Document Type : Original Research Article
1 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.
2 Department of Pathobiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran
3 Department of Applied Chemistry, Faculty of Chemistry, University of Kashan, Kashan, Iran.
4 Department of Organic Chemistry, Faculty of Chemistry, University of Kashan, Kashan, Iran
Wounds are affected by bacteria and it can delay wound healing through a complex process. External materials such as surgical suture and ex vivo pathogen can cause complications, such as infection in surgical site [1-5]. Surgical sutures are sterile filament to support the healing process. They classified according to their degradation, composition and suture, including absorbable, non-absorbable, monofilament and multifilament [6-8]. Absorbable suture are divided to natural and synthetics, digested by enzymes and hydroxylation during the wound closure process, respectively. Gut and chromic gut are absorbable sutures that remain in the cutaneous and other tissue for 21-28 days after embedding. Poor knot security and high level of proteolytic enzyme in inflammatory cells are among the limitations of these stitches[9, 10].
The presence of sutures at surgical site have some adverse effects, such as surgical infection, clinical problems, prolong hospitalization and rising of health care cost [11-13]. Chromic catgut, the catgut sutures with chromic salts to crosslink the collagen molecules, have been widely used. At the first, carbolic acid was developed in 1881, after then ethylene oxide has been replaced for suture sterilization . In recent years, suture coated with antibiotics and commercial antimicrobial suture, such as Vicryl-plus and Monocryl-plus have been used exclusively. Furthermore, polymer coating, such as carboxylic with an attaching antibiotic agent and natural polymer like chitosan and alginate, are well prospected for wound treatment [15-18].
Coating suture with nanoparticles, which can improve wound healing, surgical site infection (SSI) prevention and mechanical properties. Recently, suture has been enriched with silver nanoparticles using coating method [19, 20]. Copper nanoparticles is another type of nanostructure that its antibacterial properties has been demonstrated. The preferences of Cu nanoparticles that make it a suture investigation aspect are low production cost, wide range of antimicrobial potency, catalytic activity In addition, copper oxide and copper sulfate are well known compounds that have antibacterial properties comparable with copper metallic [21-23].
There are various methods such as chemical and physical vapor deposition, nanoimprint lithography and solid-state techniques for synthesis of the nanostructures used in the tissue engineering. The application of nanofibers was widely investigated in this research area. It has been demonstrated that the electrospun gelatin nanofibers was a good candidate for tissue scaffold. It has been reported that this scaffold exhibited desired biocompatibility and no toxicity after 24 and 48 hours in human foreskin fibroblast cells [24, 25]. The reverse micro-emulsion is another interesting technique due to its easy protocol and inexpensive requirements. In the micro-emulsion medium, some micro and nano-spherical structures are created that called micelles. Micelles are nano- capsules contain aqueous solutions. The main advantage of micro-emulsion method is the ability of the final particle sizes controlling .
In this study, copper sulfate nanoparticles (CSNPs) were formed in catgut absorbent mass. An effective method was developed for preparation of CSNPs@Catgut (the CSNPs penetrated in and coated on catgut fibers) as a suture with positively different properties from plain and chromic catgut. Catgut was immersed in a micro-emulsion solution containing CuSO4 and conditions (concentration and immersing time) were changed to find the maximum salt loading. Mechanical strength and surface morphology of enriched suture were analyzed. Cytotoxicity, antimicrobial activity and anti-inflammatory of CSNPs@Catgut were also evaluated.
Absorbable catgut plain suture as well as nylon, silk and vicryl sutures were purchased from local veterinary pharmacies (Isfahan, Iran). Copper sulfate pentahydrate (CuSO4.5H2O), Triton X-100, n-hexane, n-butanol and ethanol all were purchased from Merck.
The catgut enrichment
The catgut was enriched by CSNPs via immersing it in a reverse micro-emulsion mixture, using Triton X-100 (surfactant) and n-hexane as organic phase and n-butanol as co-surfactant. The aqueous solutions containing CuSO4.5H2O with different concentrations were prepared and added dropwise to the surfactant mixture. It was stirred for one hour vigorously until obtaining a homogenous and clear mixture. Catgut samples were immersed in solutions with different concentrations of CuSO4 (0.01-1 mole.L-1) for different times (10-1440 min) to find the optimum time for loading. Copper sulfate was loaded on catgut by micelles uptake to catgut. The swollen catguts were brought out of mixture and dried in an oven at 90oC for 48 hours. After complete drying, samples were immersed in absolute ethanol for 12 hours to dissolve micelles remnants and better participating of CuSO4 salt on catgut. They were dried again in oven at 75oC for 12 hours, so the CSNPs@Catgut were ready to use.
