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UhiC FILE COPYEFFECTS OF BIOSYNTHETIC HUMAN EPIDERMAL

o GROWZH FACTOR ON WOUND HEALING

4

6- Annual Report

October 27, 1986 ELEC.,

Dr. Gregory Schultz

Supported by:

U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21701-5012

Contract No. DAMDL7-85-C-5197

University of LouisvilleSchool of Medicine

Louisville, Kentucky 40292

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Louisville, Kentucky 40292

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Research & Development Command DAMDI7-85-C-5197

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Fort Detrick, Frederick, Mr 21701-5012 ELEMENT NO. NO. 3S162. NO. ACCESSION NO.62772A 772A874 AD 122

11. TITLE (Includie Security Classification) HrFe(U) Effects of Biosynthetic Human Epidermal Growth Factor on Wound Healing

12. PERSONAL AUTHOR(S)Gregory S. Schultz, Ph.D.

113.. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (Year, Month, Oay) 1`. PAGE COUNT

Annual Report FROM 30/QL8 TO9.2L9/84 1986 October 27 1416. SUPPLEMENTARY NOTATION

17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number)FIELD GROUP SUB-GROUP Lab animals, volunteers, Silvadene, wound healing, growth

lb ifactors

19. ABSTRACT (Continue on reverse if necessary and identify by block number) •*We are investigating the ability of

biosynthetic epidermal growth factor (EGF) to accelerating healing of cutaneous injuriesincluding middermal wounds and incisions. In preclinical animal studies, EGF significant-ly increased the rate of epidermal regeneration 2-fold in pigs following second degreethermal injuries or middermal incisions. EGF also induced hyperplasia of the epidermalcell layer which remodeled to normal skin architecture two weeks after complete healing.Two peptide growth factors structurally related to EGF, vaccinia growth factor (VGF) andtransforming growth factor-u (TGF-a), also accelerated epidermal regenera-trion in middermalburns. A single application of EGF increased the tensile strength of cutaneous incisionsin rats during the early phases of healing but only when formulated in a slow releasevehicle such as liposomes. The rapid loss of EGF from the area of li cisions when formu-lated in saline or viscous materials such as hyaluronic acid probably prevents its ac-tions. Clinical trials with EGF in donor site middermal incisions are underway to evalu-ate its clinical usefulness. '1,[ /...* .• ', ",. • - . ,;,,." --

20. DISTRIBUTION/AVAILABILITY OF ABSTRACT 21 ABSTRACT SECURITY CLASSIFICATION .

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Mrs. Judy Pawlus 301-663-7325 SGRD-WII-S

DO Form 1473, JUN 86 Previous editions are obsolete. SECURITY CLASSI"ICATION OF THIS PAGE

STA'&U• OF PROBLNMI M ' STUDY

At the present time, accelerating healing of major injuriessuch as middermal burns (second degree) or incisions is beyondthe scope of clinical medicine. However, there is no doubt thatmuch of the morbidity, mortality, and cost associated with suchmajor injuries is closely related to the length of time requiredfor them to heal. The major goals of this contract are todevelop and evaluate new methods to accelerate healing of burnsand incisions using biosynthetic peptide growth factors tostimulate normal healing processes.

BACKGROUND AND REVIEW OF APPROPRIATE LITERATURE

Epidermal regeneration. Patients with extensive second andthird degree burns are at increased risk of developinglife-threatening infections and other complications until theirepidermal layer has regenerated or been replaced with autograftskin. The limiting factor in covering major third degree burnsis the availability of autogenous skin grafts. Although cadaverand porcine skin or placental membranes are reasonable temporarysubstitutes, they are limited by the inevitable generation ofimmune responses to the foreign tissue. Similarly, syntheticskin, which has been difficult to produce in large amounts,eventually requires grafting with tho patient's own skin. Sincethe only permant solution currently available is autografting, anagent that would accelerate healing of skin graft donor siteswould decrease the morbidity and mortality of severely burnedpatients.

