Method of skin regeneration using a collagen-glycosaminoglycan matrix and cultured epithelial autograft

The present invention relates to a method of skin regeneration of a wound or burn in an animal or human. This method comprises the steps of initially covering the wound with a collagen glycosaminoglycan matrix, allowing infiltration of the grafted GC matrix by mesenchymal cells and blood vessels from healthy underlying tissue and applying a cultured epithelial autograft sheet grown from epidermal cells taken from the animal or human at a wound free site on the animal's or human's body surface. The resulting graft has excellent take rates and has the appearance, growth, maturation and differentiation of normal skin.

BACKGROUND ART 
A patient who has suffered extensive skin loss or injury is immediately 
threatened by infection and by excessive loss of fluids. To meet both of 
these needs, a large skin wound must be closed promptly by some type of 
membrane. The most direct method of accomplishing this purpose is by 
transplanting partial-thickness sections of skin to the wound, thereby 
sealing the wound and preventing fluid loss and infection. 
The transplanted section of skin can be removed ("harvested") from an 
animal of another species. This type of transplant is referred to as a 
xenograft. However, a xenograft suffers from the disadvantage that the 
transplanted skin is rejected and can only serve to cover the wound for 
three to five days. Consequently, a xenograft can only serve as a stopgap 
while the patient's skin slowly heals. 
The transplanted section of skin can also be harvested from human cadavers. 
This type of transplant is referred to as an allograft or homograft. 
However, cadaveric skin is in short supply, and allografts are often, like 
xenografts, rejected. Although immunosuppressive drugs can increase the 
period of time which an allograft may cover a wound, they also leave the 
patient vulnerable to infection. Allografts also suffer from the 
disadvantage that they expose the patient to the risk of transmission of 
diseases such as hepatitis and AIDS. 
The most desirable form of transplant is an autograft, in which skin from 
an undamaged area of the patient or identical twin is harvested and used 
to cover the wound. The risk of rejection and disease transmission is 
thereby eliminated, and the transplanted skin proliferates to form a new 
layer of dermis and epidermis. 
The harvesting operation is a painful, invasive process, which causes 
scarring. It should therefore be kept to a minimum. In addition, a 
severely burned patient may suffer skin loss or damage on nearly all of 
his or her body. This may severely limit the amount of healthy, intact 
skin that is available for autografting. When this occurs, xenografts or 
homografts may be placed across the entire wound surface to control 
infection and dehydration; they are gradually replaced as autografts 
become available. Autografts may be harvested repeatedly from a donor 
site. In such an operation, an area of xenograft or homograft is removed 
and discarded, and replaced by an autograft. Each donor site must be 
allowed to heal before another autograft is removed from it; this requires 
a substantial delay, and prolongs the recovery of the patient. 
Furthermore, the quality of the skin graft diminishes with each successive 
harvest. 
Consequently, much effort has been spent to create a skin substitute for 
the massively burned patient with limited donor sites. Attempts have been 
made to manufacture artificial skin from both biologic and synthetic 
materials with variable results. An acceptable skin substitute should 
provide both the components and functional results of normal skin. Two 
important components of the skin are the epidermis and dermis. The 
epidermis is the outer layer of skin. It consists of cells at various 
stages of differentiation and maturity. Basal cells are located at the 
lowest level (adjacent to the dermis) and are the least differentiated. 
The dermis is located below the epidermis and comprises mesenchymal cells 
and blood vessels. The junction between the dermis and epidermis is 
referred to as the basement membrane and is responsible for one of the 
most important functional results of normal skin, namely the tight 
adhesion of the dermis to the epidermis. This tight adhesion adds strength 
and durability to the skin and prevents "shearing" of the epidermis. 
"Shearing" is the "rubbing off" of the epidermis when lateral forces are 
applied to the skin, and can result in blistering and skin fragility. 
One of the most promising skin substitutes is a synthetic bilayer membrane 
(hereinafter collectively referred to as "CG bilayer"). This membrane 
comprises a bottom layer (hereinafter referred to as "CG matrix") which is 
a highly porous lattice made of collagen and glycosaminoglycan. The outer 
layer is a membrane semipermeable to moisture and impermeable to 
infectious agents such as bacteria. The CG lattice serves as a supporting 
or scaffolding structure into which blood vessels and mesenchymal cells 
migrate from below the wound, a process referred to as "infiltration". 
