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Fabricationandfeasibilitystudyofanabsorbablediacetylchitinsurgicalsutureforwoundhealing

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JinningGao

QingdaoUniversity

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WeizhiLiu

OceanUniversityofChina

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Fabrication and feasibility study of an absorbable diacetyl chitinsurgical 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

1Center of Laboratory Medicine, Qilu Hospital of Shandong University (Qingdao), Qingdao 266035, China2Laboratory of Biochemistry and Biomaterials, College of Marine Life Science, Ocean University of China, Qingdao 266003, China3Institute for Translational Medicine, The Medical College, Qingdao University, Qingdao 266021, China4Central 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 anabsorbable 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. Woundhealing 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 invarious functions as inhibitors or attractants of inflamma-tory cells and keratinocyte fibroblast, in acceleration ofmatrix turnover and collagen.2 In addition, TGF-b1 caninduce myofibroblast differentiation and collagen deposition,marked by hydroxyproline (Hyp) content in wound tissues.3

Clinically, suture is the most effective method for woundclosure with a huge market exceeding $ 1.3 billion annu-ally.4 Thus, a suitable suturing technique is essential forwound healing and prevention of excessive scar. The devel-opment of ideal absorbable suture for mechanical woundclosure with full recovery of its biological functions has longbeen a major goal in surgery and trauma. In light of thispivotal 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 othercopolymers. Currently, absorbable polymeric sutures havegot 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 sutureshave advantages on reproducible degradability in biologicalenvironment during the wound healing process.9 However,most polymeric sutures are found to cause pronouncedinflammatory response during the degradation process, add-ing severe side-effects for wound healing and much pain forpatients.10 Therefore, to solve this issue and discover newabsorbable and biocompatible sutures for clinical usage arenecessary.

Chitin, a natural polysaccharide with antimicrobial prop-erty, is one of the most promising biomaterials for effec-tively accelerating wound healing and providing protectionfrom wound infection. In particular, chitins can promotefibroblast proliferation and macrophage migration, andaccelerate vascularization and granulation during woundhealing process.3 Moreover, the non-toxic and biodegradable

*Present address: Room 220, Science Building, 5# Yushan Rd., Qingdao, ChinaCorrespondence to: B. Han; e-mail: baoqinh@ouc.edu.cn

VC 2015 WILEY PERIODICALS, INC. 1

properties of chitin make it a promising biomaterial forabsorbable suture.11 Actually, due to the excellent fiber-forming ability12 and biological activities of chitin, a fewcommercial chitin-based dressings have been developed,such as BeschitinVR (Unitika, Japan), Chitipack SVR (Eisai Co,Japan), and TegasorbVR (3M, USA).13 Previous studies haveindicated that chitin has comparable properties to collagenand lactide fibers,14 and chitin suture can be absorbed inabout four months in rat muscles.15 However, the current chi-tin suture also has limitations in mechanical strength anddegradation time, and cannot meet the requirements of someparticular surgery. To improve the quality for chitin, variousacyl derivatives of chitin have been obtained by reaction ofacetylation, 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 byMuzzarelli et al.13 and J. Matthew et al.18 have been tested inrats and obtained a good effect, demonstrating that DBC is agood candidate for further evaluation as an effective woundhealing agent. Recently, Liu et al. have demonstrated thatchitosan-halloysite nanotubes composite sponges possess ahigher mechanical strength than pure chitosan sponges.19

Diacetyl chitin (DAC) is another multifunctional acylate ofchitin. Previous studies have already revealed that acetylatedchitin fibers, with greater tenacity and elongation than chitinfibers,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 novelmonocomponent multifilament DAC suture was developed inour lab. Our study also aimed at fundamental insights on thefeasibility of DAC suture as absorbable biomaterials forwound healing. Thus, we investigated the mechanical proper-ties and degradation behavior of DAC suture, and performedfunctional study on a linear incisional wound model.

