Polymeric Materials and Artificial Organs - American Chemical Society

12 Infected Skin Wounds in Rodents Treatment with a Hydrogel Paste Containing Silver Nitrate

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P. Y. WANG Laboratory of Chemical Biology, Institute of Biomedical Engineering, Faculty of Medicine, University of Toronto, Ontario, Canada M5S 1A8

A hydrogel paste was prepared by cross-linking clinical grade dextran with epichlorohydrin. When applied to clean, p a r t i a l thickness rodent skin wounds, this paste formed a protective layer to reduce f l u i d loss, prevent eschar formation, and minimize wound contraction. For P. aeruginosa infected wound, AgNO was mixed to obtain a milky white paste which later became a non-staining, soft brown coating on wound surface. The preparation could adequately absorb wound exudates and reduce evaporative loss. P a r t i a l thickness dorsal skin wound, 3x3.5 cm , was created on anesthetized Wistar rats by dissection, and then infected with 10 bacteria/cm . I f the AgNO -dextran paste was applied to the infected wound after 24 hr, healing was delayed. At day 6, high bacteria counts were obtained in separated eschar and sometimes also in internal organs similar to infected controls using plain paste. However, when the medicated paste was applied within hours after infection, the wound healed as uninfected controls in about 10 days, and sequential samplings of the healing wound surface showed few viable organisms. The internal organs were also s t e r i l e . Therefore to be effective, the AgNO -dextran hydrogel paste should be applied to the wound surface soon after injury. 3

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Skin is a m u l t i - l a y e r e d structure. Its resistance to the p e n e t r a t i o n of harmful substances and to the l o s s of v i t a l body f l u i d s by evaporation r e s t s e s s e n t i a l l y w i t h the outermost

0097-6156/ 84/ 0256-0181 $06.00/ 0 © 1984 American Chemical Society

In Polymeric Materials and Artificial Organs; Gebelein, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1984.


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stratum corneum l a y e r which i s a heterogeneous t i s s u e comprised of dead, k e r a t i n i z e d , p a r t i a l l y d e s i c a t e d epidermal c e l l s , extractable l i p i d s , etc. Damage to t h i s l a y e r r e s u l t s i n evaporative l o s s which i n i t i a t e s a s e r i e s of body p h y s i o l o g i c a l changes as w e l l as in bacterial i n g r e s s which causes s u b s t a n t i a l h e a l i n g delay due t o t o x i n s and enzymes r e l e a s e d . Therefore, r e s t o r i n g the b a r r i e r i s an important i n i t i a l step i n the complex scheme of s k i n i n j u r y treatment. Many d r e s s i n g s have been used t o serve as a temporary b a r r i e r u n t i l n a t u r a l h e a l i n g occurs or u n t i l the wound s i t e i s ready t o r e c e i v e an a u t o g r a f t . At present, commercially a v a i l a b l e d r e s s i n g s i n c l u d e c o t t o n gauze coated w i t h petrolatum or s u l f u r - p e t r o l a t u m m i x t u r e s , non-adherent p e r f o r a t e d p l a s t i c sheets w i t h inner absorbent m a t e r i a l s , absorbent f a b r i c pads w i t h a t h i n aluminum l a y e r , water vapor permeable polyurethane sheets, nylon v e l o u r backed w i t h a s i l i c o n e membrane, e t c . I n a d d i t i o n , there are many new d r e s s i n g s i n the developmental stages ( 1 , 2 ) . R e c e n t l y , h i g h l y absorbent, c r o s s - l i n k e d dextran beads have been introduced i n the treatment of s k i n d e f e c t s due t o p e r i v a s c u l a r diseases ( 3 ) . The c l i n i c a l r e s u l t s have been reported t o be very promising, apparently because the dextran beads absorbed exudates q u i c k l y which reduced the chances f o r f u r t h e r b a c t e r i a l growth and probably a l s o helped t o expedite wound h e a l i n g . The beads are made by e p i c h l o r o h y d r i n c r o s s - l i n k i n g of an aqueous a l k a l i n e dextran emulsion i n toluene ( 4 ) . For e x t e n s i v e or i r r e g u l a r s k i n wounds, i t i s d e s i r a b l e t o have sheets or tacky pastes of the c r o s s - l i n k e d dextran material. These m a t e r i a l s have been made and c h a r a c t e r i z e d as b r i e f l y r e p o r t e d i n the previous Symposium h e l d i n March 1980 at Houston ( 5 ) . The present study evaluates the performance of a c r o s s - l i n k e d dextran paste c o n t a i n i n g 1% AgN0 on i n f e c t e d rodent s k i n wounds. 3