Epidermal growth factor (EGF), a small peptide (3458 mw)found in urine and blood of man and other animali, has potentmitogenic effects on epidermal cells in vitro (1). Multiplestudies have tested the effect of EGF on epithelial cells invivo. Brightwell et al. (2) reported that repeated Lopicaldosing of EGF accelerated epithelial regeneration of primatecorneas, and in unpublished results, EGF treatment produced totalhedling of patients with chronic corneal epithelial defects (R.Eiferman, personal communication). Results of studies investi-gating action of EGF cun healing cutaneous wounds have beeninconclusive and conflicting. Greaves (3) reported that brief (5min' exposure uf suction-',nduced epidermal wounds to EGe insaline solution once daily failed to accelerate wound closure.Arturson (4) also failed tU fird enhanced healing of midderm.~Lthermal burns in rats treated once daily for 20 seconds with EGFin saline. Thorton et al. (5) applied EGF once daily as a mistto middetmal buLns on rats and did ,ot observe an increasre inrate of healin>I. Franklin and Lynci. (6), however reportedqu, litý.tive improvement in closuer of full-thickness wounds in U•abbit Pars when treated with EGF in an ointment. The studieswhich failed to show an acceleration of healing may have not-provided adcquatc continu':z c..iz,,ourc. of Lhe remidual epidermal .LUiý: t( the EGF. Studies in vitro have clearly shown that themitogenic effect of EGF requires continuous exposure of cells toEGF for a minimui, of 6 to 12 hours (7). ,..Odes

0 .) t V . .J ci , ý r0$Jf o-61

'4

Incision healing. Treatment of sutured incisions is limitedby the inability to easily reapply material in the tract of theincision once it has been closed. Thus, a single application ofEGF in a saline vehicle might be ineffective if EGF was notretaiaed in the incision tract for an adequate period. Buckleyet al. (8) implanted polyvinyl sponge disks subcutaneouslybeneath the ventral panniculus carnosus in rats and measuredcollagen content 20 days later. Daily injections of EGF into thesponges increased collagen content 26% compared to saline injec-tion. Since the EGF rapidly disappeared from the injection site(only 10% remained after 4 hours), EGF was formulated in slowrelease pellets which were placed in the implated in the sponges.At day 7, sponges receiving 10 ug of EGF released per day con-tained 41% increase in collagen content and pronounced increasedin cellularity (DNA) and vascularity compared to saline controlsponges. Similar results were reported by Laato et al. (9)following daily injections of EGF into cylindrical hollow spongesimplanted subcutaneously on backs of rats,and by Grotendorst etal. (10) with collagen filled chambers containing EGF or plateletderived growth factor (PDGF).

RATIONAL USED IN CURRENT STUDY

Based on the in vitro and in vivo data discussed above, itseemed apparent that EGF could stimulate epidermal healing andcutaneous healing if applied in appropriate formulations. Themodel we chose for testing epidermal regeneration was middermalthemal burns and split-thickness incisions (donor site injuries)in pigs. This animal model offered several distinct advantagesover rats or guinea pigs. Pigs are large enough to sustainnumerous test burns on the same animal without imparing thegeneral health of the animal allows several test to be performedon the same animal. Thus, more direct comparisons can be madebetweern experimental and control burns by minimizing variabilitiyin healing rates between different animals. Also, pig skin isvery similar in structure and healing properties to human skinand pigs have been used extensively in burn research. Theexperimential protocol would be to test EGF in different formula-tions including saline, lanolin, and a commercially availableantibiotic cream, Silvadene. Both split thickness incisions andmiddermal thermal burns would be utilized as models.

To study healing of cutaneous incisions, we chose to useinterscapular incisions of rats. This animal model offered theadvantages of low cost per animal and the ability to produce anincision long enough to obtain miltiple samples from each inci-sion for tensile strength measurements. Also, rats have beenused extensively for studies of cutaneous incision healing. Theexperimental protocol would be to test EGF in different mehiclesincluding saline, singLye Ldme.Lar liposomes, multilamellailiposomes, and viscous polymer solutions. Tensile strengthmeasurements and histology would be perfcrmcd.

3

~~ERIN3AL N•!D

Epidermal Regeneration. Experiments were conducted as de-scribed by Brown et al. (11). Eighty four (84) split-thicknesscutaneous wounds (0.005 inches thick, lxl cm) were made on thedorsal thorax of four adult miniture pigs (Vita-Vet Laboratories,Marion, ID). Twice a day, 28 wounds were treated with EGF (10ug/ml) in lanolin or Silvadene, 28 were treated with the creamalone, and 28 were untreated. Four (4) wounds from each treat-ment group on each pig were randomly selected each day andtotally excised to a depth of 0.007 inches, including 5 mm ofsurrounding normal skin. Epidermal and dermal layers of thespecimens were separated after incubation in trypsin solutionovernight. Wounds were considered healed when no defect waspresent in the epidermal layer and not healed if any defectremained.

