Design Principles and Preliminary Clinical Performance of an Artificial

skin wounds in guinea pigs and in humans occurred. Infection, .... little contraction on that day, and the gross appearance of the graft gave no evide...
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29 Design Principles and Preliminary Clinical Performance of an Artificial Skin I. V. YANNAS—Massachusetts Institute of Technology, Fibers and Polymers Laboratories, Department of Mechanical Engineering and Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139 J. F. BURKE—Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114 M. WARPEHOSKI, P. STASIKELIS, E. M . SKRABUT, D. P. ORGILL and D. GIARD—Massachusetts Institute of Technology, Cambridge, MA 02139

We designed and successfully tested a novel family of polymeric membranes for treating patients with extensive skin loss. Stage 1 membranes consisted of a highly porous bottom layer of a covalently cross-linked collagen-glycosaminoglycan network and a top layer of a conventional silicone elastomer. Stage 2 membranes, studied so far only with guinea pigs, were prepared by seeding the bottom layer of Stage 1 membranes with autologous epidermal (basal) cells, prepared 4 h or less before grafting. Prompt and long-term closure of full-thickness skin wounds in guinea pigs and in humans occurred. Infection, exudation, and host-graft

rejection were absent while syn-

thesis of neoepidermal and neodermal tissue took place in the presence of the polymeric template.

L

oss of skin exposes an organism directly to the environment. Such exposure reveals two vital functions of skin, namely, control of bacterial

infection and of fluid loss. Unless treated, extensive skin loss can lead to

death due to either of these causes. In the United States approximately 10,000 individuals die every year due to extensive skin loss sustained in a fire, while at least 130,000 others are treated in hospitals for extensive burns. Skin loss can, of course, also result from a large number of causes not related to fire injury.

0065-2393/82/0199-0475$06.00/0 ©1982 American Chemical Society

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B e i n g a h i g h l y d i f f e r e n t i a t e d organ w i t h a m u l t i p l i c i t y of functions, skin should not be l o o k e d at s i m p l y as a m e m b r a n e that passively controls the traffic of bacteria a n d m o i s t u r e at the interface b e t w e e n the i n d i v i d u a l and the e n v i r o n m e n t . S k i n is an actively m e t a b o l i z i n g organ, and our w o r k shows that attempts to d e s i g n its r e p l a c e m e n t b e c o m e successful i f they incorporate this i m p o r t a n t fact. F o r c o n v e n i e n c e we have t e r m e d this replacement an artificial s k i n . A 10-year effort to d e s i g n an artificial skin ( i ) has y i e l d e d a bilayer p o l y m e r i c m e m b r a n e c o m p r i s i n g a top silicone elastomeric layer a n d a bottom

layer c o n s i s t i n g of a n o v e l , h i g h l y porous cross-linked c o l l a g e n -

glycosaminoglycan ( G A G ) n e t w o r k . T h e p o l y m e r i c bilayer is t e r m e d a Stage 1 m e m b r a n e , i n d i c a t i v e of its a b i l i t y to treat the needs of the patient i m m e diately after a n d u p to about 45 days f o l l o w i n g i n j u r y . A Stage 2 m e m b r a n e p r e p a r e d by s e e d i n g Stage 1 m e m b r a n e s w i t h e p i d e r m a l (basal) cells p r i o r to grafting addresses the l o n g - t e r m needs of the patient, particularly the c o n t r o l of d i s f i g u r i n g scars a n d c r i p p l i n g contractures that n o r m a l l y result w h e n a deep w o u n d is not closed w i t h an autograft. W e report the h i g h l i g h t s of o u r effort to design and study Stage 1 m e m b r a n e s w i t h animals a n d h u m a n s . A p r e l i m i n a r y account of the performance of Stage 2 m e m b r a n e s is p r e s e n t e d also.