Infiltration is responsible for creating a new dermis, referred to as the 
"neodermis", which replaces the CG matrix as it biodegrades. Epithelial 
cells from undamaged skin surrounding the edges of the wound migrate into 
CG matrix to create a new epidermis, referred as the "neoepidermis". 
Because burns and other skin wounds tend to be shallow, mesenchymal cells 
need not migrate very far to create a neodermis. However, burns often 
cover large areas of a patient's body surface. Consequently, epithelial 
cells often must migrate great distances to adequately close a wound. As a 
result, thin skin grafts are required to close the wound. Consequently, a 
need exists for new procedures which can hasten the coverage of the CG 
matrix with a neoepidermal layer. 
A second promising technology for manufacturing and applying artificial 
skin is referred to as cultured epithelial autograft (hereinafter referred 
to as "CEA"). In this method split thickness skin samples are harvested 
from a site on the patient's body surface that is wound free. The 
epithelial cells from this graft are grown in culture to give epithelial 
sheets that are applied directly to the wound bed, basal side down. 
The CEA method suffers from the limitation that it only applies a 
neoepidermis to the wound bed. There is no dermis or basement membrane 
present at the time of application, and, therefore, no basement membrane. 
Thus, there is nothing to secure the neoepidermis to the underlying 
tissue, resulting in poor take rates for CEA sheets applied directly onto 
wound beds. This is evidenced by shearing and blistering of the 
transplanted CEA. Consequently, efforts have been made to use dermal 
substrates, such as cadaveric skin to improve take rates. However, 
allograft rejection, the risk of disease transmission and limited 
availability of cadaveric skin are serious limitations on the usefulness 
of this technique. 
Despite the promise of CEA as a technique for treating wounds, improvements 
are needed if this technique is to adequately meet the needs of patients 
with wounds covering large portions of their bodies. Take rates need to be 
improved without incurring the limitations and risks involved in using 
cadaveric skin. Furthermore, a patient's wounds must either be exposed or 
temporarily covered during the approximately three week period during 
which the CEA sheets are being grown. 
SUMMARY OF THE INVENTION 
The present invention pertains to a method for regenerating skin at a burn 
or wound on a human or animal which comprises the steps of applying a 
collagen-glycosaminoglycan matrix (CG matrix) having an outer moisture 
barrier (e.g. a silicone layer) to a wound so that the semipermeable 
moisture barrier is exposed to the air. Blood vessels and mesenchymal 
cells are allowed to infiltrate the CG matrix from tissue beneath the CG 
matrix, after which the moisture barrier on the CG matrix is replaced with 
a cultured epithelial autograft (CEA) sheet. This CEA sheet is produced by 
harvesting split-thickness skin samples from an area of the human or 
animal's body surface that is wound free and culturing the epithelial 
cells from the split thickness skin samples until the cultured cells have 
reached confluence. Over time a neodermis and neoepidermis are formed at 
the burn or wound site, resulting in tissue having the appearance, 
differentiation and growth of normal skin. 
The present invention has many advantages. It allows immediate wound 
coverage, provides excellent CEA take rates and produces a result similar 
to native skin. Preparation of CEA sheets requires only that a minimal 
amount of skin be harvested from the patient, thereby reducing the 
discomfort to the patient. CG bilayers are completely synthetic and 
biodegradable over time. There is therefore no risk of disease 
transmission from donor to patient. In addition, collagen 
glycosaminoglycan can be manufactured in bulk and stored for extended 
periods of time.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention refers to a method of skin regeneration for burns or 
wounds on a human or animal. This invention overcomes many of the 
shortcomings presented by methods presently used to regenerate skin to 
cover burns or wounds. The bottom layer is initially a bi-layer comprising 
a highly porous lattice that is covered with an outer membrane that is a 
moisture barrier. The wound is covered by this bottom layer, with the 
lattice applied directly to the wound and with the moisture barrier 
exposed to the air. 
