Novel Elastomeric Fabrics for the Treatment of Hypertrophic Burn Scars Using Polyhexamethylene Biguanide Antimicrobial Agents

VARAN Nilufer Y ，KING W Martin

1 Department of Textile Engineering，Istanbul Technical University，Istanbul 34437，Turkey

2 Textile Engineering Chemistry and Science，North Carolina State University，Raleigh 27695，USA

Introduction

Pressure garment technique is a rehabilitation technique to prevent and/or reduce the formation of scar and contractures after the third and the fourth degree burn injuries.It has been found that applying pressure to this regenerating skin can accelerate the healing process，prevent the thickening，buckling and nodular formation，and decrease the inflammatory response and the amount of blood reaching the scar tissue.This reduces the degree of hypertrophia which encourages the formation of normal pliable skin tissue and reduces the extent of scarring.However，such pressure garments should be worn 24 h a day for a period of 12 to 18 months depending on the severity of the burn and the duration of the treatment.While the exact role of the pressure garment in the healing process has yet to be clarified，it is believed that by applying pressures in the range of 0.67 -6.70 kPa(5 -50 mmHg) on the newly vascularized underlying tissue gives optimal results［1-5］.

As the need for antimicrobial fabrics has increased，the criteria for effective materials have been scrutinized.Hygiene is required for the use of these garments in order to increase the effectiveness and functions［6-11］.Polyhexamethylene biguanide(PHMB)also known as polyhexanide and polyaminopropyl biguanide is a commonly used antiseptic (see Fig.1).PHMB is a potent biocide with broad spectral activity against both gram positive and negative bacteria，yet with low toxicity to higher organisms［12-14］.The minimal inhibitory concentrations range from 5 ×102(e.g.，against Staphylococcus aureus)to 1 ×104μg/L (e.g.，against Klebsiella pneumoniae).

Fig.1 General formula of PHMB

1 PHMB Antimicrobial Mechanism

PHMB is a strong，fast acting，and broad spectral biocide against bacteria.The antibacterial activity of PHMB is attributed to its interaction with cellar membrane components.It kills bacteria by puncturing their cell membranes，causing the contents to leak out (see Fig.2).It does not have the same effect on human cells so it is safe to use in contact with skin.

Fig.2 PHMB antimicrobial mechanism

PHMB has a higher activity against planktonic bacteria in studying biofilms.They are also the most effective agents against sessile bacteria found within biofilms.The effect of concentration of PHMB on planktonic versus sessile bacteria is due to either the mechanism of action or the number or disposition of cationic binding sites.Also PHMB promotes contraction and aided wound closure significantly［15-16］.

The mechanism of action of PHMB has been described in a number of articles.The maximal activity of the PHMB occurs at between pH 5 -6 and that initially the biocide interacts with the surface of the bacteria and then is transferred to the cytoplasm and cytoplasmic membrane.The cationic PHMB has little effect on neutral phospholipids in the bacterial membrane.Its effect is mainly on the acidic negatively charged species where it induces aggregation leading to increased fluidity and permeability.This results in the release of lipopolysaccharides from the outer membrane，potassium ion efflux，and eventual organism death.

Biguanide groups are the active parts of the PHMB.Compared to other biguanides，the fact that biguanide groups are separated by a C6 aliphatic hydrocarbonated chain gives PHMB a particularly high efficacy power on a wide range of microorganisms.

2 Experimental

2.1 Materials

70/30 nylon 66/spandex in powernet and flat warp knitted structures and 75/25 nylon 66/spandex in weft knitted structure fabrics were used.A powernet fabric is a raschel knit with inlaid spandex yarns.The other warp knit structure is the most popular two-bar structure locknit.The longer underlaps of the front guide bar plate on the technical back of the fabric which improves fabric extensibility，cover and handle.Spandex yarns are inserted into the structure in machine direction laying in the wale line adding extra elasticity to these units.Super stretch knits stretch 100% or more in both directions (two-way stretch nylon 66/spandex).The third selected fabric is a 1 × 1 rib double-jersey fabric.The loops are formed across the width of the fabric in weft knitting giving a high stretch in widthwise.Fabric specifications are listed in Table 1.The fabrics contain 25% or more spandex.The thickness varies from 0.50 mm to 0.62 mm and the area density varies from 164 to 277 g/m2.A PHMB antimicrobial was used for antimicrobial treatments.

Table 1 Fabric specifications

2.2 Method

2.2.1 Pretreatment

The samples were conditioned for 24 h at 20 ℃，65%relative humidity in the physical testing lab.For scouring Dupont procedure for synthetic fabrics with Lycra was followed.The procedure is given in Fig.3.The 4 000 mL stock solution was prepared with 0.5 g/L sequestering agent，2 g/L soda ash，and 2 g/L non-ionic detergent.The samples were washed at 80 ℃for 1 h，rinsed at 38 ℃ for 1.5 h，and were dried at 50 ℃for 30 min.

