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Textile Composite Wound DressingA.P.London, A.E. Tonelli, S.M. Hudson, and B.S. GuptaDepartment of Textile Engineering, Chemistry, and ScienceK.B. Wylie and G.J. Spodnick, Colleg e of V eterinary MedicineB.W. Sheldon, Department of Food Science

Non h C arolina State UniversityMain Campus DriveR!aleigh, NC 27609-8301

Abstract--Poly lactic acid (PLA), poly ecaprolactone (PCL),and chitosan(CH) are incorporated into a biodegradable woun ddressing. Physical properties of these films, includingtopography, water vapor permeability, degradation, andbacterial permeation as well as biological effects of toxicity onhuman fibroblast cells were studied. An in vivo study (currentlyin progress is proposed.

I. INTRODUCTIONHistorically, people used the materials convenientlyavailable to them for care of wounds. This includeddressings as simple as herbs combined with leavai or ragsused by primitive man to those as complex as a synthesizedpolymeric com pound with an engineered multi-layer textilestructure.Natural adhesive was used as early as 4000 years ago, asresins were applied to rags for "stickiness".l EarlySumerians (around 2100 B.C.) ecorded the first use ofband ages. They described proced ures for th e treatment ofwounds; washing, m alung plasters, and bandaging. '2Egyptians used quick setting resins as well as plasters andadhesive tapes3 and the oldest bandages were found inEgyptian tombs.4 The Greeks and Romans used herbsboiled in water or win e and applied them d irectly to wounds.This acidic herbal layer, a treatment used in many othercultures, was thoug ht to help heal wounds. Mine rals withsalts or oxides of copper and lead were also though t to aid inhealing.5For the next several centuries, dressings did not progressfar beyond the traditional rag and herb dressin g. [t wasn'tuntil after World War I1 that medical science began toinvestigate other procedures and materials for dressingapplications. One major develo pmen t in wound treatme ntwas combating infections caused by poorly launderedhospital cloths.6~ In the 19 50 s, synthetic materials werefirst used to help indu ce healing.Although dramatic advances have been m ade by mo demscience, pain a nd comfort are still issues raised by patients.Today's dressing must be periodically removed, causingtrauma to the wound. This is compou nded if the dressing in

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question contains fibrids or loose particles that can becomeincorporated into the forming epidermis.Keeping a moist environment for the wound is one ofthe major considerations in developing a d re ~ s i n g .~Byincreasing moisture at the wound/ dressing interface duringthe early stages of healing, a scab doesn't form so cells arefree to move through the exudate. Also, the secondarydamage due to dehydration which can cause excess scartissue, is reduced.*All these concerns and more must be addressed in thedesign of a new dres sing. Unfo rtunately , at present, there isno dressing available on the market which does not have tobe removed, or w hich can keep adequate moisture around thewound.

A . Properties andDesignIn this study, a dressing is proposed which encompassedthe elements of a wound dressing with those properties thatare desired (Table I). By maki ng the dressing out of apolym eric film , flexibility and comfo rt needs are met. Bymak ing the dressing biod egradab le the need to remove it iseliminated and so is the pain associated with the removalprocess. And if the films are transparent, physicia ns canmonitor healing time and check for infection.

TABLE I.PRDPERTIEE OFTWE W O W DRESSING

0-7803-2083-2195 $4.000 1995 IEEE

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The p olymers available for such a dressing include manymaterials that are used for other medical applications, suchas in drug delivery systems and sutures. These includematerials such as poly-galactic acid (PGA), poly-lactic acid(PLA), poly waprolactone (F'CL), etc. and their cc-polymers. The three materials selected were PLA, PCL andchitosan (CH). The latter is a relatively new substan ceextracted from crustacean shell (Figure I.). As the figureshows, chitosan is the N-d eacetylated form of chitin.

1CH3 1

H O = C

ti

/

'x - -'YRepeating units for chitosan and chitin wherex: 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. Itallows for the protection of the wound during the entirehealing proces s. Chitos an is degrade d and absorbed into thesystem at a high rate, while the PLA or PCL layer isdegraded and absorbed much more slowly. Therefore, whenthe chitosan is completely absorbed into the body, the PLAor PCL layer is still present and continues to protect thewound from trauma . The gauze layer will absorb the excessexudate passing through the films in the form of watervapor, in addition to keeping the dressing in place on thewound.