The surface morphology of prepared suture was characterized using scanning electron microscopy (SEM). The SEM micrographs were token using Hitachi-54160, Chiyoda-ku, Japan operated at 15 kV. The samples were placed in a vacuum chamber and coated with a thin god layer to improve their surface conductivity and subsequently obtain clear micrographs . They were presented on different magnifying and resolutions to see if copper sulfate particles grafted on catgut surface and penetrated in its bulk. The technique also was carried out to evaluate the successfulness of nano size preparation with micro-emulsion synthesis method. The X-ray diffraction (XRD) technique (X’Pert Philips 3040/60, Cu/Kα) was also used with to confirm the existence of CuSO4 phase deposited on catgut. As another characteristic test, the effect of copper grafting on tensile strength, catgut and CSNPs@Catgut were analyzed using Santam machine controller. The force required for rupture starting of each sample was measured and recorded in Newton.
Antibacterial and cytotoxicity tests
The antibacterial activity of CSNPs@Catgut suture was assessed by the zone of bacterial inhibition. It was evaluated and compared with antibacterial disk, and then the zones of bacterial inhibition in treated and untreated sutures were determined. Pieces of sutures were inoculated with approximately 1×108 to 2×108 CFU.mL-1 E. coli (ATCC25922) per plate, and incubated for 24 h at 37°C.
Viability and proliferation assays were investigated by counting live, dead (by 0.4% trypan blue solution), suspended cells in the medium, and attached cells to a petri dish, after trypsinization with hemocytometer chamber, separately. Human menstrual blood stem cells (hMBSC) were cultured and the toxicity of suture was determined by cell counts. For this purpose, the culture medium was removed from petri dish and then the petri dish was washed with PBS. The harvesting of the cells was performed by adding Trypsin/EDTA (4%) with concentration of 50 µL.cm-2 and then incubation at 38.5 ºC. After neutralizing trypsin with serum-containing culture medium, the cells washing and dilution was performed. Eventually, hemocytometer was filled and the cells were observed under an inverted microscope. The number of dead and live cells was counted in different days (5-10-15).
Animal and histological experiments
Eighteen adult female mice, weighting (25-30 g) were purchased from the pharmacy research institute (Isfahan, Iran). They were kept in standard condition and divided into control and treated groups. For this purpose, CSNPs@Catgut and simple catgut sutures were imbedded in the muscles of femur. At the end of the study, the mice (from each group) were scarified on the days 3, 7, 14, foot of mice were separated, and fixed by 85 percent paraformaldehyde. The staining of the section of muscle, including suture embedding, was performed with Hematoxylin and Eosin (H&E). The data were expressed as mean ± standard deviation for three experiments (n=3). Two-way multivariate analysis of variance was used for the results statistical evaluation.
Copper sulfate optimum loading
An experimental design was carried out to find the optimum CuSO4 micro-emulsion concentration and immersing time. A table of experimental design was created by SAS-JMP software for changing these two factors with three levels (Table 1). Salt uptake was performed and the table was completed. Data was analyzed, variations were plotted versus loading and the optimum points for each factors were derived. As shown in Fig. 1 the maximum CuSO4 was loaded during 880 min and in 0.06 M solution.
A maximum loaded amount of CuSO4 on catgut during time was observed because the suture has a limit capacity of absorbance. It seems that the suture is not saturated in time durations less than 880 min and more than that has no capacity for CuSO4 absorption. A maximum point was observed on loaded CuSO4 vs micro-emulsion concentration. It can be related to bigger sizes of micelles in concentrated solutions because of Cu2+ cations and SO4-2 anions are closer in concentrated solutions. The phenomenon can limit the diffusion of micelles into the catgut and subsequently limit the CuSO4 loading.
Morphological analysis of CSNPs@Catgut
The distribution and size evaluation of copper sulfate particles on catgut suture were investigated using SEM. Fig. 2 shows SEM micrographs of CSNPs@Catgut with different magnifying. Moreover, the presence of particles was also visible, among the filaments of the suture. The nanoparticles clearly covered the suture surface by filling the pore between fibers of the yarn. The SEM analysis showed copper sulfate particles distribution on catgut surface relatively good. The micrographs also show CuSO4 particles sizes between 50 and 100nm. It was the main idea of this research that particles attached on surface or penetrated in bulk of catgut can release slowly around the wound with absorbing the yarn. It is a fact that smaller particles have higher surface area and better performance of activation.
XRD technique was performed to confirm the deposition of CuSO4 on catgut suture.
Fig. 3 shows The XRD pattern of CSNPs@Catgut. Peaks at 2q= 19.3, 31.7 and 46.8 can be related to CuSO4 crystalline phase. The patterns also shows no crystallinity for catgut because it is relatively an amorphous phase.
Tensile strength test
The tensile strength test was performed, in order to compare the tensile strength of catgut, CSNPs@Catgut, nylon, silk and vicryl sutures. The test showed changing in tensile strength of catgut with adding CuSO4 nanoparticles. Fig. 4 shows that CuSO4 can improve the mechanical properties of yarn so that CSNPs@Catgut tensile strength be relatively comparable with nylon. The mean maximum tensile strengths at the breakdown point for CSNPs@Catgut and catgut samples were (10.0075± 0.35 N) and (4.16± 1.02 N), respectively. Therefore, catgut enrichment with CuSO4 significantly improved the strength of suture stretching. It seems that salt penetration to catgut bulk has occurred uniformly so that made the suture a semi nanocomposite structure.