Middermal thermal burns were made by 10 seconds of contactwith a brass template (3x5 cm, 430 g) heated to 70 C in aconstant-temperature water bath to the depiliated dorsal skin ofpigs (7-14 kg). After the resulting blister was removed, burnswere treated twice a day with either EGF in Silvadene, Silvadenealone, or untreated. After 7 days, the fibrinous coagulum wasremoved and burns were photographed and biopsey specimens taken.The percentage of the original burn area that appeared to bereepithelized was calculated by computerized planimetry ofenlarged photographs.

Cutaneous incision healing. Adult male Spraque Dawley ratswere anesthetized with Ketamine and Rompum and a single incision5 cm long was made in the dorsal midline at the caudal portion ofthe back of each rat. Test solutions were placed in the tract ofthe incision, and incisions were closed with five (5) evenlyplaced interrupted horizontal mattress sutures of 4-0 nylon.Rats were housed in individual cages with unrestricted feed andwater. At intervals after surgery, rats were sacrificed bycarbon dioxide inhalation, arnd incisions together with surround-ing normal skin were dissected to the panniculus carnosum, thenplaced on ice-cooled dishes until tested for tensile strength.For each incision, three strips of tissue were cut perpendicularto the original incision, and measured for tensile strength usinga commercial instrument (UNITE-O-MATIC tensiometer). The valuesof the three strips were averaged to determine the tensilestrcngth for each incision. For each experimental condition, thetensile strength was calculated by averaging the tensile strengthfor all incisions in that test group. Typically, ten (10) ratswere used for eaci experimental condition at each time point.Specimens for hi', ology were placed in 10% neutral bufferedformarin, pucui, Lhluiugh pitiftn, Sectioned, arv' sLairne2d withhematoxyline and 'vine.

Multilamel. liposomes cuntaining EGF wcrc prepared byadding lecithin (50 mg) to a 12x75 glass test tube and the hexanevehicle was evaporated under a stream of nitrogen. One ml ofphosphate buffered saline (PBS) containimg 1mg of EGF was addedand immediately vortexed and sonicated, then the liposomes were

4

separated from the solution by centrifugation at 20,000 x g for20 min or by gel filtration through a 25 cm column of Agarose 4B.The amount of EGF entraped in 11osomes was calculated by measur-ing the amount of a trace I-EGF present in the purifiedliposomes. Single lamellar liposomes also were prepared asdescribed previously (12). Briefly, a mixture of lipids contain-ing lecithin, phosphatidylglycerol, and cholesterol were added toa round bottom flask, 5ml of ether added and dried under anitrogen stream. Five ml of ether was added followed by iml ofEGF (Img/ml) in PBS then immediately sonicated. The organicphase was removed by rotary evaporation and the single lamellarliposomes were formed, then purified by gel filtration orcentrifugation as before.

To measure the retention of EGF in incisions, multilamellarand single lamellar liposomes, as well as a solution of EGF inPBS, were prepared as above with a trace amount of 125I-EGFincluded to enable measurement of EGF by scintilation counting.Incisions were made as before and a known amount of EGF wasplaced in the incision tract then the incision was closed withsutures. At intervals, the incisions and surrounding skin wereexcised and counted.

RESULTS

Epidermal regeneration. As shown in Figure 1, 50% of thesplit-thickness incisions treated with EGF in lanolin or inSilvadene were healed after two (2) days of treatment, whereasvehicle-treated or untreated groups required greater than four(4) days for 50% of the wounds to heal (p<0.05, chi-squaredanalysis). Histological evaluation of healed split-thicknesswounds were similar in appearance for all three groups woundswith a 10 to 12 cell layers of stratified epithelium characteris-tic of normal pig skin and no evidence of metaplastic transforma-tion.