MATERIALS AND METHODS

Materials and reagentsChitin with viscosity average molecular weight of 269 kDaand intrinsic viscosity 10.72 dL/g (dissolved indimethylacetamide1 5% LiCI solutions, 25�C) was suppliedby Biotemed Co., Ltd. VicrylVR Plus suture (USP 2-0) wassupplied by Ethicon, USA. Rat TGF-b1 ELISA Kit (96T) andRat Hyp ELISA Kit (96T) were from Colorfulgene Biotech-nology Co. (Wuhan, china). Adult male Sprague-Dawley rats(2006 10 g body weight) were obtained from QingdaoInstitute for Drug Control. All chemicals were analyticalgrade and received from Sinopharm Chemical Reagent Co.,Ltd (China), unless otherwise indicated.

Preparation of DACDAC was prepared as described previously in heterogeneousreaction with minor modifications, using acetic anhydride asacylating agent and perchloric acid as a catalyst.21 The briefprocess was as follows: Perchloric acid (4.0 mL) was addeddropwise in acetic anhydride (100.0 mL) at 0�C and stabilizedfor 30 min. Then chitin (10.0 g) was dispersed into the acy-

lated conditions and stirred at 0�C for 24 h. The mixture wasthen neutralized by NaOH powder and the final DAC productwas obtained after filtrated, purified (95% alcohol), and dried.

Characterization of DACChemical structure of DAC product was characterized byelemental analysis, Fourier transform infrared (FTIR), andX-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 aNicolet 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 MPDDY1291 (Koninklijke Philips Electronics NV, the Nether-lands), using conventional Bragge-Brentano geometry in q-2q configuration with Cu Ka radiation at 40 kV and 35mA.22 The XRD patterns were collected over a range from5� to 60� at a scanning rate of 2�/min and data analysiswas carried out on Jade 5.0 (Materials Data, Inc., USA).

Preparation of DAC sutureUsing 12% solution of DAC in formic acid, DAC fibers wereobtained by wet spun into coagulation baths composed ofacetone and ethanol.23 With application of modern weavingtechnology, a novel monocomponent multifilament DACsuture was obtained.

Mechanical properties of DAC sutureThere must be a proper match between the suture strengthand the tissue strength.24 To investigate the breakingstrength, breaking elongation, knot strength, knot-pullstrength, and strength with needle of DAC suture (USP 2-0),Universal Testing Instruments AGS-X (Shimadzu, Japan) wasperformed at a testing speed of 5.0 mm/s. VicrylVR Plussuture (USP 2-0), a multifilament braided PLGA sutureimpregnated with triclosan to provide antimicrobial protec-tion, was employed as control. The parameters above weremeasured ten times and the suture materials 20 cm inlength were incubated in phosphate buffer saline (PBS, pH7.2) for 30 min at 25�C before testing. For the mechanicalproperties assay, breaking strength and breaking elongationwere the tensile and elongation at which suture failureoccurred separately; strength with needle was the tensile atwhich suture with needle broke; knot strength was theamount of tensile necessary to cause a knot to slip (themiddle of the suture were tied into a knot separately beforetesting); while knot-pull strength was the breaking strengthof knotted suture in the middle. Moreover, the swelling(sutures with 25 cm in length were incubated in PBS 7.2 at25�C for 24 h), pliability and memory characteristics of DACsuture were also evaluated.

In vivo breaking strength retentionThe animal experiments in this study were carried out incompliance with the National Institute of Health Guide forthe Care and Use of Laboratory Animals. Thirty-six adultmale Sprague-Dawley rats were housed singly in standard

2 SHAO ET AL. DIACETYL CHITIN SURGICAL SUTURE FOR WOUND HEALING

cages and kept under controlled temperature and humiditywith free access to food and water.25 The rats were anesthe-tized with intraperitoneal administration of pentobarbitalsodium (3% in saline solution) at a dose of 1 mL/kg. Thedorsal side of the rats was shaved and disinfected with 75%alcohol for surgery. Sterile multifilament DAC suture (USP2-0), 10 cm in length, was implanted by sewing with a longneedle (8 cm) inserting through the left dorsal subcutis ofeach rat.26 Treated with VicrylVR Plus suture (USP 2-0) inthe 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 theimplanted sutures was determined on Universal TestingInstruments AGS-X (Shimadzu, Japan).

