Evaluation in vitro of an experimental antibacterial silk suture

Transcription

1 National University of Science and Technolgy NuSpace Institutional Repository Textile Technology Textile Technology Publications 2013 Evaluation in vitro of an experimental antibacterial silk suture Xiao-Jie, Chen Xiao-jien, C. et al Evaluation in vitro of an experimental antibacterial silk suture, pp Downloaded from the National University of Science and Technology (NUST), Zimbabwe

2 NATIONAL UNIVERSITY OF SCIENCE AND TECHNOLOGY INSTITUTIONAL REPOSITORY NUSPACE Evaluation in vitro of an experimental antibacterial silk suture Citation Published Version Citable Link Terms of Use Xiao-jien, C. et al Evaluation in vitro of an experimental antibacterial silk suture, pp This article was downloaded from NUST Institutional repository, and is made available under the terms and conditions as set out in the Institutional Repository Policy. (Article begins on next page)

3 Evaluation in vitro of an experimental antibacterial silk suture Chen Xiao-jie, Lv Ai-feng, Wang Lu *, Pethile Sibanda, Hou Dan-dan, Lu Ming-kang, Wang Wen-zu Key Laboratory of Textile Science and Technology of Ministry of Education and College of Textile, Donghua University, Shanghai, China Wu Yong-gao HORCON, Nantong, Haimen, Jiangsu, China ABSTRACT Because of good clinical performance and low cost, silk braided surgical suture becomes one of excellent and widely used sutures. But as a kind of nature protein fiber, silk easily trend to microbial infection. Beyond that, braided structure may increase the risk of hide of bacteria. Hence, the project of this study is to develop a novel antibacterial silk braided suture. Silk braided sutures were treated with antimicrobial solution which added antimicrobial agent ZL into 5% PCL solution. Then sutures were tested for in vitro efficacy against S. aureus and E. coli by a zone of inhibition assay, and sustained efficacy assay, release of antibacterial property, SEM photographs against S. aureus. Sutures with antibacterial solution exhibited distinct zones of inhibition against S. aureus and E. coli. They also kept antimicrobial property against S. aureus for 7 days, which was almost equal with coated VICRYL* Plus suture. The highest release concentration of treated suture was 1.53µg/ml, which was lower than Cmax of ZL, proved the safety of antibacterial silk suture. SEM photographs gave both the change of the surface and different bacteria growth situations on the surface before and after treating. Key words: Surgical Site Infections (SSIs); Silk braided suture; Antimicrobial agent; Sustained antibacterial efficacy; Release of antibacterial property 1. INTRODUCTION In wound closure, using sutures is a very important process. Sutures are always in the structure of monofilament or multifilament which made by natural or synthetic biomedical textile materials such as collagen, silk, nylon and PET. Though during the past three decades, a series of degradable synthetic sutures have been used in surgery successively, silk braided suture is still widely used in ocular, neural and cardiovascular as well as a variety of other tissues [1] because of its natural nature, good tying quality and moisture absorption ability. A braid is a textile structure which intersects yarns by a braiding machine which gives sutures good tenacity and knot strength [2]. But as one kind of nature protein fiber, silk easily trend to microbial infection and prolong the persistence of bacteria on wounds. Beyond that, braided structure may easily harbor bacteria into gaps and between filaments [3]. Both of these will increase the risk of surgical site infections (SSIs) which is a common infection after surgery. SSIs not only impact operation quality, increase treatment cost, but also prolong pain and hospital stays of patients. According to a report of 2009, a single SSI increases the average hospital stay of 9.7 days and the cost by $27,288 [4]. In the Guidelines for the prevention of SSIs which 1

4 published by the Centers for Disease Control (CDC), the importance role of suture in SSIs has been emphasized [5-6]. As an implant, suture is the medium for the transport of bacterial to the surgical incision. If sutures own antibacterial nature, they may prevent the adhesion and growth of bacteria on the sutures. Some researches and patents have reported that coating sutures with antibacterial agents provides an effective strategy for reducing SSIs [7]. 2. EXPERIMENTAL 2.1 Materials and Methods Suture material Silk braided sutures all in size 2-0 were supplied by HORCON, which is a Jiangsu company. Coated VICRYL* Plus antibacterial suture (Coated polyglactin 910 suture with triclosan) (ETHICON, Somerville, NJ) in size 2-0 served as the reference Antibacterial solution Polycaprolactone (PCL) particles were added into acetic acid to make a 5% concentration solution, wherein the molecular weight of PCL was ZL was one of the widely used synthetic antimicrobial agents in hospital. 10 ml PCL solution was extracted and 0.025g ZL powder was added into it, so that the concentration of ZL in solution was 2500µg/ml. The powder was distributed uniform in PCL solution after using magnetic stirring apparatus to mix 3 hours Antibacterial treatment The coating of silk braided sutures was similar with the starching of yarns, the detail procedure included: (1) the prepared antibacterial solution was poured into dipping tank 1, which needed to immerse dipping roller 3, heating machine 2 could be used at 30 ~ 40 C to keep the activity of antibacterial solution; (2) the suture was passed across the dipping roller 3, then went through the space between the rubber roller 4 and the stainless steel roller 5 below; (3) after pressed by the rubber roller 4 and the stainless steel roller 5, the suture came to guide roller 6 and 7 as a long distance for drying by room temperature; (4) the suture finally rounded on the rattler 8, the rattler 8 kept rotated 40rpm. Figure 1 Structural scheme of coating equipment 2