Antibacterial activity and Cytotoxicity
The microbiological characterizations were performed on CSNPs@Catgut and plain catguts as the control group. The zone of E. coli inhibition was compared with antibacterial disk, qualitatively (Fig. 5).
Tissue and wound healing effect
H&E staining section was compared between the control and treated group. In the treated group, inflammation was reduced and CSNPs@Catgut affected the wound healing factor. As shown in Table 3, there was a significant difference between the mean of necrosis and intensity of inflammation in all groups (control and treated) on days 3-7 and 14 (p< 0.05). The significant differences between the treated and control groups were observed on days 3-7 and 14 in terms of the intensity of inflammation. However, no significant differences were observed between the treated and control groups in the necrosis variable (p>0.05), but a significant difference was observed among 3rd, 7th and 14th days, in terms of the mean of necrosis variables.
Fig. 6 shows different characteristics between the control and treated group. So that on day 3, acute inflammation with necrosis and bleeding can be seen in the control and treatment group (Figs. 6a & 6b). This trend changed from day 7 in the treatment group with decreasing intensity of inflammation and bleeding, compared to the control group (Figs. 6c & 6d), so that the intensity of inflammation of the treatment group was less than control group (Figs. 6e & 6f).
Despite suture sterility, different approaches adapted for wound management may cause contamination, delay in wound healing and susceptibility to secondary infection. One of the approaches of wound management with a goal to improve the healing process is to prevent the bacterial adherence to the surgical suture and to fight against the formation of these contaminated points. Considering the degree of adherence of bacteria to the suture and its impact on infection and delay in wound healing, fighting against bacteria will be considered as a practical way to improve wound management. Antibiotic-resistant is another concern in the surgical site infection. Therefore, the role of metal and salt particles especially in the nano scales is inevitable for managing wound healing and infection [28, 29].
A novel copper sulfate nanoparticles/catgut (CSNPs@Catgut) with antimicrobial properties, reduced inflammation and increased wound healing was developed. The dispersion of nanoparticles on surface and in matrix of suture significantly improved its physical and chemical properties as a semi nanocomposite. The mechanical properties of the treated catgut sutures were evaluated because the reduction in the strength of the suture material can result in unexpected suture breakage, so inducing complications in healing tissues and tensile strength could reduce healing time with mechanical tissue effects. Although, types of sutures do not cause a significant difference in the process of wound healing, but it shows different tissue reaction in different conditions.
The prepared CSNPs@Catgut was characterized using SEM, XRD, tensile strength, antibacterial activity and cytotoxicity tests. SEM analysis showed the effectiveness of the dipping process, in providing a homogeneous distribution of CuSO4 on the catgut suture. XRD showed characteristic peaks of CuSO4 and confirmed the deposition its particles on catgut. The mechanical properties of the treated suture increased 2.5 times, compared to the control sample, which caused a significant difference in the strength of the material after treatment. The detail of investigation has indicated that the CSNPs@Catgut degrades in a slower rate after 7 days of the degradation process. Antibacterial activity tests demonstrated that CSNPs@Catgut can be utilized against bacterial colonization like E. coli without any cell toxicity. The histopathological study clearly demonstrated that grafting of copper sulfate nanoparticles on suture could decrease inflammation. Therefore, application of anti-inflammatory agent adapted as an effective approach to enhance healing pressure and regeneration. Suture strength, antibacterial properties and cytotoxicity positively affected the inflammatory stage of wound healing.
An effective nano-enriched suture was designed and prepared using CuSO4 nanoparticles grafting on catgut. Nanoparticles were deposited on suture using revers micro-emulsion method. Experimental design revealed loading capacity of catgut by micro-emulsion solution concentration and immersing time duration. Results of characterizations showed a complete excellence of CSNPs@Catgut compared with simple catgut in tensile strength, antibacterial activity, cytotoxicity as well as tissue and wound healing effect. It can be related to the nature of copper sulfate, particles scale, uniform particle sizes and similarly uniform particle dispersion on surface and in the bulk of catgut. The mean maximum tensile strengths for CSNPs@Catgut was more than two times higher than simple catgut. It was related to creation of a semi nanocomposite after adding CuSO4 to catgut because of good merging of particles and suture. Results also showed an obvious effect of nanoparticles on antibacterial properties of the suture. It can be depended on antibacterial nature of copper sulfate as well as high surface area of attached nanoparticles to catgut. The result of cytotoxicity test also did not show any difference between the CSNPs@Catgut and catgut. Finally, the new suture with decreasing intensity of inflammation and bleeding leaded wound healing process to good situations and made itself as a reliable candidate for wound management.
CONFLICT OF INTEREST
There is no conflict of interest among the authors.