EGF-treatment of middermal thermal burns significantly(p<0.01, Tukey's analysis of variance) increased the area ofregenerated epidermis at 7 days after injury (Table 1). Biopsyspecimens taken 7 days after burn injury confirmed completeepidermal regeneration of EGF-treated burns with stratificationof cell layers. In contrast, very little epidermal regenerationwas observed in vehicle-treated or untreated burns. Specimenstaken 35 days after burr injury appeared totally normalhistologically.

Response of middermal burns to varying doses of EGF is shownin Figure 2. All burns treated with EGF (0.1, 1, and 10 ug/ml)were significantly more healed than untreated burns, but onlyburns treated with EGF at 10 ug/ml were significantly more healedthan vehicle-treated burns (p<0.05, analysis of variance).

Treatment of middermal burns with TGF-c or VGF producedresults similar to those observed with EGF-treatment. As shownin Table 2, treatment with TGF-c or VGF resulted in substantiallymore area re-epithelized than untreated or vehicle-treated burns.Also, low levels (0.1 ug/ml) of TGF-o. appear to be more effective

than low levels of EGF in stimulating epidermal regeneration(Figure 3).

Cutaneous incision healing. Treatment of incisions at thetime of suturing with EGF in saline failed to produce an in-crease in tensile strength even at high levels of EGF (100 ug).In contrast, when repetitive doses of ZGF were given three timesa day for 5 days (75 ug per injection) via a porous indwelingcathater underlying the incision, EGF-treated incisions wereapproximately 35% stronger than saline-treated incisions at 7days (p<0,01, Figure 4). Furthermore, a single treatment with atotal of 3 ug of EGF encapsulated in multilamellar liposomes(ML-EGF) caused approximately 300% (p<0.05, T-test) increase intensile strength at 10 and 14 days post-incision compared toblank liposomes or untreated incisions (Figure 5). Histolgy of 7day incisions treated with EGF in liposomes showed hypertrophy ofthe overlying epidermis and substantially more fibroblast-likecells in the area of the incision tract compared to controlincisions. Electron microscopy of 14 day incisions treated withEGF showed many active fibroblasts present with extensive roughendoplastic reticulum and more new collagen present than controlincisions. Specimens at 28 days treated with EGF showed largenumbers of very active fibroblasts, extensive well organizedcollagen with sparse ground substance giving the overall appear-ance of a very strong, mature scar, while control specimensretained large amounts of edematous and spongy ground substanceindicating earlier stages of wound repair.

These results suggested that a major factor in the effec-tiveness of EGF in vivo is the length of exposure of incisions toEGF. When EGF formulated in saline, hyaluronic acid,multilamellar or single lamellar liposomes was placed in tractsof incisions, and the percentage of EGF remaining in the regionsurrounding the incisions was measured daily was determined, theresults shown in Figure 6 were obtained. After one day, only 20%of the EGF formulated in saline or hyaluronic acid remained inthe region surrounding the incision. EGF encapsulated in singlelamellar liposomes also was rapidly lost from the incision area,but EGF encapsulated in multilamellar liposomes was substantiallyretarded in the area of the incision with 40% still retained atthree days.

DISCUSSION AND CONCLUSIONS

The effects of EGF in vitro on epidermal and stromal cellssuggest that processes of wound healing might be accelerated invivo by exposing tissues to EGF. Our results support thishypothesis and reinforce the need to appropriately formulate EGFto provide constant exposure of target cells with EGF. Thefailure of previous studies of epidermal regeneration followingmiddermal burns or split-thickness incisions which used EGF invehicles or regimcns which limited exposure time of EGF may beexplained on this bdiis. Similar principles must Le considersdin healing cutaneous incisions. No increase in tensile strengthof incisions was found when EGF was formulated in saline or

6

E~EI I- II

hyaluronic acid which allowed rapid exit of EGF from incisions.In contrast, large increases in tensile strength were produced byEGF encapsulated in liposomes which delayed the release of EGFfrom incisions or when EGF was repeatedly infused into incisions.

The results reported here demonstrate the 'proof of princi-ple' that biosynthetic peptide growth factors can acceleratewound healing in vivo. Additional research needs to be conductedincluding evaluation of EGF in combination with other wellcharacterized growth factors such as PDGF and fibroblast growthfactor (FGF). It is probable that synergistic actions wouldoccur with combinations of growth factors producing even greateracceleration of healing. Another area for further investigationis development of better slow-release vehicles for EGF. Biode-gradable microencapsulation materials which could be designed torelease EGF at different rates would likely improve healing.Preliminary discussions with material scientists at Ethicon Inc.indicate that it is possible to incorporate EGF in microspheresof polyvicryl which can be constructed to have different rates ofbiodegradation. Additional valuable information will be obtainedwhen the limited clinical trial using EGF in Silvadene to treatskin graft donor sites begins at Emory University in late 1986under the direction of Dr. Gregory Brown.