The lattice serves as a temporary substitute for the dermis and can be any 
structure that has the following characteristic: the composition and 
structure of the lattice must be such that is does not provoke a 
substantial immune response from the graft recipient. The lattice must be 
sufficiently porous to permit blood vessels and mesenchymal cells from 
healthy tissue below the wound to migrate into the lattice. As discussed 
hereinabove, this migration is referred to as "infiltration" and is 
responsible for the generation of the neodermis. To facilitate the 
formation of the neodermis, the lattice is biodegradable. This 
biodegradation must not proceed so rapidly that the lattice disappears 
before sufficient healing occurs, i.e. before sufficient neodermis forms. 
Lattices that degrade too slowly impede cell migration and cause the 
formation of a fibrotic layer of cells surrounding the lattice. A lattice 
which biodegrades after about thirty days is preferable. 
The moisture barrier is any composition which can serve as an outer surface 
to the lattice and must be capable of being manually removed at will from 
the lattice when the CEA sheets are to be applied to the wound, as 
described hereinbelow. Compositions suitable for use as a moisture barrier 
must also have the property of being semipermeable to the passage through 
the wound of fluids from inside the body and impermeable to microorganisms 
such as bacteria and viruses from outside the body. The moisture barrier 
also imparts several desirable physical properties to the bi-layer such as 
tensile strength and suturability. 
A preferred embodiment of the present invention employs a highly porous 
lattice comprised of collagen and glycosaminoglycan (referred to 
hereinafter as "GAG"), i.e. a collagen glycosaminoglycan matrix (referred 
to hereinafter as "CG matrix") with a silicone elastomer as the outer 
membrane. A CG matrix with an outer silicone surface is prepared according 
to methods known to those skilled in the art. See U.S. Pat. Nos. 4,060,081 
(Yannas et al., 1977), 4,280,954 (Yannas et al., 1981) and 4,505,266 
(Yannas and Burke, 1985), the teachings of which are incorporated herein 
in their entirety. Various forms of GAG which may be suitable for use in 
this material include chondroitin 6-sulfate, chondroitin 4-sulfate, 
heparin, heparin sulfate, keratan sulfate, dermatan sulfate, chitin and 
chitosan. 
It is possible to control several parameters of the CG matrix (primarily 
crosslinking density, porosity and GAG content) to control the rate of 
biodegradation of the lattice. Specific conditions for forming a CG matrix 
suitable for use in the present invention are given in the 
Exemplification. However, the skilled artisan will know of other 
conditions for forming CG matrices with variations of the above-mentioned 
parameters which are similarly suitable for use in the present invention. 
In addition, certain applications of skin regeneration may require 
matrices which degrade more slowly or more quickly. The skilled artisan 
will be able to recognize applications where it is desirable to vary the 
properties of the CG matrix, and will be able to vary the parameters 
accordingly. The present invention encompasses such variations in the CG 
matrix. 
Although the research that led to this invention involved CG matrices and 
silicone outer membranes, the skin regeneration method of this invention 
is not limited to CG matrices and silicone outer membranes. Subsequent 
research may reveal other fibrous proteins, polymeric molecules or 
sintered ceramics which can be used in the present invention. Such 
lattices and materials are within the scope of this invention. 
Once the CG matrix has been prepared, the wound is readied for application 
of the covering. Areas of skin that have been destroyed or damaged are 
surgically removed to prevent it from interfering with the healing 
process. The entire area of dead and damaged skin is excised, so that 
intact epithelial cells are present at the perimeter of the wound. The CG 
matrix, with the silicone side away from the wound, is draped across the 
wound to avoid the entrapment of air pockets between the wound and the 
matrix. The membrane is sutured or stapled to the wound using conventional 
techniques and then covered with a bandage. 
After application of the CG bi-layer, blood vessels and mesenchymal cells 
from underlying healthy tissue begin, as described hereinabove, the 
process of infiltration of the grafted CG matrix. "Infiltration", as 
defined herein, further refers to allowing a sufficient period of time for 
this migration of mesenchymal cells and blood vessels. Sufficient time 
periods are those which permit subsequent application of CEA sheets with 
nearly complete take rates, as described hereinbelow. A preferred period 
of time is about ten days, but periods as low as about two to three days 
can also be used. 