Expérimentais Materials. C l i n i c a l grade dextran was purchased from Dextran Products L i m i t e d , Toronto. E p i c h l o r o h y d r i n used f o r p r e p a r i n g the hydrogel paste was s u p p l i e d by A l d r i c h Chemical Co., I n c . , Milwaukee, Wis. Concentrated sodium hydroxide s o l u t i o n , the polyhydroxy humectant, petrolatum and s i l v e r n i t r a t e are ACS or USP grade products obtained from F i s h e r S c i e n t i f i c or Canlab L i m i t e d , Toronto. The P. aeruginosa bacterium was acquired from the t e a c h i n g l a b o r a t o r y of our M e d i c a l School, and the n u t r i e n t agar p l a t e s f o r growth are prepared by our C e n t r a l S e r v i c e s at the F a c u l t y of Medicine. Hydrogel P r e p a r a t i o n . The smooth and uniform dextran paste was prepared by i n s o l u b i l i z i n g an a l k a l i n e dextran s o l u t i o n w i t h


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Infected Skin- Wound Treatment

e p i c h l o r o h y d r i n ( 6 ) . A f t e r exhaustive washings w i t h d i s t i l l e d water, and homogenization, the s o l i d content of the f r e e f l o w i n g t h i n paste was increased by evaporation t o a d e s i r e d c o n s i s t e n c y t h a t gave the best h a n d l i n g q u a l i t y . An amount of 1.25 g AgN0 was d i s s o l v e d i n d i s t i l l e d water, and added g r a d u a l l y i n s m a l l , approximately equal p o r t i o n s t o 100 g of the paste c o n t a i n i n g g l y c e r i n and s m a l l amount of petrolatum, w h i l e the paste was being kneaded. The m i l k y paste was t r a n s f e r r e d i n 10-g p o r t i o n s i n t o opaque g l a s s j a r s and capped u n t i l use. Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 10, 2015 | http://pubs.acs.org Publication Date: June 8, 1984 | doi: 10.1021/bk-1984-0256.ch012

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Volume o f Exudating F l u i d . I n order t o ensure t h a t the amount of the AgN0 -medicated paste a p p l i e d w i l l be adequate t o absorb the oozing serous f l u i d from the wound s i t e , a 3x3.5 cm wound was c r e a t e d on s e v e r a l a n e s t h e t i z e d W i s t a r r a t s (body wt.: about 400 g) by e x c i s i o n which removed t h i n s k i n l a y e r s t o expose the hypodermis. The wound was cleaned and covered w i t h a l a y e r o f impermeable p l a s t i c f i l m formed a f t e r s o l v e n t evaporation of an a e r o s o l p r e p a r a t i o n ( A e r o p l a s t ; Parke, Davis & Co., B r o c k v i l l e , O n t a r i o ) . At convenient i n t e r v a l s , the accumulated serous f l u i d under the p l a s t i c f i l m was a s p i r a t e d through a 25-gauge needle and the volume was read from the gradations on the s y r i n g e . 3