7

1. J G Rheinweld and H Green. Epidermal growth factor andthe multiplication of human epodermal keratinocytes.Nature (London) 256:421, 1977.

2. J R Brightwell, S L Riddle, R A Eiferman, P Valenzuela, P JBarr, J P Merryweather, and G S Schultz. Biosynthetic humanEGF accelerates healing of neodecadron-treated primatecorneas. Invest Ophthalmol Vis Sci 26:105, 1985.

3. M W Greaves. Lack of effect of topically appliedepidermal growth factor on epidermal growth in man invivo. Clin Exp Dermatol 5:101, 1980.

4. G Arturson. Epidermal growth factor in the healing ofcorneal wounds, epidermal wounds, and partial-thicknessscalds. Scand J Plast Reconstr Surg 18:33, 1984.

5. 1 W Thorton, C A Hess, V Cassingham, and R H Bartlett.Epidermal growth factor in the healing of second degreeburns: a controlled animal study. Burns 8:156, 1981.

6. J D Franklin and J B Lynch. Effects of topicalapplications of epidermal growth factor on wound healing.Plast Reconstr Surg 64:766, 1979.

7. G Carpenter and S Cohen. Human epidermal growth factorand the proliferation of human fibroblasts. Cell Physiol88:227,1975.

8. A Buckley, J M Davidson, C D Kemerath, T B Wolf, and S CWoodward. Sustained release of epidermal growth factoraccelerates wound repair. Proc Natl Acd Sci USA 82:7340,1985.

9. M Laato, J Niinikoski, B Gerdin, and L Lebel. Stimulationof wound healing by epidermal growth factor. Ann Surg203:379, 1986.

10. G R Grotendorst, G R Martin, D Pencev, J Sodek, and A KHarvey. Stimulation of granulation tissue formationby platelet-derived growth factor in normal and diabeticrats. J Clin Invest 76:2323, 1985.

11. G L Brown, L Curtsinger, III, J R Brightwell, D MAckerman, G R Tobin, H C Polk, Jr., C George-Nascimento,P Valenzuela, and G S Schultz. Enhancement of epidermalregeneration by biosynthetic epidermal growth factor. JExp Med 163:1319, 1986.

12. F Szoka,Jr., and D Papahadjopoulos. Procedure for prepartionof liposomes with large internal aqueous space and highcapacity by reverse-phase evaporation. Proc Nut' Acad SclUSA 75:4198, 1978.

8

to

40.

100

12: 2 "••N•o

G4O

0-

0 1 2 3 4 5 a 7DAYS AFTER W•OWCG

FiCL'RE I. Effects of hEGF and vehicles on healing of split-thickness wounds. Each pointrepresents the data from 16 wounds treated with hEGF (10 0ag/ml) in the indicated vehicles.with vehicles alone, or with no treatment. Values were compared for statistical significance byx analysis. WMB. water-miscible base (Silvadene).

TABLE I

Affect of hEGF and Vehicle on Healing of Parial-thickness BurnsTreatmen. Percentage of burn

area healed

hEGF 93 ± 6Control 36 ± 18Untreated 17 ± II

p < .o) I sirig one-way analysis of variance and "'ukev's Honest Statisticall)ilference (HSi)) test. Quantitative planinictrv nreasurements were pre-.fornied oin photographs of partial-Wlhickness horns that were treated for 7d w'ith hFF(; in Silkadiee (10 pg/nil), Silvadene alone (cont rol) or in-

r.reated. \'ahies are mean ± SD for eight , ounds.

9 a

I I

400

20.

Contrl W'W' O.V,.'n i I~Kb#nv

DOSE OF hEOF

FIGURE 2. Response of partial-thickness burns to varying doses of hEGF. Partial-thicknessburns (3 X 3 cm) were treated for 7 d with Silvadene alone, Silvadene containing hEGF (10$Ag/ml, I ,sg/ml, 0.1 #g/ml), or were not treated. The percentage of original burn area thathadc healed was measured by quantitative planimetry of photographs. Each data point is themean : SID for four burns. WMB, water miscible base (Silvadene).

