Textile Composite Wound Dressing

Textile Composite Wound Dressing A.P.London, A.E. Tonelli, S.M. Hudson, and B.S. Gupta Department of Textile Engineering, Chemistry, and Science

K.B. Wylie and G.J. Spodnick, College of Veterinary Medicine B.W. Sheldon, Department of Food Science Nonh Carolina State University Main Campus Drive R!aleigh, NC 27609-8301

Abstract--Poly lactic acid (PLA), poly ecaprolactone (PCL), and chitosan(CH) are incorporated into a biodegradable wound dressing. Physical properties of these films, including topography, water vapor permeability, degradation, and bacterial permeation as well as biological effects of toxicity on human fibroblast cells were studied. An in vivo study (currently in progress is proposed.

question contains fibrids or loose particles that can become incorporated into the forming epidermis. Keeping a moist environment for the wound is one of the major considerations in developing a d r e ~ s i n g . ~By increasing moisture at the wound/ dressing interface during the early stages of healing, a scab doesn't form so cells are free to move through the exudate. Also, the secondary damage due to dehydration which can cause excess scar tissue, is reduced.* All these concerns and more must be addressed in the design of a new dressing. Unfortunately, at present, there is no dressing available on the market which does not have to be removed, or which can keep adequate moisture around the wound.

I. INTRODUCTION Historically, people used the materials conveniently available to them for care of wounds. This included dressings as simple as herbs combined with leavai or rags used by primitive man to those as complex as a synthesized polymeric compound with an engineered multi-layer textile structure. Natural adhesive was used as early as 4000 years ago, as resins were applied to rags for "stickiness".l Early Sumerians (around 2100 B.C.)recorded the first use of bandages. They described procedures for the treatment of wounds; washing, malung plasters, and bandaging. 2' Egyptians used quick setting resins as well as plasters and adhesive tapes3 and the oldest bandages were found in Egyptian tombs.4 The Greeks and Romans used herbs boiled in water or wine and applied them directly to wounds. This acidic herbal layer, a treatment used in many other cultures, was thought to help heal wounds. Minerals with salts or oxides of copper and lead were also thought to aid in healing.5 For the next several centuries, dressings did not progress far beyond the traditional rag and herb dressing. [t wasn't until after World War I1 that medical science began to investigate other procedures and materials for dressing applications. One major development in wound treatment was combating infections caused by poorly laundered hospital cloths.6~ In the 1950s, synthetic materials were first used to help induce healing. Although dramatic advances have been made by modem science, pain and comfort are still issues raised by patients. Today's dressing must be periodically removed, causing trauma to the wound. This is compounded if the dressing in

A . Properties and Design In this study, a dressing is proposed which encompassed the elements of a wound dressing with those properties that are desired (Table I). By making the dressing out of a polymeric film, flexibility and comfort needs are met. By making the dressing biodegradable the need to remove it is eliminated and so is the pain associated with the removal process. And if the films are transparent, physicians can monitor healing time and check for infection. TABLE I. PRDPERTIEE OFTWE W O W DRESSING

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The polymers available for such a dressing include many materials that are used for other medical applications, such as in drug delivery systems and sutures. These include materials such as poly-galactic acid (PGA), poly-lactic acid (PLA), poly waprolactone (F'CL), etc. and their ccpolymers. The three materials selected were PLA, PCL and chitosan (CH). The latter is a relatively new substance extracted from crustacean shell (Figure I.). As the figure shows, chitosan is the N-deacetylated form of chitin.

were both dissolved in methylene chloride and cast onto 4" glass petri dishes.