Summary of Design Principles D e s i g n Stages. A staged design c o r r e s p o n d i n g to patient survival and patient r e h a b i l i t a t i o n , respectively, was u s e d . Stage 1 is a w o u n d closure that prevents, i n a single a p p l i c a t i o n , bacterial infection and f l u i d loss even w i t h the largest, full-thickness injuries over a p e r i o d of not less than 30 days. Stage 2 m e m b r a n e s are advanced versions of Stage 1 m e m b r a n e s capable, i n a d d i t i o n , of c o n t r o l l i n g scar f o r m a t i o n . T h e P h y s i c a l C h e m i s t r y o f W o u n d C l o s u r e . T h e efficient w e t t i n g of the w o u n d b e d b y the graft is essential. W i t h o u t such w e t t i n g , m i c r o s c o p i c air pockets lodge themselves at the g r a f t - w o u n d b e d interface and f o r m sites of bacterial p r o l i f e r a t i o n (1 ). W e t t i n g of a freshly excised w o u n d b e d can be achieved b y r e d u c i n g the b e n d i n g r i g i d i t y of the m e m b r a n e to a l e v e l that is sufficiently l o w to ensure d r a p i n g over the r o u g h w o u n d b e d as w e l l as over surfaces of negative c u r v a t u r e (e.g., clavicle, p o p l i t e a l region). In a d d i t i o n , the surface e n e r g y of the g r a f t - a i r surface must be no h i g h e r than that of the w o u n d - a i r surface. O n c e a c h i e v e d , this air-free g r a f t - w o u n d interface must be m a i n t a i n e d . Two major events can destroy the intimate interfacial contact. T h e first is a history of m e c h a n i c a l loads, p r i m a r i l y shear forces and p e e l forces, to w h i c h a graft is a c c i d e n t a l l y exposed d u r i n g c l i n i c a l manipulations of the patient. T h e second is a force a r i s i n g f r o m shrinkage of the graft due to d e h y d r a t i o n or, conversely, an i n t e r n a l p e e l force a r i s i n g f r o m s w e l l i n g (edema) at the g r a f t - w o u n d b e d interface caused b y the i n a b i l i t y of tissue mositure to escape.

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T h e s e p h y s i c o c h e m i c a l properties w e r e i n c o r p o r a t e d into a bilayer p o l y m e r i c m e m b r a n e . T h e top (silicone) layer renders the graft suturable a n d the grafted site aseptic, w h i l e the thickness of the layer is adjusted to p r o v i d e the r e q u i r e d o p t i m a l m o i s t u r e flux rate (about

1 mg/cm /h). T h e 2

bottom

( c o l l a g e n - G A G ) layer is susceptible to degradation by e n z y m e s , for e x a m p l e , collagenases, released b y the w o u n d and s u r r o u n d i n g d e r m a l tissue. Because this layer is h i g h l y porous, it is p o p u l a t e d r a p i d l y b y m e s e n c h y m a l cells that synthesize n e w connective tissue. M a t c h i n g (2, 3) of the t i m e constants for biodégradation a n d n e w tissue synthesis appears to be responsible for the o b s e r v e d d e v e l o p m e n t of substantial p e e l strengths, a m o u n t i n g to about 45 N/m (45 g/cm) 10 days after grafting. T h e c o n t r o l l e d b i o c h e m i c a l interaction b e t w e e n the c o l l a g e n - G A G layer a n d the w o u n d b e d appears to be i n d i s pensable i n m a i n t a i n i n g the i n t e g r i t y of the g r a f t - w o u n d b e d b o n d , t h e r e b y p r o t e c t i n g the w o u n d f r o m infection and f l u i d loss. L i m i t i n g D i m e n s i o n s o f the G r a f t .