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Evaporative Loss Through Hydrogel Coating. S i m i l a r wounds were induced on the d o r s a l s k i n of a n e s t h e t i z e d W i s t a r r a t s as d e s c r i b e d i n the preceding S e c t i o n and the medicated hydrogel paste was spread evenly t o a t h i c k n e s s of 3-5 mm. The paste c o a t i n g extended about 0.5 cm beyond the margin o f the wound, and a f t e r 4-6 h r when the paste became much l e s s t a c k y , the evaporative l o s s o f the covered wound was measured over the paste c o a t i n g w i t h a hygrometer ( 7 ) . The c o n t r o l was e i t h e r a s i m i l a r open wound o r a l a y e r o f the hydrogel paste spread t o comparable t h i c k n e s s on a t h i n polyethylene sheet and allowed t o become l e s s tacky before hygrométrie measurements were taken. E v a l u a t i o n on I n f e c t e d Wound. Twenty-two male W i s t a r r a t s were d i v i d e d i n t o 3 groups o f 6, 10, and 6 animals. A wound of 3x3.5 cm s i z e was i n f l i c t e d on the back o f each o f the 6 r a t s i n the f i r s t group as d e s c r i b e d above. Two animals o f the f i r s t group o f 6 had the d o r s a l wound covered w i t h the medicated paste about 3 hr a f t e r i n j u r y , which was the l e n g t h of time r e q u i r e d i n l a t e r experiments t o apply 1 0 b a c t e r i a / cm g r a d u a l l y t o the whole wound s u r f a c e . The other 2 r a t s had the wound exposed f o r about 24 h r , and then covered w i t h the medicated paste i n order t o observe the e f f e c t o f wound d e s i c a t i o n on subsequent h e a l i n g o r b a c t e r i a l i n g r e s s . The remaining 2 o f t h i s group had the wound i n f e c t e d w i t h about 10 P. aeruginosa b a c t e r i a / c m , and a f t e r 16 h r , the 2

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i n f e c t e d wound was covered w i t h a l a y e r of p l a i n paste. A l l the paste c o a t i n g was o v e r l a i d w i t h a 2-ply c o t t o n gauze to reduce s h i f t i n g of the e l a s t i c paste due t o movement of the animal. The wound on the next group of 10 animals was a l s o i n f e c t e d w i t h about 1 0 b a c t e r i a / c m which took about 2.5 hr to apply from a suspension o f P. aeruginosa i n n u t r i e n t b r o t h grown to s a t u r a t i o n . A f t e r another 0.5 h r , the i n f e c t e d wound was covered w i t h the medicated paste and the animals were returned t o t h e i r separate cage. For the l a s t group of 6 W i s t a r r a t s , the i n f e c t e d wound was l e f t exposed, and then t r e a t e d w i t h the medicated paste a f t e r about 24 h r . At day 6 a f t e r i n j u r y o r at more frequent i n t e r v a l s , the wound s u r f a c e , the t h i n eschar l a y e r ( i f any), and the i n t e r n a l organs were samp l e d f o r b a c t e r i a l content by m e c h a n i c a l l y d i s i n t e g r a t i n g the t i s s u e i n n u t r i e n t broth and p l a t i n g on n u t r i e n t agar p l a t e s , f o l l o w e d by i n c u b a t i o n at 37°C f o r 24-48 hr and counting.


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Results Exudate Volume from Skin Wound. I n the f i r s t of the 2 experiments the d o r s a l s k i n wound on the a n e s t h e t i z e d r a t was covered immediately w i t h the A e r o p l a s t impermeable f i l m . Table 1 shows that about 20% of the t o t a l volume of the exudated f l u i d was c o l l e c t e d i n 1.5 h r , and by 6 h r , as much as about 50% was c o l l e c t e d . A f t e r 30 h r , the oozing had e s s e n t i a l l y ceased, and the f i n a l volume of 2.2 ml serous f l u i d was c o l l e c t e d i n 47 hr f o r the 3x3.5 cm wound. No f u r t h e r increase i n volume was observed even a f t e r another 24 h r . 2

In the second experiment, the wound was exposed f o r 3 hr before the impermeable f i l m was a p p l i e d . The f l u i d was found t o ooze much slower and the f i n a l t o t a l volume was one h a l f that of the f i r s t experiment (Table I ) . Evaporative Loss from Covered Wound. The e v a p o r a t i v e l o s s of the d o r s a l wound covered w i t h the medicated paste was found t o be 52 mg H20/cm /hr a f t e r 6 hr which was about 50% l e s s than an open wound ( 1 ) . The value decreased t o about 20 mg H 0/cm /hr i n 24 h r and soon a t t a i n e d an almost steady r a t e of 12.5 mg H 0/cm /hr f o r the next s e v e r a l days. When the evaporative l o s s of the paste spread over a p l a s t i c sheet was determined 6 hr a f t e r spreading by the same procedure, the values was 39 mg H20/cm /hr which decreased t o 12.5 mg H 0/cm /hr at 18 h r . A f t e r 24 h r , i t became 7 mg H 0/ cm /hr, and remained at t h i s l e v e l f o r some time due t o the humectant present i n the paste. 2

