Skin Regeneration with a Bioreplaceable Polymeric Template - ACS

13 Skin Regeneration with a Bioreplaceable Polymeric Template I. V. YANNAS, D. P. ORGILL, and E. M. SKRABUT Massachusetts Institute of Technology, Cambridge, MA 02139

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J. F. BURKE Massachusetts General Hospital, Boston, MA 02114 Previously we have described a biodegradable polymeric template which can induce wound tissue to synthesize new skin (1,2). This template, a highly porous, crosslinked collagen-glycosaminoglycan (CG) network, i s currently used to treat excised skin wounds i n patients who have suffered extensive burns (3,4). We now report certain structural and functional properties of the newly synthesized t i s s u e . This preliminary character ization of the regrown organ suggests its close s i m i l a r i t y to, as well as certain d i s t i n c t differences from the intact skin adjacent to it. The polymeric template was a b i l a y e r membrane c o n s i s t i n g of a 0 . 5 mm-thick top l a y e r of p o l y ( d i m e t h y l s i l o x a n e ) and a 1.5-mm-thick l a y e r of a h i g h l y porous c r o s s l i n k e d c o l l a g e n - c h o n d r o i t i n 6 - s u l f a t e (CG) network. The method of p r e p a r a t i o n has been described elsewhere i n d e t a i l ( 5 - 7 ) . P r i o r to g r a f t i n g the polymeric template was seeded w i t h autologous b a s a l c e l l s , implanted i n t o the CG l a y e r using a c e n t r i f u g a t i o n procedure which has been d e s c r i b e d ( 2 ) . A f u l l - t h i c k n e s s s k i n wound, measuring 3 χ 1,5 cm or 4 χ 4 cm, was prepared under a s e p t i c c o n d i t i o n s by e x c i s i n g the s k i n down t o , but not i n c l u d i n g the panniculus carnosus of the guinea p i g . The s u r g i c a l procedure f o r preparing a s k i n d e f i c i t has been described (8). Immediately f o l l o w i n g e x c i s i o n of the s k i n , a g r a f t which had been cut to f i t w i t h i n the wound perimeter was placed on the wound bed and was sutured to immediately adjacent s k i n as de s c r i b e d (8). A f t e r c a r e f u l bandaging of the g r a f t e d area (8), the animals were placed i n cages and were fed Charles R i v e r Guinea P i g Formula. One week f o l l o w i n g g r a f t i n g the wounds were unbandaged and photographed. When the wound had j u s t been covered by a confluent neoepidermis, u s u a l l y between 10 and 14 days, the sutures were cut o f f and the s i l i c o n e l a y e r was removed at v i r t u a l l y zero 0097-6156/ 84/ 0256-0191 $06.00/ 0 © 1984 American Chemical Society

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


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POLYMERIC MATERIALS AND

ARTIFICIAL ORGANS

peel s t r e n g t h . The moisture f l u x r a t e was determined w i t h an Evaporimeter (Servo Med, Stockholm) a f t e r the s i l i c o n e l a y e r had been removed. The probe was placed a l t e r n a t e l y on the wounded area and on i n t a c t s k i n , p r e v i o u s l y shaven, about 5 cm away from the wound perimeter. A simple n e u r o l o g i c a l t e s t (pin p r i c k ) was o c c a s i o n a l l y administered to the area of the wound and to an i n t a c t s k i n area. V a s c u l a r i z a t i o n of the g r a f t e d area was confirmed by observing blanching f o l l o w i n g a p p l i c a t i o n of hand pressure. At v a r i o u s time i n t e r v a l s , animals were s a c r i f i c e d and specimens of the wound contents as w e l l as of an i n t a c t s k i n area about 5 cm from the wound perimeter were e i t h e r removed f o r mecha n i c a l t e s t i n g , or were f i x e d p r i o r to processing f o r h i s t o l o g i c a l s t a i n i n g . T e n s i l e specimens were stored i n p h y s i o l o g i c a l s a l i n e at 4°C and were s t r e t c h e d i n an I n s t r o n U n i v e r s a l Tester Model TM at 100% min^ at room temperature w i t h i n 24 h of s a c r i f i c e . Specimens f o r h i s t o l o g i c a l study were s t a i n e d w i t h hematoxylin and eosin and viewed i n a l i g h t microscope. 1