gauzebandage

E L or PLA Chitosan

1 H

CH3 I O=C

wound

1 Figure II. Sandwich construction of proposed dressing /

B . Phase Two - Degradation

ti L

'x

-

-'Y

Degradation rate is an important physical property of biodegradable dressings and can adversely affect healing if not controlled. All polymers degrade at different rates depending on the surrounding environment. Chitin for instance degrades differently depending on the molting cycle of the particular crustacean species and ratio of degradation to synthesis of new cuticle.9 Enzymes contribute to the majority of biodegradation of natural substances. Chitin endocuticles (the inner layers) are degraded by the chitonase enzyme in nature. The degraded cuticle is recycled to form the new shell and is exposed as the old exocuticle is sloughed off during molting. Samples with a 70% deacetylation can be degraded by the lysozyme enzyme if it contains a certain sequence of N-acetylated residues. lo Comparative viscosities were used initially to determine this aspect. Films were exposed to a biological saline solution for a period of 1-3 weeks. They were then redissolved to measure their viscosities and compare these to the initial viscosities of the undegraded films. The CH films decomposed in 10-14 days, but the PLA and PCL films acted as hydrogels and results were unsatisfactory, as the excess cross-linking between the polymer and saline will decrease degradation rates. A study by Schindler et.al. shows that the degradation times of PLA and PCL based sutures and drug delivery systems, are approximately 1 year. The films used in this study are extremely thin (0.008-0.035 mm as measured by a Thwing-Albert Thickness tester); the degradation times in the presence of enzymes and bacteria should be low and lie in the range of 2-6 weeks. The exact degradation times of the PLA, PCL and CH films will be. determined during the in vivo study currently in progress.

Repeating units for chitosan and chitin where x: predominant unit for chitosari (x > 70%) y: predominant unit for chitin (x < 50%)

Figure I. Structures of Chitin and Chirosan

The sandwich design adopted is shown in Figure 11. It allows for the protection of the wound during the entire healing process. Chitosan is degraded and absorbed into the system at a high rate, while the PLA or PCL layer is degraded and absorbed much more slowly. Therefore, when the chitosan is completely absorbed into the body, the PLA or PCL layer is still present and continues to protect the wound from trauma. The gauze layer will absorb the excess exudate passing through the films in the form of water vapor, in addition to keeping the dressing in place on the wound.

11. PHYSICAL PROPERTIES A. Phase One - Film Construction All films were solvent cast. Chitosan was dissolved in 5% acetic acid and cast onto 1x3 ft. polycarbonate sheets. Chitosan acetate salts are highly water soluble, so the prepared films were soaked until neutral in a methanol/sodium hydroxide bath. The PCL and PLA films

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C. Phase Three - Surface Topography and Sterilization

As stated previously, water vapor permeability is important in the transport of exudate from the wound. The test method was based upon ASTM E-96-92: Standard Test Methods for Water Vapor Transmission of Materials. Each individual film and sandwiched films were glued onto metal weighing pans containing 35 mL of distilled water. The pans were placed upon a rotating platform in a room at a constant standard temperature (24" C) and RH (50 %). After 2 hours, at which time equilibrium should be reached, the pans were removed and weighed. After 24 hours, the pans were re-weighed and weight loss tabulated. Three separate runs were completed for each sample and water vapor permeability was calculated using the following equation:

The surface topography of the fdms was examined by SEM before and after sterilization with ethylene oxide (EO). The non-conductivity of the films caused charging, so samples were Au-Pd sputter-coated and overlaid with silver colloidal paste. The pre-sterilized films showled little topography, with minor ridges and bumps, possibly due to particulates. After sterilization the films were much smoother and had relatively few visual surface characteristics. The porous nature of the films was an important consideration in this phase; films had to have poi-es large enough to allow water vapor to transport through, but not large enough to allow bacteria to penetrate into the: wound. Results of the SEM analysis revealed the absence of microscopic pores large enough for bacteria, which are 2-3 pm, to penetrate the films.

I

(1) Water Vapor Permeability

Average weight loss ) 1Area (m2) 124 hours = g/m /24 hours where Area = 1 ~ :r2 r =3.15 cm; 10,OOO cm2/A=l/m2 Average weight loss = 3 runs13

$

D.Phase Four - Bacterial Migration The question of bacterial migration becomes quite important when a biodegradable dressing is used. In the beginning , the dressing may be a good barrier to microorganisms, but as time goes on and the dressing degrades, microbes have an open window to infect the wound. For this reason and because chitosan has a short lifespan, the sandwiched layer approach was thought to be the mast appropriate. As the chitosan degrades into the wound, the middle layer of polymeric film protects the wound from the anticipated microbial invasion. The study was conducted at the Department of Focd Science under the direction of Dr. Brian Sheldon. T:he films were secured on BHI agar plates supplemented with nalidixic acid by placing a poly-vinyl chloride (PVC, 1 in.) ring over the films, resulting in a compression of the films into the agar. The films, separately and sandwiched, were then inoculated with 100 pL of nalidixic acid resistant strains of Staphylococcus aureus or Escherichia coli and incubated at 37°C for 5 days and monitored at 24 hours intervals. While no growth was detected underneath the films, the problem of the bacteria solution running off the film onto the agar was quite prevalent despite the use of the PVC retainer ring. Bacterial growth was detected on the edges of all the films with the exception of the PLA and PLA/CH plates, but again this was due to run-off. Because there was no detectable growth beneath the film, the results were viewed as acceptable.