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Wound Covered w i t h Medicated Paste. The u n i n f e c t e d c o n t r o l wounds covered by the medicated paste a f t e r 3 hr healed w i t h about 10-15% c o n t r a c t i o n along the wound margins i n about 10


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Table I . Serous F l u i d Oozed from 3x3.5 cnr D o r s a l Wound

Time (hr)

Accumulated Volume (ml)

(Wound covered Immed.) 1.5 3 5 6 10 16 22 27 30 47 71

0.4 0.7 0.9 1.0 1.3 1.5 1.7 1.8 1.9 2.2 2.2

(Wound covered a f t e r 3 h r ) 2.5 5 10 16 23 35 46 65

0.1 0.2 0.4 0.6 0.8 1.0 1.1 1.2

days. The 2 animals w i t h the wound exposed f o r 24 hr before treatment healed i n over 12 days when t h i n l a y e r of eschar, formed due t o d e s i c a t i o n before the paste was a p p l i e d , separated from d i f f e r e n t areas of the healed s k i n s u r f a c e . There were a l s o c o n t r a c t i o n s , but wound margin d i s t o r t i o n was more apparent. No b a c t e r i a l growth was found on a l l the u n i n f e c t e d c o n t r o l wound s u r f a c e . For the 2 i n f e c t e d c o n t r o l s , 1 d i e d a f t e r 11 days, and the other d i e d at day 17. The wounds showed v a r i o u s extent of h e a l i n g , but high b a c t e r i a l counts were found on the wound s u r f a c e and i n the i n t e r n a l organs. The summary o f these r e s u l t s i s shown i n Table I I . The animals w i t h wound i n f e c t e d by P. aeruginosa and covered a f t e r 3 h r w i t h the medicated paste healed u n e v e n t f u l l y i n about 11 days (Table I I I ) as the u n i n f e c t e d c o n t r o l s w i t h



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Table I I . C o n t r o l Group w i t h AgN3 Containing Paste on Wound

Duration before (or f o r ) * No. o f Treatment Rats (hr)

24

(2.5)*

Healing (day)

Ag+ Paste

~10

15% c o n t r a c t i o n

+

-12

Edge d i s t o r t i o n + contraction

Ag Paste 10* Bact. per cm

2

vary 16

Bact. Counts (No./Cm )

Treatment

Plain Paste

2

>10* (wound surf.)

Observation

Died day 11 & 17



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Table I I I . AgN3 Containing Paste on I n f e c t e d Wound

Duration before (or f o r ) * No. o f Treatment Rats (hr)

10

Treatment

Healing (day)

9

(2.5)* — > 1 0 Bact. per cm 0.5

(2.5)*

24

2

-11

Bact. Counts (No./Cm ) 2

Observation

~10 (wound surf.; day 6)

3

Some contraction 4 distortion

8

Some contraction 4 distortion

+

>Ag Paste 9

>10 Bact. per cm

2

~22

~10 (eschar; day 14)

+

>Ag Paste


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minimal wound c o n t r a c t i o n (Table I I ) . Sampling of wound surface a t days 2 and 6 showed 10 b a c t e r i a / c m , but the t h i n l a y e r o f eschar, formed due t o the 24-hr wound exposure before being covered w i t h the medicated paste, contained >10 organisms/g. I n 1 r a t , the i n t e r n a l organs and blood sample were a l s o found t o g i v e very high counts o f v i a b l e b a c t e r i a . 3