Results Not l a t e r than 7 days a f t e r g r a f t i n g , i s l a n d s of new epidermis had formed between the s i l i c o n e l a y e r and the CG l a y e r of the g r a f t , w h i l e the host epidermis was invading the area j u s t below the s i l i c o n e l a y e r at the s i t e of the wound perimeter. Between 10 and 14 days, the neoepidermis had become f u l l y confluent over the e n t i r e wound area. The neoepidermis formed by p r o l i f e r a t i o n of the seeded b a s a l c e l l s at the s i l i c o n e - C G i n t e r f a c e became d i s t i n c t l y k e r a t i n i z e d , as viewed h i s t o l o g i c a l l y , by days 12 to 14. The c o l l a g e n - c h o n d r o i t i n 6 - s u l f a t e l a y e r was invaded by a v a r i e t y of mesodermal c e l l s and s y n t h e s i s of new c o l l a g e n f i b e r s became h i s t o l o g i c a l l y evident between days 14 and 18. By day 28 the morphology of the newly synthesized c o l l a g e n f i b e r s had become w e l l e s t a b l i s h e d i n the l a y e r of neodermis. The neodermis showed h i s t o l o g i c a l and c l i n i c a l evidence of being r i c h l y v a s c u l a r i z e d by day 7, or e a r l i e r . Simple n e u r o l o g i c a l t e s t i n g gave p o s i t i v e r e s u l t s before day 21. A p r e l i m i n a r y comparison of p r o p e r t i e s of newly synthesized (regenerated) s k i n and i n t a c t (normal) s k i n shows s e v e r a l c l o s e s i m i l a r i t i e s . However, d i f f e r e n c e s are a l s o apparent, s t r i k i n g among them being the absence of s k i n accessory organs, i n c l u d i n g h a i r (the guinea p i g has no sweat g l a n d s ) . Figures 1,2,3A and 3B. Discussion P r e l i m i n a r y c h a r a c t e r i z a t i o n shows that newly synthesized s k i n i s s t r i k i n g l y s i m i l a r , though not i d e n t i c a l , to i n t a c t s k i n . Ongoing s t u d i e s are d i r e c t e d towards biochemical c h a r a c t e r i z a t i o n of macromolecular components i n new s k i n , d e t a i l e d morphological a n a l y s i s and e l u c i d a t i o n of the k i n e t i c s of s y n t h e i s of new organ. These p r e l i m i n a r y r e s u l t s suggest t h a t the polymeric temp-


13.

YANNAS ET A L .

PARTIAL


193

A Bioreplaceable Polymeric Template

REGENERATION OF SKIN

.silicone

epidermis

( ι Π ι i dermis

intact skin

CG full - thickness wound

grafted

epidermis L A

WW VM

neodermis

partly confluent

fully confluent neoepidermis early synthesis of neodermis

Figure

J.

neoepidermis

CG

neoepidermis

wound

neoepidermis

MJMJJJSJM1 neoepidermis

1111 (\

1. Schematic r e p r e s e n t a t i o n

sequence which l e d t o s y n t h e s i s

neodermis partly regenerated skin (no hair follicles)

of e x p e r i m e n t a l

o f new s k i n i n the guinea

Pig-



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POLYMERIC MATERIALS A N D ARTIFICIAL ORGANS

Figure 2. A r e c t a n g u l a r segment of regenerated guinea p i g s k i n (perimeter marked by arrows) surrounded by i n t a c t , p a r t l y shaven s k i n . O r i g i n a l s i z e of e x c i s e d , f u l l thickness wound was 3 χ 1.5 cm.



YANNAS ET AL.