Another aspect of the present invention refers the regeneration of skin at 
a burn or wound site on a human or animal using seeded CG matrices. 
"Seeded CG matrices" refer to CG matrices into which epidermal or dermal 
cells harvested from a wound free site on the patient's body surface have 
been introduced. Each epidermal or dermal cell that survives the seeding 
process can reproduce and multiply, thereby hastening the formation of a 
neoepidermis and/or neodermis. Preferred epidermal and dermal cells are 
keratinocytes and fibroblasts, respectively. Seeded CG matrices are 
described in U.S. Pat. No. 4,060,081, the teachings of which are 
incorporated herein by reference in its entirety. Matrices which have been 
seeded are referred to as "cellular" while unseeded matrices are referred 
to as "acellular". 
Seeded CG matrices may be autologous, i.e. matrices seeded with cells 
obtained from the human or animal having the burn or wound, or they may be 
heterologous, i.e. seeded with cells obtained from a donor. In addition, 
cells being used to seed a CG matrix may undergo genetic manipulation in 
order to prevent rejection or to change the cell's phenotype in some 
beneficial manner. Genetic manipulation includes introducing genetic 
matter into the cells so that the protein gene product or products are 
expressed in sufficient quantities to cause the desired change in 
phenotype. An example of suitable genetic matter includes the gene 
encoding for a growth factor along with the requisite control elements. 
Once infiltration has occurred the wound is ready for the application of a 
CEA sheet onto the CG matrix. The CEA sheet eventually forms a 
neoepidermis. The CEA sheets are prepared by surgically removing 
split-thickness skin samples from the patient in an area of the patient's 
body surface (hereinafter referred to as the "donor site") that is wound 
free. This procedure can take place prior to, concurrent with or after the 
application of the CG bilayer to the wound bed. The epidermis is 
enzymatically and/or mechanically removed from the dermis. The epidermal 
layer is then mechanically and/or enzymatically separated into small 
pieces of epidermis and preferably into individual cells. The epidermal 
cells are then placed in culture media and allowed to reproduce until all 
available intercellular space has been eliminated and the cells have 
formed sheets from about one to seven layers thick. Cultured epithelial 
cells that have reached this stage are said to have "reached confluence". 
This process takes approximately three weeks. The epithelial cells may be 
subcultured a number of times to produce additional cultured grafts. 
Subculture takes a primary CEA, separates and resuspends individual cells 
to be recultured. This can allow for increases of up to four orders of 
magnitude in areas which can be covered by cultured grafts. A specific 
procedure for preparing CEA is included in the Exemplification. 
Preparations of CEA are well known in the art, and the skilled 
practitioner will know of many variations of the specific procedure 
disclosed in the Exemplification. In addition, there may be certain 
applications of this invention where variations in the method of preparing 
CEA will lead to more desirable results. The skilled artisan will be able 
to recognize these applications and make the appropriate changes. All such 
variations in the preparation of CEA that can be used in conjunction with 
CG matrix to form an artificial skin bilayer are within the scope of this 
invention. See Teppe, Roberto George Casper, "Cultured Kerotinocyte 
Grafting: Implications for Wound Healing," Profschrift, Denhang, 
Netherlands, 1993, the teachings of which are hereby incorporated by 
references in their entirety. 
After CEA sheets have reached confluence, grafting onto the CG matrix can 
be performed. The moisture barrier (e.g. silicone outer layer) is manually 
removed from the CG matrix. The surface layer of vascularized matrix may 
be excised with a dermatome or surgical blade. Hemostasis is achieved with 
electrocautery, direct pressure or topical hemostatic agents, if desired. 
The CEA sheets are removed from the culture flask. Petroleum impregnated 
gauze is secured to the surface of the sheets with surgical clips. The 
sheets are then placed on the matrix sites with the basal cell layer side 
down and secured to the matrix site with sutures or surgical staples, 
thereby keeping the CEA sheet firmly adherent to the matrix site. The 
wound is covered with dry sterile gauze which is changed periodically and 
the CEA is allowed to adhere. An autograft that "takes" is indicated by 
visually observing epidermis on the wound surface which persists for 
several days post-grafting. The extent to which a graft takes can be more 
precisely determined by the amounts of wound surface area which is 
epithelized, i.e., how much of the wound surface area is covered by 
neoepidermis. This can be determined by histological means and is 
described more fully in the Exemplification. A graft which takes is 
typically characterized by the pressure of epithelial cells covering the 
neodermis. Eventually there is a formation of a basement membrane at the 
junction of the neoepidermis and dermis, including components such as 
anchoring fibrils. This results in a tight union between the neoepidermis 
and neodermis. 