M i g r a t i o n of cells f r o m the w o u n d

b e d i n a d i r e c t i o n r o u g h l y n o r m a l to the plane o f the m e m b r a n e m u s t be c o n s i d e r e d i n light of two major parameters: m e a n pore size and thickness. If the characteristic p o r e size is lower than about 5 μπι, the advance of m i g r a t i n g cells is l i m i t e d b y the biodégradation rate of the c o l l a g e n - G A G matrix. O n the other h a n d , i f the thickness of a sufficiently porous nonvasc u l a r i z e d layer exceeds a certain l i m i t , the m o t i l i t y of m i g r a t i n g fibroblasts is l i m i t e d by the diffusivity of c r i t i c a l nutrients o r i g i n a t i n g at the w o u n d b e d COIn the event of adequate vascularization of the c o l l a g e n - G A G layer, f o l l o w i n g m i g r a t i o n of e n d o t h e l i a l cells i n it, n u t r i e n t transport can p r o c e e d via b l o o d capillaries. A s i m p l e mathematical m o d e l based o n the analysis of T h i e l e (4), and m o d i f i e d b y W a g n e r (5) and W e i s z (6), r e l a t i n g the reactivity and diffusive flow i n porous catalyst particles can be u s e d (I ) to gain insight into this process. T h e l i m i t i n g d i m e n s i o n o f the graft i n the plane is d e t e r m i n e d p r i m a r i l y by the m i g r a t i o n v e l o c i t y of e p i t h e l i a l c e l l sheets advancing f r o m the w o u n d edge. A t approximate speeds of about 0.25 mm/day, e p i t h e l i a l c e l l sheets advancing f r o m opposite w o u n d edges are observed to cover the surface of a w o u n d w i t h a l e n g t h of 3 c m w i t h i n about 60 days, a p e r i o d that is barely acceptable c l i n i c a l l y . T h i s m e c h a n i s m w o u l d be inadequate as a means of e p i t h e l i a l i z i n g massive w o u n d s of about 30 c m r e s u l t i n g f r o m extensive b u r n s . O n e successful approach that we used involves s e e d i n g of the collag e n - G A G layer, before grafting, w i t h autologous e p i d e r m a l cells. F o l l o w i n g grafting, these cells proliferate i n the sterile i n t e r i o r of the graft a n d f o r m sheets of mature, k e r a t i n i z e d e p i d e r m i s . T h i s approach appears to o v e r c o m e the l i m i t a t i o n w i t h respect to l e n g t h of w o u n d that can be grafted effectively by a single a p p l i c a t i o n . T h e role of contraction as a process that accelerates w o u n d closure was n e g l e c t e d i n this qualitative discussion. C o n s t r a i n t s o n the S e l e c t i o n o f C h e m i c a l C o m p o n e n t s .

The physico-

c h e m i c a l , m e c h a n i c a l , and b i o c h e m i c a l attributes of an effective l o w e r layer

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appear to b e m e t b y a 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-sulfate network (7). A d e t a i l e d rationale for use o f these m a c r o m o l e c u l a r components is p r e s e n t e d e l s e w h e r e (J).

Experimental The materials used and the detailed procedures employed in preparation of Stage 1 membranes are described elsewhere in detail (7-10). Stage 2 membranes were prepared by seeding the collagen-GAG layer of Stage 1 membranes with autologous epidermal (basal) cells. Epidermal cells were harvested from the animal, and basal cells were separated by the method of Prunieras et al. (11,12). A suspension of cells was seeded into the porous collagen-GAG layer by a variety of methods, including inoculation using a hypodermic syringe and a centrifugal force field to drive the cells into the porous membrane. The entire procedure starting with cell harvest from the animal and ending with grafting of the seeded membrane on the animal lasted 4 h. Promptness of wound closure following skin injury was thereby maintained. The animal model used was a 3 X 1.5-cm, full-thickness excised skin wound on the guinea pig, described elsewhere (1). Human subjects, all victims of extensive burns, were grafted with segments of the bilayer membrane following primary excision of burned eschar. Techniques previously used with autografting (13) were generally used. Grafts were applied on the excised surface, free from devitalized tissue, on which meticulous hemostasis had been obtained. Following placement on the wound bed, grafts were carefully sutured under slight tension, avoiding wrinkling of the thin membrane.