2.2.2 Antibacterial treatment

Pad-dry-cure method was used to apply PHMB antimicrobial by using a laboratory type padding machine，with temperature at 40 ℃and pH 6.5.The fabrics were squeezed to a wet pickup of 53% for powernet warp knitted fabrics，66%for flat warp knitted fabrics，and 84% for weft knitted fabrics.The padding temperature was 20 ℃，the pressure was 1 bar，and the machine speed was 1.5 m/min.A laboratory dryer was used for drying at 100 ℃for 1 min and curing at 150 ℃for 1.5 min.The samples were treated with 4% PHMB antimicrobial agent.

Fig.3 Scouring of fabric samples

2.2.3 Colorfastness to laundering

The treated samples were washed to determine the antimicrobial effects of fabrics after laundering by following AATCC Test Method 61 (2A)-2010 “Colorfastness to Laundering:Accelerated”to evaluate the washing durability of the treated fabrics”［17］.Treated fabrics were subjected to 5，10，and 50 consecutive launderings.

2.3 Testing and analysis

2.3.1 Fourier transform infrared spectroscopy (FTIR)

In order to identify the infrared absorption spectrum and detect any changes in the structure of the PHMB treated samples during treatment，treated samples were scanned using a Nicolet Nexus 470 Spectrophotometer with AVATAR Omni Sampler for Attenuated Total Reflectance (ATR)mode.The specimen was mounted onto the surface of the germanium (Ge)crystal in the ATR assembly.A total of 64 scans were aggregated between 1 000 cm-1and 4 000 cm-1with each spectrum having a 4 cm-1resolution.The aggregated scans showing the absorbance across the infrared spectrum was acquired using OMNICTMsoftware.

2.3.2 Scanning electron microscopy (SEM)

SEM analyses were performed on three different sample sets to see if the PHMB solution appeared on the fabric and fiber surfaces.Images were acquired from a JEOL JSM 5900-LV SEM using an accelerating voltage of 15 kV.Specimens were mounted on aluminum stubs using conductive carbon tape.They were then coated with gold/palladium using a HummerTM6.2 Sputter Coating System (Anatech，CA，USA)to obtain a conductive coating about 10 nm thick.Untreated control samples were used to make a comparison.

2.3.3 Pressure measurements using wireless pressure sensors

A static mannequin and pressure garments designed for leg(from knee to ankle)for each type of fabric were used for pressure measurements before and after antimicrobial treatments.Measurements were recorded using calibrated pressure sensors that were connected to a data acquisition and management software program by wireless transmitters.Static mannequin and software program with wireless pressure sensors are shown in Fig.4.The measurements were taken on each garment from calf and ankle before and after the treatment.Pressure measurements from calf and ankle by using wireless pressure sensors are shown in Fig.5.

2.3.4 Antibacterial activity

AATCC Test Method 100-2004 “Assessment of Antibacterial Finishes on Textiles”was followed to determine antimicrobial activity［17］.Assessment of antibacterial activity finishes on textile material is determined by the degree of antibacterial activity intended in the use of such materials.Staphylococcus aureus ATCC 6538 (1.60 × 108CFU/L)microorganism was used as a test inoculum.Untreated control samples，treated samples and treated after 5，10，and 50 washed samples were tested.

Fig.4 Preparation of experimental design of (a)static mannequin and (b)software program with wireless pressure sensors

Fig.5 Taking pressures using wireless pressure sensors from (a)calf and (b)ankle

3 Results and Discussion

3.1 FTIR analysis

Figure 6 shows FTIR spectra of untreated and treated fabric samples with PHMB.PHMB has a strong absorbance between 1 200 and 1 700 cm-1.Spectral comparisons of (a)treated powernet warp knitted showed the absorption bands (1 250 cm-1and 1 350 cm-1);(b)untreated powernet warp control fabric showed no absorption bands between 1 200 cm-1and 1 700 cm-1;(c)treated flat warp knitted fabric showed the absorption bands (1 250 cm-1and 1 350 cm-1);(d)untreated flat warp control fabric showed no absorption bands between 1 200 cm-1and 1 700 cm-1;(e)treated weft knitted fabric showed the absorption bands (1 250 cm-1and 1 350 cm-1);and (f)untreated weft control knitted fabric samples showed no absorption bands between 1 200 cm-1and 1 700 cm-1.

The infrared absorption spectra of the treated samples are obtained by FTIR analysis and the spectrum search in the FTIR has confirmed that all samples show the presence of the antimicrobial agent PHMB，as indicated by the fabric samples showing absorption bands between 1 200 cm-1and 1 700 cm-1.

3.2 SEM analysis

SEM images of the untreated samples are shown in Fig.7.The bonding of 4 -8 fibers together and forming a compact and brighter structure after the treatments shows that the antimicrobial treatments are successful.SEM images of treated fabric samples with PHMB antimicrobial solutions are shown in Fig.8.