The invention will now be further and specifically described by the 
following examples. 
EXEMPLIFICATION 
The effectiveness of the present method of skin regeneration was tested on 
Yorkshire pigs by applying CEA sheets to four different types of wounds. 
In one type of wound a CEA sheet was applied onto a CG substrate. A CEA 
sheet was also applied directly to full thickness wounds freshly excised 
to subcutaneous fat and to full thickness wounds freshly excised to 
fascia. Excising a wound to fascia refers to a wound in which the 
epidermis, dermis and subcutaneous fat layers have all been removed. The 
ability of the CEA sheets to take to these three types of wounds was 
tested on three animals, each of which had all three types of wounds. In 
the fourth wound type, CEA sheets were applied directly to a wound seven 
days after the wound was created by excising to subcutaneous fat. This 
type of wound, referred to as "granulating", was tested on two separate 
animals which were free from the other wound types. 
Split thickness skin samples were harvested from three Yorkshire pigs 
according to the technique described below. The skin samples from each pig 
were then cultured separately to prepare CEA sheets according to the 
procedure described below. Fourteen days after the harvesting of the 
split-thickness skin samples, 4.times.4 cm full thickness wounds excised 
to fat using a surgical scalpel were made on the dorsums of the three 
pigs. A total of sixteen wounds were made on all three pigs. CG bilayers, 
prepared according to the procedure described below, were applied to the 
freshly excised wounds, silicone side up. The membrane was then secured 
with sutures. 
Twenty-four days after the taking of the split thickness skin samples, the 
CEA sheets had reached confluence and were ready for application to the 
animals' wounds. In preparation for the CEA grafting, the silicone layer 
on the CG matrices that had been grafted onto the wounds of the subject 
pigs was manually removed. A total of twelve 4.times.4 cm full thickness 
wounds, using standard surgical techniques, were then made on the dorsums 
of the subject pigs. Eight of these wounds were excised to subcutaneous 
fat, the other four were excised to faschia. 
The CEA sheets prepared from skin harvested from the subject pigs were 
removed from culture and secured to petroleum impregnated gauze with 
surgical clips. They were cut to a suitable size and then applied directly 
to the three types of wounds, basal side down. Each animal was grafted 
only with CEA sheets derived from cells harvested from that particular 
animal. Dry sterile gauze was placed over the sheet and secured to the 
matrix or wound with sutures or stainless steel staples. Dry sterile gauze 
dressings were applied and changed daily. 
Seven days after the grafting of the CEA sheets, the animals were examined 
for the completeness of graft take. This was first done visually. Areas of 
CEA that appeared pink, with a translucent surface, were judged to have 
taken. How completely the grafts had taken was also determined by 
quantitative histology. Regularly spaced biopsies were taken from each 
wound. The biopsies were spaced so as to be representative of the wound 
area. The neoepidermal was examined along the entire cross section of each 
wound biopsy. Hematoxylin and eosin sections were analyzed along the 
entire cross section for the presence of epithelial cells on the neodermal 
surface. The percentage of epithelial coverage on the dermal layer is the 
histological take. The gross and histological takes are given below in the 
Table. 
The experimental protocol for determining the effectiveness of CEA take 
when CEA sheets are applied to granulating wounds was the same as 
described hereinabove except for the following modification. Four full 
thickness wounds 4.times.4 cm in size were excised to subcutaneous fat on 
the dorsum of each animal seventeen days after harvesting the split 
thickness skin samples and allowed to granulate. Granulating wounds 
underwent periodic dressing changes with a petroleum impregnated gauze. 
Grafting of the CEA sheets took place seven days later. 
HARVESTING OF SKIN 
A fasted Yorkshire pig (15-20 kg) is suspended in a Panepinto body sling 
and anesthetized with 1.0-2.5% Halothane delivered in conjunction with a 
30:50 mixture of nitrous oxide and oxygen via a facial mask. A 
pulseoxymeter is used to monitor heart rate and blood oxygen levels during 
the procedure. 