In vivo biocompatibility and biodegradabilityThirty adult male Sprague-Dawley rats were housed andanesthetized as described above before operation. The glu-teal side of the rats was shaved and disinfected with iodinesolution before surgery. Sterile multifilament DAC suture(USP 2-0), each �3 cm long, was implanted into the leftside of the gluteal muscle. To avoid secession of the sutureconstruction, both ends of the DAC suture were tied into aknot 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 afteroperation, 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 andsuturingAdult male Sprague-Dawley rats were housed and anesthe-tized as described above before operation. The back hair ofthe rats were shaved and disinfected with iodine solution.Three centimeters long, a linear paravertebral incision wasmade with a sterile surgical blade through the full thicknessof the skin.27,28 The wound closure was performed withsterile multifilament DAC sutures (USP 2-0) of 0.5 cm apart,and treated with VicrylVR Plus suture (USP 2-0) wasemployed as control. The rats were housed individually forinvestigation of wound healing process at each time point.

Macroscopic evaluation and wound breaking strengthThe wound area of 12 rats was macroscopically evaluatedfor 21 days continuously after treatment. The followingparameters were determined: wound healing rate, woundbreaking strength, suture retention, presence of ulcers, andscars. Rats with complete skin reconstruction and wound

healing at 14 days was considered to be successful case forwound healing rate. Wound breaking strength was the ten-sile strength of a healing wound at which separation of thewound edges occurs. Thus, it was determined 7 days and14 days after operation using a tensiometer (Meixun,Wuhan, China). Skin ulcers and scars were examined andscored as absent and present.

Hyp and TGF-b1Myofibroblast differentiation and collagen deposition wereassayed with Hyp, which was mainly secreted by the fibro-blasts and gave rise to collagen secretion, using a Rat HypELISA Kit (96 T). In addition, another relevant cytokine TGF-b1, which was critical to wound contraction and marked bysynthesis of collagen and fibronectin, was determined inwound tissue with a Rat TGF-b1 ELISA Kit (96 T). At weeks 1and 2 after operation on the linear incisional wound model,wound skin tissue (1.0 g) of each linear incision was collectedwith 5.0 mL PBS (pH 7.4), completely homogenized for 30 swith a Tissue Tearor (IKA, Germany), and centrifuged withSigma 3K 30 (USA) at 3000 rpm for 20 min. The supernatantwas collected to measure the levels of Hyp and TGF-b1 byELISA, using commercially available assay systems accordingto the manufacturer’s instructions. Rats treated with VicrylVR

Plus suture (USP 2-0) were employed as control.

Histological examinationAt weeks 1, 2, and 3 after operation on the linear incisionalwound model, seven rats were sacrificed separately and thetrauma samples were cut into 5 lm for histopathologicalexamination. HE-staining was carried out for investigationof inflammatory reaction, collagen arrangement, sutureretention, re-epithelialization, and re-growth of skin appen-dages in wounding area.

Statistics analysisStatistical analysis of data was performed by one-wayANOVA on SPSS (version 20.0).

RESULTS

Characterization of DAC productFigure 1 displays the synthetic route of DAC from chitin. Thebasic 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

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shown above, the DAC product was water-insoluble and theintrinsic viscosity in formic acid at 25�C was 2.06 dL/g. Theresults of CNH elemental analysis was also given, suggesting thedegree 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 almostdisappeared, indicating they were esterified and the DS wasapproximately to 2. New strong characteristic peaks at1747 cm21 and 1232 cm21, which attributed to the estersof fatty acids, revealed the acetylation of chitin at 3 and 6positions. Furthermore, the intensification of bands at790 cm21 and 740 cm21 corresponding to methyl groupsconfirmed the chemical structures of the DAC product.21

XRD (Figure 3), another independent approach, wasemployed to further characterize DAC. The spectroscopyshowed the characteristic structure for the pure chitin pow-der at peak 20.10� . The spectrum of DAC resembled that ofchitin. However, two stronger peaks of DAC spectrum at9.36� and 19.32� attributed to acetylation,29 revealed thechemical structures of the DAC product.

Mechanical characterization of DAC sutureThe monocomponent DAC suture was manufactured bymodern multifilament braiding technique with a diameter

range 0.30–0.35 mm, with no coating materials or dyes, asshown in Figure 4(A). There are several characteristicswhich are essential for all sutures, such as physical andmechanical properties, handling characteristics, biologicaland degradation behaviors. In light of the significance ofmechanical properties to a suture, relevant mechanicalparameters were investigated and the results are given inTable II. Figure 4(B) shows the load–displacement profile ofDAC suture (USP 2-0) and the control (VicrylVR Plus, USP 2-0) at testing speed of 5 mm/s. The results indicated thatVicrylVR Plus suture had advantage in mechanical strengthover DAC suture, about 20% higher. Both the two suturescould 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 forknotting.

