Nanofiber Micro-Dispersed Oxidized Cellulose as a Carrier for Topical Antimicrobials: First Experience

Abstract

Background:

Micro-dispersed oxidized cellulose, already used for hemostasis, might be helpful for introduction of an antimicrobial drug.

Aim:

To examine the effect of topically applied gentamicin attached to a new biodegradable carrier formed by micro-dispersed oxidized cellulose in microfiber and nanofiber form for treatment of acute wound infection and to assess the influence of this carrier on healing.

Materials and Methods:

A model of a full-thickness infected dermal wound was created in 12 female domestic pigs. The effectiveness of topical gentamicin delivered with micro-dispersed oxidized cellulose carrier was tested in acute wound infections caused by Staphylococcus aureus, Pseudomonas aeruginosa, or Escherichia coli.

Results:

The effectiveness of nanofiber micro-dispersed oxidized cellulose with gentamicin was proved according to culture findings. When assessed macroscopically, 100% of wounds treated by the nanofiber product had no signs of local infection. When microfiber micro-dispersed oxidized cellulose was used, cultures demonstrated residual bacteria in 94.4% of treated incisions despite the absence of clinically recognized infection.

Conclusions:

Micro-dispersed oxidized cellulose carrier with a sufficient concentration of an attached antibiotic appears to be effective for the treatment of full-thickness skin infections. The positive influence of the product on the healing of a dermal incision was shown, and a good hemostatic effect was confirmed.

Micro-dispersed oxidized cellulose^® (HemCon Medical Technologies, Inc., Portland, OR) (MDOC) is a random copolymer of polyanhydroglucose and polyanhydroglucuronic acid used for its hemostatic effect. We decided to combine this ability with the antimicrobial effect of gentamicin. The effect of topical gentamicin attached to microfiber and nanofiber forms of MDOC was tested on full-thickness porcine dermal incisional infections caused by Staphylococcus aureus, Pseudomonas aeruginosa, or Escherichia coli.

Materials and Methods

Materials

Micro-dispersed oxidized cellulose is an ionogenic polysaccharide that functions as a carboxylate ion exchanger and, in the form of the sodium-calcium salt of polyanhydroglucuronic acid (PAGA), is absorbed completely by the host. As a carboxylate polymer, MDOC creates intermolecular complexes (IMC) with positively-charged, low-molecular-weight substances or polymers; i.e., it can work as a carrier of substances such as basic antibiotics, including gentamicin.

Micro-dispersed oxidized cellulose is not a film-forming or fiber-forming substance of itself. For the production of MDOC nanofibers of 50–500 nm and microfibers of 50 micrometer, it is necessary to add biocompatible and absorbable polymers that create fibers or act as a binding material for prepared nanofibers. Medicinal glycerin, which has an impact on the physical properties of microfibers or nanofibers, has been successful as a softening agent for these systems. The typical composition of the nanofibers is listed in Table 1. Fibers of this composition were always fully absorbable after their application. The formats of antimicrobial MDOC non-woven nanofabrics applied to contaminated incisions were the same as the basic nanofiber composition mentioned above, only gentamicin sulfate was attached to PAGA during the manufacturing process. Therefore, instead of the sulfuric acid in gentamicin sulfate, the anion (polyanhydroglucuronate) was created by PAGA with the composition shown in Table 1.

Table 1.

Composition of Original and Antimicrobial Nanofibers of Micro-Dispersed Oxidized Cellulose

Original
Polyvinyl alcohol (PVA)	50% w/w
MDOC (sodium-calcium salt of PAGA)	33% w/w
Glycerol (in 100% form)	17% w/w
Antimicrobial fibers
PVA	42.80
MDOC (Lot No.72105)	28.60
Glycerol	14.30
Gentamicin sulfate	14.30 LOT No. A7694, Product No. G-1067 (FLUKA)

At the beginning of testing, when we tried to prepare nanofabrics, it was impossible to achieve a higher surface density than 15 g/m². This means that, at a concentration of 14.3% w/w of gentamicin in the MDOC nanofabrics, the content of drug per 100 cm² was a maximum of 21.45 mg, which is insufficient for its intended effect (the brand name formulation Garamycin Schwamm^® contains 130 mg of gentamicin for the same area). Therefore, microfibers were prepared from long-fiber, medicated cotton wool and transferred to raw cellulose. After hydrolysis, gentamicin sulfate was added to the reaction mixture during homogenization at 20°C. The fibers were created by washing with hydrous alcohol and dehydrated using concentrated ethyl alcohol. Afterward, they were dried to a constant weight in a laminar box.

The technology of the production of MDOC nanofabrics with a surface density as high as 150 g/m² was developed further. Thus, a uniform concentration of gentamicin in an amount as high as 150 mg on an area of 100 cm² was achieved; i.e., the concentrations are comparable to those of the lots of commercial gentamicin–collagen implants used in the topical treatment of infected incisions (e.g., Garamycin Schwamm^®, Collatamp-G^®, Gentacoll^®, Sulmycin Implant^®).