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Discussion At p r e s e n t , the best way t o t r e a t i n j u r e d s k i n has not y e t been established. Some p r e f e r exposure of the wound t o form eschar which p r o t e c t s the i n j u r e d s i t e . Others promote the use of d r e s s i n g s t o prevent e v a p o r a t i v e l o s s and t o reduce the chances of wound c o n t r a c t i o n s or i n f e c t i o n . Most d r e s s i n g s are designed t o a s s i s t wound h e a l i n g by i n c o r p o r a t i n g a medicament to reduce i n f e c t i o n , u s i n g s p e c i a l polymer f i l m l a y e r t o prevent adhesion t o g r a n u l a t i o n s , having v a r i o u s extent of fluid transmission or absorption to decrease exudate accumulation a t wound s i t e , e t c . Table I shows t h a t a 3x3.5 cm d o r s a l s k i n wound on a W i s t a r r a t can ooze out 2.2 ml of serous f l u i d i n about 3 days. This f l u i d volume i s reduced t o 1.2 m l , i f the wound i s exposed f o r 3 hr before treatment. Beyond the 3 h r exposure, there may be f u r t h e r r e d u c t i o n i n o o z i n g , but the chance of eschar formation w i l l a l s o i n c r e a s e . V a r i o u s compositions o f the AgN03~medicated dextran hydrogel paste have been evaluated. The one w i t h a good s p r e a d a b i l i t y and f l u i d a b s o r p t i o n has been used i n the present study. Measurements by a simple hygrométrie method showed t h a t the medicated paste on the wound reduced evaporative l o s s by almost 50% as compared t o an open wound which had a r a t e of 93 mg H20/cm /hr (7). On subsequent days, the r e l a t i v e l y steady value o f 12.5 mg H20/cm /hr was about 2x higher than the e v a p o r a t i v e l o s s of the paste spread over a p l a s t i c sheet used as a c o n t r o l . The higher e v a p o r a t i v e r a t e of the paste on the wound s i t e might be due t o the t r a n s m i s s i o n o f excess moisture from the wound s u r f a c e . The oozing serous f l u i d had apparently been f i r s t absorbed i n t o the paste, because no exudate accumulation was ever observed under the paste c o a t i n g . The humectant i n the paste which r e t a i n e d moist u r e o b v i o u s l y must have kept the wound s u r f a c e s u f f i c i e n t l y moist t o prevent eschar formation. U n i n f e c t e d wounds, exposed f o r 24 h r and then covered w i t h the medicated paste, were observed t o r e q u i r e s e v e r a l days 2

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longer to h e a l than s i m i l a r wounds covered by the paste a f t e r 3 hr. I t appeared t h a t d e s i c a t i o n of the i n j u r e d t i s s u e s might have caused f u r t h e r damage to lower t i s s u e l a y e r s . On the i n f e c t e d wound, longer exposure before a p p l y i n g the medicated paste a l s o seemed to a l l o w the organisms to penetrate the damage t i s s u e s which f u r t h e r caused s u b s t a n t i a l l y delayed h e a l i n g and i n f e r i o r scar appearances. To achieve the best r e s u l t s , the present study i n d i c a t e s t h a t a medicated dextran hydrogel paste should be a p p l i e d soon a f t e r i n j u r y , but p r e f e r a b l y a f t e r about 3 hr when reduced exudate oozing i s w i t h i n the absorbing c a p a c i t y of the paste having the o p t i m a l handling c h a r a c t e r i s t i c s as w e l l .

Literature Cited 1. Nathan, P.; Law, E.J.; MacMillan, B.G.; Murphy, D.F.; Ronel; D'Andrea, M.J.; Abrahams, R.A. Trans. Amer. Soc. Artif. Intern. Organs. 1976, 22, 30. 2. Yannas, I.V.; Burke, J.F. J. Biomed. Mater. Res. 1980, 14, 65. 3. Arturson, G. Burns., 1977, 3, 112. 4. Flodin, G.M.; Ingleman, G.-A. U.S. Patent No. 3,042,667 (1962). 5. Wang. P.Y.; Samji, N.; Polymer Sci. Technol. 1980, 14. 29. 6. Wang, P.Y. Brit. Patent S. No. 2,099,704 (1982). 7. Wang, P.Y.; Evans, D.W.; Samji, N.; Llewellyn-Thomas, E. J. Surg. Res. 1980, 28, 182. RECEIVED

April 23, 1984


Polymeric Materials and Artificial Organs - American Chemical Society

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