11. PHYSICAL PROPERTIESA. Phase One - Film Construction

All films were solvent cast. Chito san was dissolved in 5%acetic acid and cast onto 1x3 ft . polycarbonate sheets.Chitosan acetate salts are highly water soluble, so theprepared films were soaked until neutral in amethanol/sodium hydroxide bath. The PCL and PLA films

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

gauzebandageE L r PLAChitosan

wound

Figure II. Sandwich construction of proposed dressing

B . Phase Tw o - DegradationDegradation rate is an important physical property ofbiodegradable dressings and can adversely affect healing ifnot controlled. All polymers degrade at different ratesdepending on the surrounding environment. Chitin forinstance degrad es differently depend ing on the moltin g cycleof the particular crustacean species and ratio of degradationto synthes is of new cuticle.9Enzymes contribute to the majority of biodegradation ofnatural substances. Chitin endocuticles (the inner layers) aredegraded by the chitonase enzyme in nature. The degradedcuticle is recycled to form the new shell and is exposed as

the old exocuticle is sloughed off during molting. Sampleswith a 70 % deacetylation can be degraded by the lysozymeenzym e if it contains a certain sequence of N-acetylatedresidues. loComparative viscosities were used initially to determinethis aspect. Films were exposed to a biological salinesolution for a period of 1-3 weeks. They were then re-dissolved to measure their viscosities and compare these tothe initial viscosities of the und egraded films.The CH films decomposed in 10-14 days, but the PLA andPCL films acted as hyd rogels and results were un satisfactory,as the excess cross-linking between the polymer and salinewill decrease degradatio n rates. A study by Schind ler et.al.shows that the degradation times of PLA and PCL basedsutures and drug delivery systems, are approximately 1 year.The films used in this study are extremely thin (0.008-0.035m m as measured by a Th wing-Albert Thickness tester); thedegradation times in the presence of enzym es and bacteriashould be ow and lie in the range of 2-6 weeks. The exactdegradation times of the PLA, PCL an d CH films will be.determined during the in vivo study currently in progress.

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C. Phase Three - Surface Topo graph y and Sterilization

Average weight loss WVP: g/mL/24(g) hoursArea = 320.79 /m2 ~

The surface topography of the fdms was examined bySEM before and after sterilization with ethylene oxide (EO).The non-conductivity of the films caused charging, sosamples were Au-Pd sputter-coated and overlaid with silvercolloidal paste. The pre-sterilized films showled littletopography, with minor ridges and bumps, possibly due toparticulates. After sterilization the films were muchsmoother and had relatively few visual surfacecharacteristics.The porous nature of the films was an importantconsideration in this phase; films had to have poi-es largeenough to allow water vapor to transport through, but notlarge enough to allow bacteria to penetrate into the: wound.Results of the SEM analysis revealed the absence ofmicroscopic pores large enough for bacteria, which are 2-3pm, to penetrate the films.

PLAPCLCHPLA/CH

D. hase Four - Bacterial Migration

1.53 170.021.63 522.893.59 1151.64.28 89.92

The question of b acterial migration becom es quiteimportant when a biodegradable dressing is used. In thebeginning , he dressing may be a good barrier to micro-organisms, 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, thesandwiched layer approach was thought to be the mastappropriate. As the chitosan degrades into the woun d, themiddle layer of polymeric film protects the wound from theanticipated microbial invasion.Science under the direction of Dr. Brian Sh eldon. T:he filmswere secured on BH I agar plates supplemen ted withnalidixic acid by placing a poly-vinyl chloride (PVC, 1 in.)ring over the films, resulting in a compression of the filmsinto the agar. Th e films, separately and sandw iched, werethen inoculated with 100pL of nalidixic acid resistantstrains of Staphylococcus aureus or Escherichia coli an dincubated at 37C for 5 days and monitored at 24 hoursintervals.While no g rowth was detected underneath the films, theproblem of the bacteria solution running off the film onto theagar was quite prevalent despite the use of the PVC retainerring. Bacterial growth was detected on the edges of all thefilms with the exception of the PLA and PLA/CH plates, butagain this was due to run-off. Because there was nodetectable growth beneath the film, the results were viewedas acceptable.