L

Most WVP rates for the samples (Table 11.) were comparable to those of film dressin s available today (436 862 g/m2/24 hours, or 18 - 35 g/m hour). An uncovered wound loses water vapor at the rate of 3400 - 5200 g/m2/24 hours (140-215 g/m2/hour). Excess exudate must therefore be removed by syringe aspiration as no matter how permeable the dressing may be, not all of the exudate will be removed from the wound by water vapor transport. l2

f

Average weight loss (g) PLA

PCL CH PLA/CH

1.53 1.63 3.59 .28

WVP: g/mL/24 hours Area = 320.79 /m2 170.02 522.89 1151.64 89.92

111. IN VITRO STUDIES The in vitro studies were conducted to determine the cytotoxicity of the films as a precursor to the in vivo study. There were two studies completed. The first dealt with any toxic affects the films might have on the cells. The second

E. Phase Five - Water Vapor Permeability

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~

study determined whether or not the films would promote cell growth on their surfaces. All films were EO-sterilized.

IV. CONCLUSION

Based on the findings, it can be concluded that the design considered was sound and a good candidate for further analysis. Topography of the films exhibited few pores. Therefore the results of the bacterial permeation experiment was not surprising. Both the WVP and bacteria studies gave good results, the films were permeable to water vapor, but provided a barrier to bacteria so the in vitro study was warranted. The in vitro study also showed promising results, as the fdms would support cell growth without cytotoxicity. The in vivo study, currently in progress, will be the culmination of these studies and will indicate whether further testing of the films using different conditions and manipulation of variables should be undertaken.

A. Cytotoxicity In this phase of the study, each film was exposed to a biological nutrient media for a period of 72 hours. Three cell plates, one for each polymer, were seeded with 75,000 cells/well and monolayers formed over a 72 hour period. The media was then introduced to the wells and incubated for 72 hours and 3 weeks. The cells were then harvested, stained for viability, and counted. Cell counts from the PLA and PCL extracts were high, but the CH counts were low. The study was repeated with yirradiated CH films which gave cell counts comparable to those of the PCL and PLA films. Therefore, it was determined that the EO sterilization had produce some effect on the CH film behavior.

REFERENCES

[ 11 G. Majno, M.D., The Healing Hand: Man and Wound in the Ancient World, Harvard University Press, Cambridge, 1975, p. 14. [2] Ibid., p. 46. [3] L. Magner, A History of Medicine, Marcel Dekker Inc., New York, 1992, p. 34. [4] G. Majno, M.D., The Healing Hand, 1975, p. 73. [5] L. Magner, A Histom of Medicine, 1992, p.73. [6] Ibid., p. 301. [7] S . Thomas, Wound Management and Dressings, The Royal Pharmaceutical Press, London, 1990, p. 9, reference 4. [SI Ibid., p. 10-11. [9] S. Salmon, The Biosynthesis and Morphology of Chitin, C V Polysaccharides, 1994, p. 35, reference 5132. [lo] Ibid., p. 37. [ I l l Schindler, A. et al., Bidemadable Polymers for Sustained Drug Delivery, Contemporary Topics in Polymer Science, Vol. 2, pg 251[12] S. Thomas, Wound Management and Dressings, 1990, p. 28.

B. Cell Growth Study

Films were first cut into small circles (10 mm) and secured with hemoclips onto sterile plastic 13 mm discs. The films/discs were then placed into the bottom of cell plate wells and covered with media. Cells were harvested and the cell suspension was then introduced into each well and incubated for 1 week or until a monolayer was formed. The plates were then examined to determine adherence of the cells as: no adherence to well or film surfaces, cell adherence only to well surface, or cell adherence to both well and film surfaces. Histologic evaluation was completed to qualitatively determine fibroblast morphology and fibroblast/film interactions. After 7 days, the cells had formed a monolayer on all of the wells and discs. On the PCL and PLA films, a monolayer was formed, but on the EO sterilized CH films cells if present were few and far between.

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