5 1- dipping tank; 2- heating machine; 3- dipping roller; 4- rubber roller; 5- stainless steel roller; 6,7- guide roller; 8- rattler 2.2 Evaluation of antibacterial suture Zone of inhibition assay Sutures before and after treatment both were evaluated by zone of inhibition assay [8-9]. In this paper, gram-positive Staphylococcus aureus ATCC and gram-negative Escherichia coli ATCC were selected as tested bacterial. Organisms were culture in tryptic soy broth (TSB) at 37 C and shook in shake bed for 14h to form challenge inocula which was then measured by ultraviolet spectrophotometer (TU-1901) to guarantee each 1ml inocula contains approximately 10 8 colony-forming units (CFU) of S. aureus or E. coli. Melted tryptic soy agar (TSA) at 60 C was poured into sterile dishes and allowed to solidify. Then sterile soft cotton buds dipped with challenge inocula coated TSA dishes wellproportionately. The cut suture pieces (5cm) were placed on the middle of the surface of each dish. Then the dishes were cultivated for 18h in a 37 C incubator. After incubation, the zones of inhibition of each dish were measured with an electro-vernier caliper. According to ISO 20645: 2004 Textile fabrics- determination of antibacterial activity- agar diffusion plate test [10], inhibition zones were calculated by the following formula, data was means of double samples and each sample was measured in three different places. Where H the inhibition zone in mm; D the total diameter of specimen and inhibition zone in mm; d the total diameter of specimen in mm Sustained efficacy assay If suture samples proved to be antimicrobial after zone of inhibition assay, they were transferred daily onto new petri dishes growing a similar number of bacteria. The assay was terminated when the zone of inhibition was disappeared [11] Release of antibacterial property A standard curve of absorbance - concentration can be achieved by using a series of solution with known concentration and measured absorbance at each known concentration point. Then depending on this standard curve, the concentration of the sample can be detected by measuring the absorbance of the sample [12]. The detail procedure included: (1) the maximum absorption wave length was 289 nm by using TU-1901 to test the spectrum of the antimicrobial solution; (2) a standard curve of absorbance - concentration was achieved by using a series of PBS buffer with different concentration and measured absorbance at each concentration point, the relationship between absorbance and concentration was: A=0.0512C (A-absorbance, C- concentration); (3) the suture with antibacterial treatment was placed in 20ml PBS buffer whose ph was 7.2, then placed in a 37 C incubator, and replaced with a new buffer every 24h, the absorbance of buffer after removing suture was tested. 3

6 Figure 2 Spectrum of the antibacterial agent Figure 3 The standard curve of absorbance concentration SEM Untreated and treated sutures were placed in petri dishes equipped with 20ml TSB medium respectively, inoculated 0.1ml S. aureus whose concentration was 10 5 cfu/ml, and then the dishes were cultivated at 37 C for 18h in the incubator; after washed by sterilized PBS buffer whose ph was 7.2, 10% formaldehyde was added dropwise and bacteria were fixed at 4 C for 30min in the fridge. 70% and 99% alcohol were added dropwise on the suture in order, then sutures were dried in super clean bench at room temperature for 1h. The dried sutures were observed under a field emission scanning electron microscope (SEM, HITACHI/SU8010). 3. RESULTS AND DISCUSSION 3.1 Zone of inhibition assay The results of zone of inhibition assays of untreated and treated sutures against S. aureus and E. coli are listed in Table 1. Both the treated sutures exhibited distinct zones of inhibition against S. aureus and E. coli, and the inhibition zones were greater than 1mm. In contrast, the untreated sutures had no zone. Fig.2 shows the representative pictures. According to ISO 20645: 2004 [10], this indicates treated sutures show good effect against S. aureus and E. coli. Table 1 Zone of inhibition assay Suture Inhibition zone (mm) S. aureus E. coli Untreated 0 0 Treated 7.27± ±0.60 4