The study was conducted at the Department of Focd

E. Phase Five - Water Vapor Permeability

As stated previously, water vapor permeability isimportant in the transport of exudate from the wound. Thetest method was based upon ASTM E-96-92: Standard TestMethod s for Water Vapor Transmission of Materials.Each individu al film and sandwiched films were gluedonto m etal weighing pans containing 35 mL of distilledwater. The pans were placed upon a rotating platform in aroom at a constant standard temperature (24" C) and RH (50%). After 2 hours, at which time equilibrium should bereached, the pans were removed and weighed. After 24hours, the pans were re-weighed and w eight loss tabulated.Three separate runs were completed for each sample andwater vapor permeability was calculated using the followingequation:

I (1) Water Vapor PermeabilityAverage weight loss ) 1Area (m2) 124 hours =$/m /24 hourswhere Area = 1~ :2 r =3.15 cm ; 10,OOO cm2/A=l/m2Average weight loss = 3 runs13

L

Most WVP rates for the samples (Table 11.)werecomparable to those of film dressin s available today (436 -wound loses water vapor at the rate of 3400 - 5200 g/m2/24hours (140-215 g/m2/hour). Excess exudate must thereforebe removed by syringe aspiration as no matter howpermeable the dressing may be, not all of the exud ate will beremoved from the wound by water vapor transport. l2

862 g/m2/24 hours, or 18 - 35 g/m f our) . An uncovered

111 IN VITRO STUDIESThe in vitro studies were conducted to determine thecytotoxicity of the films as a precursor to the in vivo study.There were two studies completed. The first dealt with anytoxic affects the f i lms might have on the cells. The second

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

A . CytotoxicityIn this phase of the study, each film was exposed to abiolog ical nutrient media for a period of 72 hours. Threecell plates, one for each polymer, were seeded with 75,000cells/well and monolayers formed over a 72 hour period.The media was then introduced to the wells and incubatedfor 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, butthe CH counts were low. The study was repeated with y-irradiated CH films which gave cell counts comparable tothose of the PCL and PLA films. Therefore, it wasdetermined that the EO sterilization had produce some effect

on the CH film behavior.B . Cell Growth Study

Films were first cut into small circles (10 mm) an dsecure d with hem oclips onto sterile plastic 13 mm discs.The films /discs were then placed into the bottom of cell platewells and cove red with media. Cells were harvested and thecell suspension was then introduced into each well andincubated for 1 week or until a monolayer was formed.The plates were then examined to determine adherence ofthe cells as: no adherence to well or film surfaces, celladherence only to well surface, or cell adhe rence to both welland film surfaces. Histologic evaluation was completed toqualitatively determine fibroblast morphology andfibroblast/film interactions.After 7 days, the cells had formed a monolayer on all ofthe wells and discs. On the PCL and PLA films, amonolayer was formed, but on the EO sterilized CH filmscells if present were few and far between.

IV. CONCLUSIONBased on the findings, it can be concluded that the designconsidered was sound and a good candidate for furtheranalysis. Topo graph y of the films exhibite d few pores.There fore the results of the bacterial perme ation expe rimen t

was not surprising. Both the WVP and bacteria studies gavegood results, the films were permeable to water vapor, butprovided a barrier to bacteria so the in vitro study waswarranted. The in vitro study also sho wed promising results,as the fdms w ould supp ort cell growth without cytotoxicity.Th e in vivo study, currently in progress, will be theculmination of these studies and will indicate whetherfurther testing of the films using different conditions andmanipulation of variables should be undertaken.

REFERENCES

[11 G. Majno, M.D., The Healing Hand: Man andWound in the A ncient World, Harvard University Press,Cambridge, 1 975, p. 14.[2] Ibid., p. 46 .[3] L. Magner, A History of M edicine, Marcel Dekker Inc.,New Yo rk, 199 2, 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 M anageme nt and Dressings,The Royal Pharmaceutical Press, London, 1990 , p. 9,reference 4.[SI Ibid., p. 10- 11.[9] S . Salm on, The Biosynthesis and Morphology of Ch itin,Polysaccharides, 1994, p. 35, reference 51 32 .[lo] Ibid., p. 37.[ I l l Schindler , A. et al., Bidem adable Polymers forSustained Drug Delivery, Contemporary Topics inPolymer Science, Vol. 2, pg 251-[12] S. Thomas, Wound M anagemen t and Dressings,

C V

1990, p. 28.

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