Results G r a f t i n g o f F u l l - T h i c k n e s s W o u n d s i n G u i n e a P i g s . Results obtained by grafting m o r e than 120 animals w i t h Stage 1 membranes showed clear differences b e t w e e n the p e r f o r m a n c e o f the bilayer m e m b r a n e d e s c r i b e d here a n d that o f the allograft. B y D a y 14, the allograft was generally w e l l o n its way to b e i n g r e j e c t e d as t h e w o u n d was u n d e r g o i n g strong contraction. B y contrast, 3 X 1 . 5 - c m w o u n d s c o v e r e d w i t h the bilayer m e m b r a n e showed little contraction o n that day, a n d the gross appearance o f the graft gave no evidence of rejection. H i s t o l o g i c a l study of the region grafted w i t h the bilayer m e m b r a n e s h o w e d n o e v i d e n c e o f rejection over the entire p e r i o d o f observation o f grafted animals (up to 400 days). N o i m m u n o s u p p r e s s i o n was used. Significant delay i n onset o f w o u n d contraction o c c u r r e d w i t h w o u n d s grafted w i t h Stage 1 m e m b r a n e s c o m p a r e d to the ungrafted controls. W e observed a delay i n " h a l f - l i f e " o f the 3 X 1 . 5 - c m w o u n d (the t i m e necessary for the w o u n d area to contract to 5 0 % o f the o r i g i n a l area) f r o m about 13 days w i t h the ungrafted controls to about 28 days w i t h grafted w o u n d s . T h e histological e v i d e n c e shows that e p i d e r m a l m i g r a t i o n consistently o c c u r r e d over, rather than u n d e r , the b o t t o m ( c o l l a g e n - G A G ) layer o f Stage 1 m e m b r a n e s . C o v e r a g e o f t h e b o t t o m layer o f the 3 X 1 . 5 - c m graft b y the advancing e p i d e r m i s was c o m p l e t e b y D a y s 30 to 40. T h e top (silicone) layer of the graft was e j e c t e d spontaneously at about the same t i m e , revealing a scar, w h i l e t h e histological e v i d e n c e clearly i n d i c a t e d c o m p l e t i o n of e p i thelialization over t h e e n t i r e area.

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R e c e n t l y , Stage 2 m e m b r a n e s w e r e u s e d successfully i n a n i m a l studies to seed the w o u n d b e d w i t h autologous e p i d e r m a l (basal) cells (see E x p e r i m e n t a l section). W h e n transferred to the w o u n d b e d , w h i c h was m a i n t a i n e d sterile b y the grafted m e m b r a n e , i n o c u l a t e d basal cells m i g r a t e d extensively and f o r m e d c o n f l u e n t sheets o f m a t u r e , k e r a t i n i z e d e p i d e r m i s at the interface b e t w e e n the silicone layer a n d the c o l l a g e n - G A G layer. M o s t o f the e p i d e r mal sheet f o r m a t i o n o c c u r r e d i n locations that w e r e clearly r e m o v e d f r o m the w o u n d edge. F o r m a t i o n o f m a t u r e , k e r a t i n i z e d e p i d e r m i s o c c u r e d i n less than 2 weeks f o l l o w i n g grafting o f the full-thickness w o u n d b y the c e l l i n o c u l a t e d p o l y m e r i c m e m b r a n e . T h i s f i n d i n g suggests that the a d d i t i o n a l m a n i p u l a t i o n o f i n o c u l a t i n g Stage 1 m e m b r a n e s w i t h autologous e p i d e r m a l cells extends almost i n d e f i n i t e l y the area o f skin loss that can be closed b y a single a p p l i c a t i o n o f this b i l a y e r m e m b r a n e . G r a f t i n g o f H u m a n Subjects.

Seven extensively b u r n e d (50 to 9 5 %

b o d y area) h u m a n subjects, 5- to 60-year-old males and females, w e r e grafted w i t h rectangular pieces o f cell-free Stage 1 m e m b r a n e , r a n g i n g f r o m 5 X 10 to 15 X 25 c m . T h e grafts r e m a i n e d i n place f r o m 25 to 46 days. D u r i n g this p e r i o d no i n f e c t i o n o r i n f l a m m a t i o n was n o t e d , a n d no i m m u n o s u p p r e s s i o n was u s e d . O c c a s i o n a l l i f t i n g o f the edge o f the graft was treated as w i t h an autograft (13), b y d e b r i d i n g the edge. W h e n e v e r the graft was next to intact e p i d e r m i s , the e p i d e r m a l edge m i g r a t e d b e t w e e n the two layers o f the m e m b r a n e s over a distance o f a few m i l l i m e t e r s . B e t w e e n 25 a n d 46 days f o l l o w i n g grafting the silicone layer was r e m o v e d , a n d t h i n layers o f autoe p i d e r m a l grafts w e r e harvested a n d p l a c e d o n top of the n e o d e r m a l tissue that h a d r e p l a c e d the o r i g i n a l c o l l a g e n - G A G layer. T h i s p r o c e d u r e left m i n i m a l scarring b o t h at the d o n o r site a n d at the graft site. A d e t a i l e d discussion of the h u m a n studies appears elsewhere (14).