Breaking strength retention in vivoThe basic parameters of the DAC suture and the controlsuture are given in Table III. The duly degradability insidea biological environment is pivotal for an ideal absorbablesuture. Here, the breaking strength retention rate of DACsuture was further assessed in subcutis of rats. As shownin Figure 5, during a 6-week period observation for DACsuture group, a strength loss of 23.1% was detected dur-ing the initial 7 days, which had a significant differencewith control (32.6% at 7 days). After one week of implan-tation, a relative slow strength loss occurred and DACsuture remained approximately 63% of the originalstrength at 14 days. DAC suture was completely absorbedat 42 days with excellent biocompatibility and no brokenor liquefaction. On the other hand, strength of the controlshowed 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 liquefiedsignificantly at 21 days observation time, leading toobvious tissue reactions and entirely loss of suturestrength. All of these results indicated that DAC suture hadhigher breaking strength retention rate than control invivo. Therefore, it is believed that in the critical post-operative period (traditionally within 3 weeks), DAC suturewas able to maintain its physical and biochemical functionsfor longer time.

Biocompatibility and biodegradability in muscleTo investigate the degradation behavior and tissue reactionof the monocomponent multifilament DAC suture, micro-scopic observation was performed. DAC suture (USP 2-0)and the control suture were completely absorbed at 35–42days and 42–49 days, respectively (Figure 6). At 7 dayspost-implantation, both the two sutures were surroundedby a large amount of inflammatory sells [Figure 6(A,F)]. Athick collagen-like capsule with macrophages and fibro-blasts was observed and the suture kept its original shapein 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

At 14 days observation, only a small amount of inflamma-tory cells at the contact region were observed surroundingboth the suture strands. In the DAC group [Figure 6(B)],the suture kept its original shape, yet limited fissures wereobserved. And the diameter of the DAC fiber reduced tosome extent, indicating a gradual degradation process hasoccurred. In contrast, the collagen-like capsule stillremained in control group, and the arrangement of themultifilament fibers inside was broken and some fiberswere partly absorbed [Figure 6(G)]. As the degradationproceeded, no remarkable tissue reaction was observed forDAC suture at 21 days and the diameter of DAC fibers fur-ther reduced [Figure 6(C)]. In the control group, the areaof visible fibrous region decreased to approximately half ofthe 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 obviousinflammatory reactions or other tissue reactions wereobserved [Figure 6(E,J)].

Macroscopic observation and wound breaking strengthA linear full thickness wound model was surgically createdand the feasibility of the DAC suture for wound closure andfurther healing was evaluated by comparison with VicrylVR

Plus. The operation process of suturing is shown in Figure7(A). DAC suture could efficiently close the wound with nosuture breaking or knot slipping. During a 3-week periodobservation, the wound healing process after treated withDAC suture [Figure 7(B)] and the control suture [Figure7(C)] was separately recorded daily. It should be stated thatDAC suture broke approximately at 6 days and disappearedmacroscopically at 11 days. On the other hand, tensilestrength 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 situationwas 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 thecomplete skin regeneration was accomplished at about 20days. 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 breakingstrength of the DAC group and the control group at 7 daysand 14 days (Figure 8) were significantly higher than thatof 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

DACVicrylVR Plus

(Edicon, USA)

U.S.P. size 2-0 2-0Breaking

strength (MPa)189.95 237.65

Breakingelongation (%)

16.77 25.13

Knot-pullstrength (MPa)

144.37 194.06

strength withneedle (MPa)

135.88 169.57

Knot strength(MPa)

97.52 104.88

Swelling (%) 134.65 18.63Pliability 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

DACVicrylVR Plus

(Edicon, USA)

Composition Diacetyl chitin (DAC) Poly (LL to GL) (PLGA)U.S.P. size 2-0 2-0Diameter 0.30–0.35 mm 0.30–0.35 mmLength 30 mm 90 mmFormation Multifilament braided Multifilament braidedCoatings Not coated CoatedDyed Not dyed Dyed (purple)

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sutures could enhance wound healing acceleration. In par-ticular, there was also significant difference between thetwo sutures (p< 0.05) that DAC group showed higherwound breaking strength.