Experimental protocol

Twelve female domestic pigs (35–45 kg) were used. Each experiment took seven days. After intramuscular administration of ketamine 30 mg/kg (Narkamon^®, Zentiva, Czech Republic), azaperone 40 mg/kg (Stressnil^®, Janssen Pharmaceutica, Belgium), and atropine 0.5 mg (Atropin Biotika A.U.V.^®, Biotika, Slovakia), the animal was put under general intravenous anesthesia and maintained with ketamine. After preparation of the operative field, eight full-thickness dermal defects, 5 cm long with side incisions and fascial injury, were created in the paravertebral area (four incisions on each side). Contusion of the margins using Péan forceps was performed to imitate the most common incision type in routine practice. After that, 0.5 mL of a bacterial suspension at a density of 10⁸ colony-forming units/mL was inoculated into seven incisions; the last one was left clean as a control. Each microbiological agent was tested separately in two animals for the MDOC in microfiber and nanofiber form with gentamicin and in 12 treated sites (two were left clean and two were infected controls). After 45 minutes, beds of MDOC with gentamicin (5 × 1.6 cm containing either 1.67 mg of gentamicin attached to a microfiber carrier or 10.83 mg attached to a nanofiber one) were placed in six infected incisions. The sites of treatment were not rotated. The incisions were left for open healing as is standard for contaminated or dirty incisions. We preferred open healing because of the possibility of better evaluation of the incisions macroscopically by the clinician and repeated harvesting of microbiological swabs. The entire operative area was covered by gauze and a surgical towel. After 24, 48, and 168 h, swabs were obtained, and macroscopic assessment by the clinician was performed. At the end of the experiment, some tissue samples of the incision margins were taken for histopathologic examination, and the animal was destroyed by intravenous application of T-61^® (Intervet Canada Ltd., Kirkland, Quebec, Canada). During the experiment, the animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Experimental Animals.”

Results

The results of cultivations on blood agar of swabs from the incisions taken after 24, 48, and 168 h were compared. In S. aureus and P. aeruginosa infections, 50% of the sites treated by nanofiber MDOC with gentamicin were negative; all swabs taken from infected incisions treated with microfiber MDOC were positive at the end of the experiment. The results for E. coli infections were the same, despite the low concentration of gentamicin attached to microfiber MDOC (Table 2).

Table 2.

Number (Percent) of Treated Infected Incisions Having Negative Cultures

	Escherichia coli			Staphylococcus aureus			Pseudomonas aeruginosa
	24 h	48 h	168 h	24 h	48 h	168 h	24 h	48 h	168 h
Microfiber MDOC + gentamicin	6/12 (50%)	10/12 (83)	12/12 (100%)	10/12 (83)	6/12 (50)	0/12	8/12 (67)	3/12 (25)	0/12
Nanofiber MDOC + gentamicin	12/12 (100)	12/12 (100)	12/12 (100)	12/12 (100)	12/12 (100)	6/12 (50)	12/12 (100)	12/12 (100)	6/12 (50)

MDOC = micro-dispersed oxidized cellulose.

Although the microbial results, with the exception of the E. coli infections, are not comparable, macroscopic assessment showed minimal differences between the tested materials. Nanofiber MDOC with gentamicin was fully absorbed at 94.4% of the treated sites after 48 h and at 100% after 168 h. All the incisions were macroscopically clean, healed with a crust, and did not show signs of local infection after 48 and 168 h. Microfiber MDOC with gentamicin was absorbed completely in 80.6% of all treated incisions, and 94.4% of them were free of signs of local infection after 168 h (Table 3).

Table 3.

Macroscopic Assessment after 168 h by Group (%)

	Escherichia coli			Staphylococcus aureus			Pseudomonas aeruginosa
	I	II	III	I	II	III	I	II	III
Microfiber MDOC + gentamicin	9/12 (75)	3/12 (25)	0/12	11/12 (92)	0/12	1/12 (8)	9/12 (75)	2/12 (17)	1/12 (8)
Nanofiber MDOC + gentamicin	12/12 (100)	0/12	0/12	12/12 (100)	0/12	0/12	12/12 (100)	0/12	0/12

Group I = clean incision, test material fully absorbed; group II = clean incision, test material not fully absorbed; group III = surgical site with signs of local infection (pus, Celsus's signs of inflammation), test material not absorbed.

MDOC = micro-dispersed oxidized cellulose.

Discussion

Surgical site infections (SSI) are the second most frequent health care-associated infections, and many researchers are involved in the development of new site care products and new technologies for incision healing. These infections can lead to greater morbidity, longer hospital stays, higher hospital costs, and death. Topical administration of antibiotics for prophylaxis is supported by some studies in clean incisions [1]. Topical antimicrobials can be used in the treatment of secondarily infected incisions, and the result of the treatment is equivalent to systemic administration of antibiotics in the case of minor infected sites [2]. The more important role of topical antibiotics is in the treatment of chronic infections [3].