Discussion E x p e r i m e n t s w i t h animals have s h o w n that closure o f 3 X 1 . 5 - c m , f u l l thickness skin w o u n d s w i t h Stage 1 m e m b r a n e s provides p r o m p t , reliable p r o t e c t i o n against i n f e c t i o n a n d f l u i d loss b y a single application. Subsequent removal is not necessary. T h e s e findings are the first successful effort to p r o m p t l y close large, f u l l - t h i c k n e s s w o u n d s , not r e q u i r i n g r e p l a c e m e n t or the use o f an autograft. P r o m p t n e s s o f c l o s u r e o f f u l l - t h i c k n e s s skin w o u n d s is essential. F a i l u r e to close large w o u n d s satisfactorily w i t h i n 3 - 7 days following i n j u r y increases mortality significantly (15). R e c e n t l y , c u l t u r e d autologous e p i d e r m a l cells (16) a n d a r e c o n s t i t u t e d collagen lattice p o p u l a t e d w i t h c u l t u r e d autologous fibroblasts a n d e p i d e r m a l cells (17) w e r e grafted onto full-thickness skin wounds i n h u m a n s 5 weeks f o l l o w i n g c e l l harvesting (16), or w e r e grafted o n rats at least 2 weeks f o l l o w i n g c e l l harvesting (17). Because they r e q u i r e c u l t u r i n g o f cells p r i o r to grafting, these i n t e r e s t i n g p r o c e d u r e s (16,17) do not appear to m e e t t h e c r i t e r i o n o f p r o m p t n e s s o f w o u n d closure f o l l o w i n g injury.

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I n contrast, Stage 1 m e m b r a n e s are available i n sterile containers a n d are ready for use i n a matter o f m i n u t e s . Stage 2 membranes (see E x p e r i mental section) r e q u i r e no m o r e than a 4 - h interval b e t w e e n harvesting o f cells and grafting, because cells seeded into the membranes before grafting are c u l t u r e d i n s i d e the graft, w h i c h is m a i n t a i n e d sterile b y the silicone m e m b r a n e , rather than outside o f it. T h i s seeding p r o c e d u r e y i e l d s , therefore, a graft that induces the w o u n d tissue itself to generate n e w tissue i n vivo. T h e r e is no d e p e n d e n c e o n an ex vivo tissue c u l t u r e m e d i u m to generate such tissue d u r i n g a l e n g t h y p r o c e d u r e before grafting. Promptness of treatment is t h e r e b y assured b y u s i n g these bilayer p o l y m e r m e m b r a n e s . L o n g - t e r m f u n c t i o n is also an i m p o r t a n t characteristic of a skin graft. C u r r e n t l y , cadaver s k i n , m a i n t a i n e d i n a frozen skin bank (18), is used successfully as an adequate b u t t e m p o r a r y w o u n d closure. C a d a v e r skin is n o r m a l l y used w i t h i m m u n o s u p p r e s s i o n , i n o r d e r to delay host-graft rejection, b u t such t r e a t e m e n t greatly increases the risk o f infection. I n contrast w i t h cadaver grafts, p o r c i n e skin grafts are c o m m e r c i a l l y available, b u t they are n o r m a l l y r e m o v e d b e t w e e n the t h i r d a n d n i n t h days. Several m e m b r a n e s based o n synthetic a n d natural p o l y m e r s have b e e n u s e d , b u t failure to control infection has b e e n a consistent p r o b l e m . A u t o g r a f t i n g remains a standard treatment because it p r o v i d e s for functional replacement o f skin over an i n d e f i n i t e p e r i o d o f t i m e . Nevertheless, harvesting of a splitthickness autograft is a serious operation a n d , i n cases w h e r e the i n j u r y is massive, sufficient autograft is unavailable. L a s t l y , harvesting o f a splitthickness autograft n o r m a l l y leaves a scarred d o n o r site. Stage 1 m e m b r a n e s are not rejected a n d therefore appear to be s u p e r i o r to cadaver a n d p o r c i n e s k i n grafts. I n studies o f Stage 1 m e m b r a n e s w i t h extensively b u r n e d h u m a n s , the silicone (top) layer was electively r e m o v e d b e t w e e n 25 and 46 days after grafting and r e p l a c e d b y a u t o e p i d e r m a l grafts. T h i s p r o c e d u r e eventually y i e l d e d a relatively scar-free d o n o r site a n d a largely scar-free treatment site that appeared to r e q u i r e no f u r t h e r manipulation. Stage 2 m e m b r a n e s , s t u d i e d so far o n l y w i t h animals, d o not appear to r e q u i r e eventual r e p l a c e m e n t o f the silicone layer b y a u t o e p i d e r m a l grafts. Instead, these advanced m e m b r a n e s i n d u c e w o u n d tissue to construct b o t h n e o e p i d e r m a l and n e o d e r m a l tissue, t h e r e b y l e a d i n g to an apparently functional reconstruction o f skin (19). T h e r e f o r e , Stage 2 m e m b r a n e s appear to compare favorable i n p e r f o r m a n c e w i t h the autograft itself. D e v e l o p m e n t o f these m e m b r a n e s is i n progress.

