan,
and their derivatives. J Macromol Sci Part C Polym Rev 2000;40:
69–83.
16. Nrsm N, Nooucrn J, Tokura S, Shiota H. Studies on chitin. I. Acet-
ylation of chitin. Polym J 1979;11:27–32.
17. Tokura S, Somorin O. Studies on chitin. V. Formylation, propiony-
lation, and butyrylation of chitin. Polym J 1981;13:24l–245.
18. Rhett JM, Ghatnekar GS, Palatinus JA, O’Quinn M, Yost MJ,
Gourdie RG. Novel therapies for scar reduction and regenerative
healing of skin wounds. Trends Biotechnol 2008;26:173–180.
19. Liu M, Shen Y, Ao P, Dai L, Liu Z, Zhou C. The improvement of
hemostatic and wound healing property of chitosan by halloysite
nanotubes. RSC Adv 2014;4:23540–23553.
20. Tokura S, Nishi N, Somorin O, Noguchi J. Studies on chitin. IV.
Preparation of acetylchitin fibers. Polym J 1980;12:695–700.
21. Draczynski Z. Synthesis and solubility properties of chitin acetate/
butyrate copolymers. J Appl Polym Sci 2011;122:175–182.
22. Huang Y, Song L, Huang T, Liu X, Xiao Y, Wu Y, Wu F, Gu Z.
Characterization and formation mechanism of nano-structured
hydroxyapatite coatings deposited by the liquid precursor plasma
spraying process. Biomed Mater 2010;5:054113.
23. Nishimura S-I, Nishi N, Tokura S. Adhesion behaviour of murine
lymphocytes on the surface of fibrous chitin and its derivatives.
Int J Biol Macromol 1985;7:100–104.
24. Patel KA, Thomas W. Sutures, ligatures and staples. Surgery
(Oxford) 2008;26:48–53.
25. Kong X, Han B, Li H, Liang Y, Shao K, Liu W. New biodegradable
small diameter artificial vascular prosthesis: A feasibility study.
J Biomed Mater Res Part A 2012;100:1494–1504.
26. Im JN, Kim JK, Kim H-K, In CH, Lee KY, Park WH. In vitro and in
vivo degradation behaviors of synthetic absorbable bicomponent
monofilament suture prepared with poly (p-dioxanone) and its
copolymer. Polym Degrad Stab 2007;92:667–674.
27. S€untar I, Akkol EK, Keles¸ H, Oktem A, Bas¸er KHC, Yes¸ilada E. A
novel wound healing ointment: A formulation of Hypericum per-
foratum oil and sage and oregano essential oils based on tradi-
tional Turkish knowledge. J Ethnopharmacol 2011;134:89–96.
28. S€untar I, Koca U, Keles H, Akkol EK. Wound healing activity of
Rubus sanctus Schreber (Rosaceae): Preclinical study in animal
models. Evid Based Complement Alternat Med 2011;2011:816156.
29. Garg S, Jana AK. Characterization and evaluation of acylated
starch with different acyl groups and degrees of substitution. Car-
bohydr Polym 2011;83:1623–1630.
30. Minagawa T, Okamura Y, Shigemasa Y, Minami S, Okamoto Y.
Effects of molecular weight and deacetylation degree of chitin/chi-
tosan on wound healing. Carbohydr Polym 2007;67:640–644.
31. Pillai C, Paul W, Sharma CP. Chitin and chitosan polymers: Chem-
istry, solubility and fiber formation. Prog Polym Sci 2009;34:641–
678.
32. Benhabiles M, Salah R, Lounici H, Drouiche N, Goosen M,
Mameri N. Antibacterial activity of chitin, chitosan and its oligom-
ers prepared from shrimp shell waste. Food Hydrocolloid 2012;29:
48–56.
33. Muzzarelli R, Muzzarelli C. Chitosan Chemistry: Relevance to
the Biomedical Sciences. Polysaccharides I. Berlin Heidelberg:
Springer; 2005. pp 151–209.
34. Kurita K. Chitin and chitosan: Functional biopolymers from
marine crustaceans. Marine Biotechnol 2006;8:203–226.
35. Tachibana M, Yaita A, Taniura H, Fukasawa K, Nagasue N,
Nakamura T. The use of chitin as a new absorbable suture mate-
rial—An experimental study. Jpn J Surg 1988;18:533–539.
36. M€akel€a P, Pohjonen T, T€orm€al€a P, Waris T, Ashammakhi N.
Strength retention properties of self-reinforced poly L-lactide (SR-
PLLA) sutures compared with polyglyconate (Maxon sup R/
sup) and polydioxanone (PDS) sutures. An in vitro study. Bio-
materials 2002;23:2587–2592.
37. Shigemasa Y, Saito K, Sashiwa H, Saimoto H. Enzymatic degrada-
tion of chitins and partially deacetylated chitins. Int J Biol Macro-
mol 1994;16:43–49.
38. Azab AK, Doviner V, Orkin B, Kleinstern J, Srebnik M, Nissan A,
Rubinstein A. Biocompatibility evaluation of crosslinked chitosan
hydrogels after subcutaneous and intraperitoneal implantation in
the rat. J Biomed Mater Res A 2007;83:414–422.
39. Pi~neros-Fernandez A, Salopek LS, Rodeheaver PF, Drake DB,
Edlich R, Rodeheaver GT. A revolutionary advance in skin closure
compared to current methods. J Long Term Eff Med Implants
2006;16:19–27.
40. Jayakumar R, Prabaharan M, Sudheesh Kumar P, Nair S, Tamura
H. Biomaterials based on chitin and chitosan in wound dressing
applications. Biotechnol Adv 2011;29:322–337.
41. Bakkers J, Semino CE, Stroband H, Kijne JW, Robbins PW,
Spaink HP. An important developmental role for oligosaccharides
during early embryogenesis of cyprinid fish. Proc Natl Acad Sci
1997;94:7982–7986.
42. Varki A. Does DG42 synthesize hyaluronan or chitin? A contro-
versy about oligosaccharides in vertebrate development. Proc
Natl Acad Sci USA 1996;93:4523.
10 SHAO ET AL. DIACETYL CHITIN SURGICAL SUTURE FOR WOUND HEALING