Ne 100-

0

-4

505.

0.2

C0

A B C D E

Figure Rs Relative abilities of various treatment regimens to facilitate

epithelial wound healing. The growth factors were all testA at 0.1 '4g/ml.

This concentration has previously been shovn to be suboptimal for EGF (2).

Conditions arei A. Untreated; B. SilvadLneR ,1lone; C. SilvacdeneR and EGF;

D. SilvadeneR and rGF; FE. SlivadeneR and VGF. Results were scored at 9-10

days and are the average of two or more experiments using different test

animals. Error bars are mean t standard deviation.

1 fl

Table 2. pithelial regeneration In mld-dermal burns treated vith TGF-m or VGV

Percent of original burn area with regenerated epithelium

VGr (•*/ml)

Vehicle Untreated 0.1 0.5 1.0

right oide 68 t 6 10 1 1 96 t 2 95 t 1 85 t 3

left side 76 *1 6 * 4 92 t 1 90 * 2 681*

animal 2

rTCF-a fiut/al)

.Vehicle Untreated 0.1 1.0 10

right side 43 t 5 30 t 6 98 * 1 73 t 1 90 ± 1

hTGF-a (ulmi).

Vehicle Untreated 0.1 1.0 5.0

left aide 58 t 2 17 t 3 86 * 4 84 4 66 ± 6

Kid-dorsal thermal injuries were made on the dorsal thorax of anesthetized

female Yorkshire pigs (30 lbs) whose backs had been shaved and dipilitated

with commercial hair cream remover (2). A brass template (3 x 3 Co, 147 jm)

was equilibrated in a 701C water bath and placed in fLire contact with the skin

for exactly 10 eec and the resulting blister was removed. Five mid-dermal

burns were placed on each side of the spine and were separated from each other

by approximately one inch. Burns were treated twice a day with approximately

3 ml of vehicle cream (Silvadenes) alone or containing growth factor or were

untreated. Synthetic TGF-a, both human and rat, was purified to homogeneity

by reverse phase chromatography as previously described (ý). VGF was purified

11

0

II

PB E6a ?1UJ

b!

Figure 4. Tensile strength of incisions treated with EGF.Single incisions 5 cm long were made to the panniculuscarnosum in the dorsal midline at the caudal portion of ratsand closed with five interrupted sutures. EGF (75 i•q orsaline was injected three times daily for five days via anindwelling perforated catheter underlying each incision,then incisions excised and tensile strength measuret. at 7and 14 days. Values represented are mean and standarddeviation of ten incisions. Paired-T test used for statis-tical comparison.

12

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INTEPOCAPUuAN FAT VI*ONS

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figure 5. Tensile strength of incis'ions treated with2O7-liposomes. Single incisions S am long were Made to thinpanniauluu carnoiwi in the dorsal midline at the caudalportion of rats and tG7 13 "q) encapsulated in multilamiellarliposomes or blank ILposomes placed in the tract Mf theincision which was than closed with five interrupted su-tures. Incisions were excised at weekly intervals Andtested for tensils strength. Values represented are themean of seven inc~isions and T-text wasn used for statisticalcomparisons.

13!

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PE 9 RAN INMWOUNDAsA MWONO VEHIL

* LMUA&ELLAR., U.POSOMISo NiALELAMLLAR LPOISOMS0 SAluLAW NEoo

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FliG (-, UAIAI3SUM Of aV gINAINIMAN INCISIN SITe. to Vivo. IODIIAT9D EF IN

MATILAIM LIMONSII. UNIALMH.I M LIPOWIIS, OR SALIN WAS PLACEO INTO TE TrACT

OF A 4 0 INTKNCAU•.LM INCISION WHICH WAS CLOIUD USING 4.0 NYLON IN A RUNNING

SUTUNE, AT INMICATIO TIlES, ANIMIALS WIN SACRIFICED AND 0.5 m Of TISSUE WAS UICISED

FOUR IAOI10 Of WOUND. TIiM NOAS WllEDO AND IODINA1D t11 WAS MEASURED USING

A CNIUM GAPMA COUNTER,

14