To quantify the performance of the sutures for full thick-ness wound, wound healing rate, skin ulcers, and scarswere investigated by macroscopic evaluation at 14 dayspost-operation. Skin ulcers were observed in DAC group (2/12) and control (3/12), and hypertrophic or keloid scarswere evaluated in DAC group (1/12) and control group (2/12). Rats with complete skin reconstruction and woundhealing at 14 days was considered to be successful case forwound 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 indicatedthat this monocomponent multifilament DAC suture rapidlyrestored the structural and functional properties ofwounded skin and had advantage over the control.

Inflammation and collagen arrangementHistological examinations were performed at weeks 1, 2,and 3 post-procedures to investigate the healing processand the structure, in terms of inflammatory reaction, cell

proliferation, collagen arrangement, suture retention, andre-epithelialization. The results showed that the suturematerial and the wound site were surrounded by a largeamount of macrophages and neutrophile granulocytes inboth the two groups at 7 days [Figure 9(A,D)]. As the skinreconstruction proceeded, DAC fiber were hydrolyzed gradu-ally by glycosidases and became oligomers and monomersand the fiber fragment could still be observed around thewound site at 14 days [Figure 9(B)]. Beside this, the inflam-mation reaction in regenerated tissue almost disappearedand numerous types of cell differentiation were observedpossibly induced by the products of DAC, leading to acceler-ation of the wound healing process. While for control groupat 14 days [Figure 9(E)], there were still a massive quantityof inflammation cells and fibroblasts surrounding thewound site, and extensive suture strands could also beobserved. At 21 days histological examination, there was noremarkable tissue reaction or fiber fragment observed inboth the DAC group [Figure 9(C)] and the control group[Figure 9(F)]. The fibroblasts differentiation and collagendeposition 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 groupat 21 days [Figure 9(C)], more activated fibroblasts in thewound site were observed than the control group. In partic-ular, the collagen fibers in the wound site tended to runparallel to the epidermal layer as the normal skin in DACgroup, 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 phenomenonmight explain the higher wound breaking strength for DACgroup.

Re-epithelialization and re-growth of skin appendagesGenerally, keratinocytes at the wound edge migrate over theepidermis to differentiate the new outer layer in a processtermed 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

with the epidermic cells fully differentiated and basal cellsclosely arranged. Also, the superficial crust disappeared anda flat horny layer could be observed. However, in the controlgroup 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 thanthat in control group. At week 2 after treatment, a smallamount of sebaceous gland cells and hair follicle cells wereobserved 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 theDAC suture stimulated the re-epithelialization and thegrowth of skin appendages.

Hyp and TGF-b1 determinationTo investigate myofibroblast differentiation and collagendeposition in the wound site, a Rat Hyp ELISA Kit (96 T)was employed. It was demonstrated that the contents ofHyp 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.]

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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 towound contraction and marked by synthesis of collagen andfibronectin, was also studied to quantify the acceleration ofDAC suture to wound healing [Figure 10(B)]. At week 1after operation, the level of TGF-b1 in DAC group was sig-nificantly higher than control (p< 0.05). The data obtainedat week 3 indicated there was no significant differencebetween the two groups (p> 0.05). According to all theseresults, it was out of doubt that DAC suture enhanced colla-gen deposition and relevant cytokines secretion, leading toacceleration of the wound healing process.

DISCUSSION

To meet different surgical needs, the development of newabsorbable and biocompatible sutures with proper mechani-cal strength, modest inflammatory response, and significant

wound healing promotion is necessary and urgent. In lightof the excellent fiber-forming ability,31 antimicrobial proper-ties,32 wound healing promotion, and other outstanding bio-chemical activities, chitin and its derivatives have beeninfluential in the development of interest in wound healingdevices. Actually, one of the most promising biomaterialsthat can accelerate wound healing process is chitin, which isa natural polysaccharide containing N-acetyl glucosaminemoiety.33 The latter is also found in certain human glyco-proteins in connective tissues like keratin sulfate. It hasbeen reported that several chitin sutures have remarkableproperties over other fibers for biomedical applications.34