Figure 3A. H i s t o l o g i c a l s e c t i o n of i n t a c t dermis. Key: Ε epidermis; D, dermis; and H, h a i r f o l l i c l e . (Mag. 275X, photograph reduced 15%)

Figure 3B. H i s t o l o g i c a l s e c t i o n of new s k i n . Key: NE, Neoepidermis; and ND, Neodermis. (Mag. 275X, photograph reduced 15%)


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POLYMERIC MATERIALS AND ARTIFICIAL ORGANS

l a t e used i n t h i s work s t i m u l a t e s the wounded mammalian t i s s u e i n a novel way. The r e s u l t of such s t i m u l a t i o n i s not scar, as i s the case when t h i s template i s not used. Future work w i l l address the question of the extent to which such an unexpected outcome r e s u l t s from r e p e t i t i o n of c e r t a i n l a t e stages of the ontogenetic development of s k i n .


Table 1. Comparison of New Skin t o I n t a c t Skin i n the Guinea P i g

Property Moisture p e r m e a b i l i t y , i n v i v o , gm/cm /h Mechanical p r o p e r t i e s , i n v i t r o t e n s i l e strength, Pa Second d e r i v a t i v e of s t r e s s s t r a i n curve Histological

t e s t (pin p r i c k )

Vascularization test Color

31 x 10

6

New Skin 4.7 ± 1.0 14 χ 10

+

+

+ + + + wavy 20-40 0.8-1.3

+ +

6

studies

M u l t i l a y e r e d k e r a t i n i z i n g epidermis I n t a c t dermal-epidermal j u n c t i o n Skin accessory organs (eg., h a i r ) Dermal v a s c u l a r i z a t i o n Collagen morphology Epidermal t h i c k n e s s , ym Dermal t h i c k n e s s , mm Neurological

Intact Skin 4.5 ± 0.8

(blanching)^

6

+ l e s s wavy 30-40 0.9-1.4

+

+

+

+

white

white

Measured value remained i n v a r i a n t , w i t h i n experimental e r r o r , between 1 and 10 months f o l l o w i n g g r a f t i n g . ^Performed 10 months f o l l o w i n g g r a f t i n g . °Positive r e s u l t s obtained by day 21. ^ P o s i t i v e r e s u l t s obtained by day 14. e

C o l o r changes i n the g r a f t were as f o l l o w s : r e d , up t o about 2 months; pink to o f f - w h i t e about 2-5 months; w h i t e , a f t e r about 5 months.


13.


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197

Acknowledgments


We thank V . M . Ingram, J . C . Murphy, F . O . Schmitt, W. Schoene and D . F . Waugh f o r u s e f u l d i s c u s s i o n s as w e l l as I . Blank f o r the use of the Evaporimeter and J . C . Murphy f o r the use of a Z e i s s l i g h t microscope. This research was p a r t l y supported by N a t i o n a l I n s t i t u t e s of Health Grant GM 23946; by the Department of Mechanical Engineering, MIT; and by the O f f i c e of the Dean of Engineering, MIT.

Literature Cited 1. Yannas, I.V. and Burke, J.F. J. Biomed. Mater. Res. 1980, 14, 65-81. 2. Yannas, I.V., Burke, J.F., Orgill, D.P. and Skrabut, E.M. Science, 1982, 215, 174. 3. Burke, J.F., Yannas, I.V., Quinby, W.C. Bondoc, C.C. and Jung, W.K. Ann. Surg., 1981, 194, 413. 4. Yannas, I.V,, Burke, J.F., Warpehoski, M. Stasikelis, P. Skrabut, E.M., Orgill, D. and Giard, D.J. Trans. Am. Soc. Artif. Org. 1982, 27, 19. 5. Yannas, I.V., Burke, J.F., Gordon, P.L., Huang, C. and Rubenstein, R.H. J. Biomed. Mater. Res. 1980, 14, 107-131. 6. Yannas, I.V., Burke, J.F., Huang, C. and Gordon, P.L. Polymer Prepr. Am. Chem. Soc. 1975, 16(2), 209-214. 7. Dagalakis, N. Flink, J. Stasikelis, P. Burke, J.F. and Yannas, I.V. J. Biomed. Mater. Res. 1980, 14, 511-528. 8. Yannas, I.V., "The Surgical Wound", Dineen, P. Ed.; Lea and Febiger, Philadelphia, 1981, Chap. 15, p. 171. RECEIVED March

19,1984


Skin Regeneration with a Bioreplaceable Polymeric Template - ACS

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