One of the most common topical antibiotics in surgery is gentamicin because of its effectiveness against a broad spectrum of gram-positive and gram-negative bacteria [4,5]. The other advantages of topical gentamicin are its high concentration in incisional fluid and minimal concentration in serum. High local gentamicin concentrations, about 75–200 times the minimum inhibition concentration (MIC), were observed in incisional fluid compared with serum (1–4 mg/L after 24 h) with both concentrations being safely below the toxic threshold [4,6 –8].

Most published articles on topically administered gentamicin with a collagen carrier describe results for the prophylaxis of SSI in cardiac surgery [4 –10]. Gentamicin–collagen implants of various brand names (e.g., Collatamp-G^®, Gentacoll^®, Sulmycin Implant^®) that contain 130 mg of gentamicin and 280 mg of collagen were put underneath the sternum or between its edges before sternotomy closure. In some studies, more than one implant of gentamicin–collagen was applied [8,9,11]. In the randomized controlled study by Friberg et al., the incidence of SSI in 2,000 patients undergoing open-heart surgery was reduced significantly, from 9% to 4.3% in the gentamicin group, but there was no effect on the occurrence of osteitis or mediastinitis [11]. The difference in deep SSI in all groups was not of statistical significance, except in groups of patients with a body mass index >25 kg/m² or diabetes mellitus [9,11]. On the other hand, there were significantly more reoperations for bleeding in the gentamicin–collagen group (4.0% vs. 2.3%). This result was not explained, but it can be argued that the difference may be attributable to bleeding from the bone marrow from a gap between the sternum halves when two gentamicin–collagen layers were inserted [11]. In general, topically administered gentamicin–collagen implants are recommended for antibiotic prophylaxis in cardiac surgery.

The use of topical gentamicin as prophylaxis also is recommended in some clean procedures in orthopedic and general surgery. Eveillard et al. proved the effectiveness of gentamicin-impregnated cement in the prevention of deep site infection after primary total knee arthroplasty. The infection rates were 1.21% for patients who had antimicrobial cement and 9.52% for those who did not [12]. Musella et al. recommended the use of gentamicin-laced collagen tampons in groin hernia repairs if polypropylene mesh is inserted under the aponeurosis of the external oblique muscle. In the gentamicin group, 1/301 patients (0.3%) developed a postoperative site infection compared with 6/294 (2.0%) in the control group [13].

In clean-contaminated and contaminated procedures, the results are ambiguous. After one week of treatment, Buimer et al. describe a significant reduction in postoperative complications (dehiscence, infection) in patients treated with an enclosure of gentamicin after a primary excision for hidradenitis suppurativa (35%) compared with patients treated only with primary excision (52%). However, after three months, complications in both groups were similar [14].

A number of studies evaluated the effectiveness of topical gentamicin in colorectal surgery procedures. Sites after stoma closure and perineal incision sites after abdominoperineal excisions were contaminated, and the incidence of SSI was high. Haase et al. did not show any differential effect between topically and subcutaneous gentamicin implants with regard to the prevention of SSI after loop ileostomy closure [15]. However, good results with topical administration of gentamicin were achieved in other randomized controlled studies [16 –18]. Grüssner et al. found a reduction of pathogens in cultures and a lower infection rate in perineal sites after abdominoperineal excisions. In total, bacteriologic efficacy amounted to 83.7% in the treated group vs. 60.4% in control subjects. The difference in infection rates was significant as well, 6% vs. 20.8% [16]. Similar results were reported in the studies of Rosen et al. and Gomez et al., although with a significantly higher percentage of primary incision healing in the gentamicin group (87%) than the control group (46%), and the rate of infection was 9% vs. 44% in the control subjects [17,18].

We decided to use a new biodegradable carrier formed by micro-dispersed oxidized cellulose in microfiber and nanofiber form. This cellulose has a good hemostatic effect and facilitates blood clot formation, proved prior to our study, and is produced as SEAL-ON^TM (HemCon Medical Technologies, Inc.), which has been shown to stop bleeding.

Micro-dispersed oxidized cellulose is fully resorbed within the first 48 h in 94% of sites when the nanofiber form is used and in 80.6% when the microfiber form is used. In contrast, collagen matrix, used in many topical antibiotic products, is fully biodegradable and resorbed within 1–8 weeks, depending on the vascularity of the tissue [14].

When gentamicin is attached to the MDOC carrier in concentrations comparable to those in these products (130 mg/100 cm²), we found its ability to be resorbed more quickly superior for topical administration in the treatment of soft tissue infections. We also did not notice any adverse effects from the treatment with gentamicin, in accordance with all previous published results.

Conclusions

Topical gentamicin attached to micro-dispersed oxidized cellulose, especially in nanofiber form, seems to be effective in the treatment of soft tissue infections thanks to its antimicrobial effect, excellent resorption of the carrier, healing effect, and influence on blood clot formation.

Footnotes

Acknowledgment

The study was supported by the Faculty of Military Health Sciences University of Defence in Hradec Králové, Czech Republic, research project No. 0000503.

Author Disclosure Statement

There are no conflicts of interest.

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