An ideal suture should have proper mechanical charac-teristics, especially enough tensile strength, pliability forease of handling, and knot security. In this study, we foundthat DAC suture met the strength requirement for conven-tional suturing. Investigation into the breaking strengthretention of the DAC suture in vivo revealed that DAC sutureremained approximately 63% of the original strength at 14days, which was higher than chitin suture reported (45% at14 days).35 Therefore, it is believed that in the critical post-operative period (traditionally within 3 weeks), DAC sutureis able to maintain its physical and biochemical functions,showing the potential of replacing traditional short- ormiddle-term absorbable sutures. It should be noted thatsuitable percentage elongation is necessary for an idealsuture. Since high percentage elongation can give an indica-tion of the potential gap formation that may occur under aparticular load, while low percentage elongation may causedifficulty for knotting.36 The result indicated that the break-ing elongation of DAC suture was approximately 17%, whichwas flexible to tie in a stable knot. Previous studies haveshowed that DAC fiber has greater tenacity and elongationthan 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

groups are totally esterified.23 However, the multifilamentstructure 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, whichwas similar to VicrylVR Plus suture and 80 days faster thanchitin suture reported previously.15 The fast absorptionbehavior of DAC suture may be attributed to the acetylationand/or the fabrication of the suture. Particularly, the mono-component multifilament DAC suture has relatively largerspecific 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 byglycosidases such as chitinase and lysozyme,37 leading tothe fast absorption behavior. In addition, DAC sutureretained approximately 63% of the original strength at 14days. All these results revealed the potential of DAC sutureused in short- or middle-term wound support. Certainly, notype of suture is suitable for every situation, and varioussutures are being designed all the time. A previous studyhas 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 prolongedsupport, such as bone. Another synthetic absorbable bicom-ponent monofilament suture (MonoFlex), composed ofpoly(p-dioxanone) and its copolymer, has been prepared forextended wound support.26

In this study, it should be noted that VicrylVR Plus sutureshowed pronounced inflammatory reaction compared withDAC suture, which elicited unremarkable tissue response.Previous studies have also reported that traditional absorb-able polymeric sutures may cause unavoidable inflammatoryreaction.38,39 DBC, another acylated derivative, also showshigh biocompatibility in the form of films and non-wovensin previous studies.13,40

Considering the mechanical properties and biocompati-bility, this monocomponent multifilament DAC suture wasfound to be potential applied as an ideal absorbable suture.Particularly, present evidence pointed out that DAC suturecould significantly accelerate the wound healing processwith 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 tothe epidermal layer as the normal skin in DAC group. As aresult, DAC suture showed higher wound breaking strength.The mechanism of the promotion of skin regeneration byDAC suture may be attributed to the outstanding biochemi-cal significance of chitins. As the acetylated chitin fibers aregradually degraded by glycosidases such as chitinase andlysozyme,37 they turn into oligomers and monomers in thewound area. It has been reported that chitins and its biode-gradable substances could accelerate fibroblast proliferationand macrophage migration, and they could also promotegranulation, cytokine production, and vascularization, lead-ing to better organization and smooth wound healing.3 Theresults in contents of Hyp and TGF-b1 in the wound sitewere consistent with previous studies. Specifically, com-pared with the control group, DAC suture tended to enhancemuch smoother re-epithelialization. Recent evidence pointsto 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 hyaluronandevotes to the correct tissue reconstitution of the woundsin the fetus heal.

CONCLUSIONS

A novel monocomponent multifilament absorbable suture,DAC suture, was fabricated and characterized by multipleapproaches including elemental analysis, FTIR, and XRD.Investigations into the mechanical properties revealed theenough 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 completelyabsorbed in 42 days with no remarkable tissue reaction invivo. Particularly, it was demonstrated that DAC suture sig-nificantly accelerated the wound healing process on a lin-ear full thickness wound model, with higher woundbreaking strength and less wound healing time. Thus, thedata obtained above indicated the potential application ofthis novel DAC suture for short- or middle-term woundsupport, such as epithelial and connective tissue. However,the mechanism of the promotion action of DAC suture fortissue regeneration needs to be verified and more time isneeded.

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

ACKNOWLEDGMENT

The authors are grateful to Biotemed Co., Ltd. for providing thechitin.

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