Acknowledgment T h i s w o r k was s u p p o r t e d i n part b y N I H grants H L 14322, G M 23946, and G M 21700. W e thank V . M . I n g r a m , F . O . Schmitt, a n d R. L . Trelstad for useful discussions.

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Literature Cited 1. Yannas, I. V.; Burke, J. F. J. Biomed. Mater. Res. 1980, 14, 65-81. 2. Yannas, I. V.; Burke, J. F.; Umbreit, M . ; Stasikelis, P. Fed. Proc., 1979, 38, 988. 3. Yannas, I.V.; Burke, J. F.; Huang, C.; Gordon, P. L. J. Biomed. Mater. Res. 1975, 9, 623-628. 4. Thiele, E . W. Ind. Eng. Chem., Ind. Ed. 1939, 31, 916-920. 5. Wagner, C. Z. Phys. Chem. (Leipzig) 1943, 193, 1-5. 6. Weisz, P. B.; Prater, C. D. In "Advances in Catalysis and Related Subjects;" Frankenburg, W. G . ; Komerewskv, V. I.; Rideal, E . , Ed. Academic: New York, 1954. Vol. 6, pp. 143-196. 7. Yannas, I. V.; Burke, J. F.; Gordon. P. L.; Huang, C.; Rubenstein, R. H . J. Biomed, Mater. Res. 1980, 14, 511-528. 8. Dagalakis, N.; Fink, J.; Stasikelis, P.; Burke, J. F.; Yannas, I. V.J.Biomed. Mater. Res. 1980, 14, 511-528. 9. Yannas, I. V.; Burke, J. F.; Huang, C.; Gordon, P. L. Am. Chem.Soc.,Polymer Prepr. (Chicago, Aug., 1975), 16(2), 209-214. 10. Yannas, I. V.; Burke, J. F.; Gordon, P. L.; Huang, C. US Patent 4060081, 1977; Yannis, I. V.; Gordon, P. L.; Huang, C.; Silver, F. H.; Burke, J. F., U.S. Patent 42 80954, 1981. 11. Prunieras, M . ; Delescluse, C.; Regnier, M . J. Invest. Dermatol. 1976, 67, 58-65. 12. Regnier, M . ; Delescluse, C.; Prunieras, M . Acta Dermatovener (Stockholm) 1973, 53, 241-247. 13. Burke, J. F.; Bondoc, C. C.; Quinby, W. C. J. Trauma 1974, 14, 389-394. 14. Burke, J. F.; Yannis, I. V.; Quinby, W. C.; Bondoc, C. C.; Jung, W. K. Ann. Surg. 1981, 194, 413-428. 15. Shires, G. T.; Black, E. A. J. Trauma 1979, 19, 855-936 16. O'Connor, N. E.; Mulliken, J. B.; Banks-Schlegel, S.; Kehinde, O.; Green, H . Lancet 1981-I, 75-78. 17. Bell, E.; Ehrlich, H . P.; Buttle, D. J.; Nakatsumi, T. Science 1981, 211, 1052-1054. 18. Bondoc, C. C.; Burke, J. F. Ann. Surg. 1971, 158, 371. 19. Yannis, I. V.; Burke, J. F.; Orgill, D. P.; Skrabut, Ε. M . Science 1982, 215, 174-176. R E C E I V E D for review January 16, 1981.

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