Fig.6 FTIR spectra of the fabric samples after treatment with PHMB:(a)treated powernet warp;(b)untreated powernet warp control;(c)treaed flat warp;(d)untreated flat warp control;(e)treated weft;(f)untreated weft control knitted fabrics samples

SEM images of the treated samples show that the bonding of the fibers，compact structure and alignment of the fibers strongly confirming the antimicrobial treatments using PHMB antimicrobial agents are achieved.

Fig.7 SEM images for untreated samples

Fig.8 SEM images for treated fabric samples with PHMB(magnification ×1 100)

3.3 Pressure measurements

The pressures were taken from calf and ankle and were measured in the range of 4.8 -6.1 mmHg，and the mean scores were obtained between 5.0 -6.0 mmHg (100 mmHg =13.3 kPa)(see Table 2).Higher pressures were measured for ankle before and after treatment.The Laplace equation was originally described to define the relationship between the pressure of a closed elastic membrane and the tension within the membrane［18］.The localised static pressure exerted on the leg by a compression system can be calculated overall using Laplace's law (see Eq.(1)).where P is the interface pressure in mmHg，100 mmHg =13.3 kPa;T/W is the tension (N)by width unit;R is the radius of the leg (m);and α is the proportionality coefficient.

Table 2 Pressures for the treated samples Unit:mmHg

In practice，the radius of the leg cannot be easily measured，so the circumference is used instead;in addition，it is necessary to take into account the bandage application method;finally，the pressure is expressed in mmHg，which gives the final equation (see Eq.(2)).

where P is the interface pressure under the bandage (in mmHg)，also called the compression force;T is the tension of the bandage (kgf)(1 kgf/cm2=98 kPa)，which depends on the material and its stretch %;N is the number of layers(defined by the overlap and the number of bandages);C is the circumference at the measurement point (cm);and W is the bandage width (cm).

Laplace's law states that sub-bandage pressure (P)is directly proportional to bandage tension (T)and inversely proportional to the circumference (C)of the limb to which it is applied.As the circumference of the limb increases(from C1 to C2)，the pressure decreases (see Fig.9)［19］.

Fig.9 Illustration of Laplace's law

Mean scores of final pressures showed a small significant increase for fabric samples after treated with PHMB.Weft knitted fabrics gave the highest pressure values before and after antimicrobial treatments (see Fig.10).This is attributed to more elastic structure of weft knitted fabrics than warp knitted fabrics which causes a small but significant shrinkage during processes.

Fig.10 Mean scores of final pressures for the treated samples with PHMB (error bars:±standard deviation)

3.4 Antibacterial activity

The percentage reduction of bacteria for treated samples with PHMB is shown in Table 3.Equation (3)was used to calculate the percentage reduction of bacteria.PHMB treated samples showed excellent antimicrobial activity for all fabric samples.The antimicrobial activity for each fabric sample was preserved even after 5 washes.After 10 and 50 washes a very good antimicrobial activity was observed with a small but significant decrease for fabric samples.

where R is the reduction(%);A is the number of bacteria recovered from the inoculated treated sample;C is the number of bacteria recovered from the inoculated untreated control sample.

Table 3 Percentage reduction of bacteria for the treated samples

A comparison of antimicrobial activity for fabrics treated with PHMB antimicrobial agent procedure can be seen in Fig.11.

Fig.11 A comparison of antimicrobial activity for PHMB antimicrobial agent procedure in terms of fabric structure

4 Conclusions

It is aimed to progress the rehabilitation performances of burn pressure garments by imparting durable antimicrobial properties with the control of exerted pressures before and after antimicrobial treatments.Nylon 66/spandex fabrics in three different structures were treated using a PHMB antimicrobial agent procedure.

The pressures are measured between 5.0 - 6.0 mmHg which is an acceptable optimal medical range (0 -15 mmHg)for low pressured garments that reduces the degree of hypertrophy，encourages the formation of normal pliable skin tissue，and reduces the extent of scarring.All fabric samples showed excellent antimicrobial activity.It remained the same even after 5 washes for all types of fabrics.After 10 and 50 washes，a small but significant decrease was observed for all fabric samples.All samples preserved their strong antibacterial activity up to 50 launderings.

Novel elastomeric fabrics for use in burn pressure garments having durable (up to 50 washes)antimicrobial property were developed using PHMB antimicrobial agent procedure.50 launderings equal to 1 year for home use and 2 months for hospital use (laundering conditions in hospitals are more aggressive).These treatments also yield good results to prevent odor， decrease infection by preventing and/or blocking microbial growth according to the antimicrobial mechanism and support reducing of scarring by providing a hygienic environment around the scar.Antimicrobial finish inhibits odorcausing microorganisms from colonizing and growing in the textile and thus prevents microorganisms from degrading the product.These are also good results to decrease costs by providing an appropriate level of rehabilitation.

Acknowledgements

Financial support of this research was provided by NC State University College of Textiles and the Scientific and Technological Research Council of Turkey.

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