7 a. b a. b 1) S. aureus 2) E. coli Figure 4 Zone of inhibition assay a. untreated, b. treated 3.2 Sustained efficacy assay Figure 5 Sustained efficacy assay of coated VICRYL* Plus suture and coated ZL suture Fig. 5 shows the results of sustained efficacy assays of coated VICRYL* Plus suture and coated ZL suture against S. aureus. The zones of inhibition of the antibacterial silk braided suture lasted for 7 days, which consisted with the skin wound healing (7 ~10 days). Inhibition zones fitted well as a logarithmic curve which decreased gradually with time, controlled the release of ZL ideally. Compared with successful commercial production, both the days of sustained efficacy and the variation trend of release are equal well. 3.3 Release of antibacterial property For ZL, the single maximum dose always is 200mg, then the peak concentration (Cmax) is 2.92±0.54µg/ml 1 ~2h later after dose. Cmax means the highest concentration of plasma after the absorption of drug in the body. If Cmax is higher than the safe range, it may cause toxic reaction. In other words, Cmax is an important indicator to measure the absorption and safety of drug in the body [13]. As shown in Fig. 6, the release concentration decreased from the 1 st to the 5 th days, and on the 6 th day it became 0. This result was in keeping with sustained efficacy assay of coated ZL suture. The highest release concentration was 1.53µg/ml which was lower than the Cmax 5

8 of ZL. So that we can say that during the use of this antibacterial silk braided suture, it would not cause drug toxicity. Figure 6 Sustained efficacy assay of antibacterial silk braided suture 3.4 SEM a b c d Figure 7 SEM photographs shows the surface changes: a. untreated( 150) b. treated( 150); and the growth of bacterial on the surface of sutures: c. untreated( 2000)d. treated( 2000) Fig. 7 gave a typical SEM microstructure of surface changes and growth of bacterial of the antibacterial silk braided suture. From Fig. 7 a, it obviously showed that there were so many hairiness on the surface of untreated suture because of the uneven tension during the 6

9 braided process of multi-strand silk raw. From Fig. 7 b, The surface of the suture became uniform and smooth by the relatively high press of rollers and the coating of antibacterial solution. There was also less space between monofilaments, so that it decreased the risk of enter and adhesion of bacterial. Clusters of cocci were seen on the surface of untreated suture clearly in Fig. 7 c, while almost none bacteria could be seen on the surface of treated suture in Fig. 7 d. Thus it proved that treated silk braided suture owned antibacterial property which could resist the adhesion and growth of bacteria effectively. 4. CONCLUSIONS Silk braided sutures treated with antibacterial solution which adds ZL into 5% PCL solution provides in vitro excellent antimicrobial efficacy against S. aureus and E. coli compared with untreated sutures. These antibacterial sutures demonstrates a similar sustained antibacterial efficacy against S. aureus with coated VICRYL* Plus antibacterial suture. The release of ZL is also in the safe range. Our finding shows the potential of a new kind of antibacterial silk braided suture. REFERENCES 1. Gregory h. Altman, Frank Diaz, Caroline Jakuba, et al, Silk-based Biomaterials, Biomaterials, 24, 2003, S. Viju, G. Thillagavathi, Fabrication and Characterization of Silk Braided Sutures, Fibers and Polymers, 13, 2012, Michelle J. Henry-Stanley, Donavon J. Hess, Aaron M. T. Barnes, et al, Bacterial Contamination of Surgical Suture Resembles a Biofilm: Surgical Infections, 11, 2010, De Lissovoy G, Fraeman K, Hutchins V, et al, Surgical Site Infection: Incidence and Impact on Hospital Utilization and Treatment Costs, Am J Infect Control, 37, 2009, C. D. Owens, K. Stoessel, Surgical Site Infections: Epidemiology, Microbiology and Prevention, Journal of Hospital Infection, 70(52), 2008, FAN Chao-gang, The New Interpretation of The Guideline for Prevention of Surgical Site Infection, Journal of Chinese Gastrointestinal Surgery, 15(6), 2012, David Leaper, Andrew J McBain, Axel Kramer, et al, Healthcare Associated Infection: Novel Strategies and Antimicrobial Implants to Prevent Surgical Site Infection, Ann R Coll Surg Engl, 92, 2010, Stephen Rothenburger, Daniel Spangler, Spangler Bhende, and Daniel Burkley, In Vitro Antimicrobial Evaluation of Coated VICRYL* Plus Antimicrobial suture (Coated Polyglactin 910 with Triclosan) using Zone of Inhibition Assays, Surg Infect, 3(11), 2002, Ming Xin-tian, Stephen Rothenburger, Yang Da-chuan, In Vitro Antibacterial Efficacy of MONOCRYL Plus Antibacterial Suture (Poliglecaprone 25 with Triclosan), Surg Infect, 8, 2007, International Organization for Standardization, Textile Fabrics- Determination of Antibacterial Activity- Agar Diffusion Plate Test, ISO 20645:

10 11. MING Xin-tian, Stephen Rotehenburger, Michele M. Nichols, In Vivo and In Vitro Antibacterial Efficacy of PDS* Plus (Polidioxanone with Triclosan) Suture, Surg Infect, 9(4), 2008, Gupta, B., R. Jain, H. Singh, Preparation of Antimicrobial Sutures by Preirradiation Grafting onto Polypropylene Monofilament, Polymers for Advanced Technologies, 19(12), 2008, QIAN Zhi-yu, Pharmacology, Chinese Medical Science and Technology Press, 2009,