The dorsal hair of the scapular area is clipped with shears and the 
remaining stubble is removed with shaving cream and a razor. The area is 
then washed for three minutes with germicidal soap and sterile water. A 
three minute application of 70% isopropanol completes the surface 
preparation. 
The donor area is then sterile draped and sterile mineral oil is used for 
lubrication. A Goulian knife with a 0.010" shim is used to remove strips 
of donor skin. The harvested skin is then placed in sterile vials of 
phosphate buffered saline (PBS) supplemented with an 
antibiotic/antimycotic solution for travel to the culture facility. 
PREATION OF CEA 
The skin samples are washed twice in fresh PBS, placed in 0.25% dispase 
solution (single layer with no folds or overlap) and either placed at 
37.degree. C. for two hours of 4.degree. C. over night. 
After dispasing, the skin samples are again washed in PBS and the epidermis 
is mechanically separated from the dermis (with a pair of dissecting 
forceps) and placed in a 0.5%/0.01% solution of trypsin and EDTA. The 
dermis is scraped with a scalpel to remove any basal cells and then 
discarded. The epidermal sheets are placed in a single layer without 
overlap; the sheets are then incubated with the trypsin solution for 30 
minutes to separate the cells. 
The epidermal samples are finely shredded with forceps and the cell 
solution is resuspended for one minute. The trypsin activity is 
neutralized by adding an equivalent amount of fetal bovine serum (FBS) 
supplemented medium (all references to medium refer to 20% FBS 
supplemented Waymouth's medium, see below). The cell solution is 
resuspended for four minutes and filtered through a 100 .mu.m sieve into a 
new dish. The original dish is washed with 5 ml of medium and filtered to 
reduce the cell loss during dish transfer. The suspension is placed in a 
sterile centrifuge tube and centrifuged for five minutes at 1200 rpm 
(5.degree. C.); the dish can again be rinsed with 5 ml of medium to 
dislodge extra cells. 
After centrifuging, the supernatant is removed, the pellet is re-suspended 
in 10 ml of medium, and the cell suspension is placed on ice. The cells 
are counted; trypan blue is used to exclude nonviable cells. 
The suspension is then diluted to 1.times.10.sup.6 cells/ml and 10 ml of 
the solution is plated in 75 cm.sup.2 culture flasks. The flasks are 
placed in an incubator (37.degree. C.) with 5% CO.sub.2 and 90% humidity. 
Culture medium is changed every other day. Toward the end of the culture 
period (approximately three weeks), the medium may need changing daily. 
CULTURE MEDIUM WITH SUPPLEMENTS 
500 ml 1x Waymouth's Medium MB 752/1 
114 ml Fetal Bovine Serum (Sigma F-2442) 
5 ml 100x MEM Nonessential Amino Acids 
2 ml L-arginine Stock (11.4 g/100 ml) 
1 ml Sodium Pyruvate (11.0 g/100 ml) 
1 ml Putrescine--HCl Stock (116.11 mg/100 ml) 
2 ml Insulin (2.5 mg/ml in 4 mM HCl) 
1 ml Hydrocortisone (5 mg/ml in 95% EtOH) 
10 .mu.l Cholera Toxin (5 mM) 
10 ml 100x Antibiotic/Antimycotic (GIBCO) 
Amphotericin B (25 ug/ml), Penicillin G Sodium 
(10000 units/ml), Streptomycin Sulfate 
(10000 ug/ml). 
PREATION OF CG MATRIX 
Bovine hide collagen, 0.5% by weight is dispersed in 0.05M acetic acid and 
coprecipitate is concentrated by centrifugation and excess acetic acid is 
decanted. The concentrated coprecipitate is poured into flat stainless 
steel freezing pans to a volume of 0.3 ml per square centimeter and placed 
on the cooled (-30.degree. C.) shelf of a freeze-drier. The frozen aqueous 
component of the coprecipitate is sublimated under vacuum to produce a 
highly porous matrix 2-3 mm thick. The constituent molecules of the matrix 
are cross-linked using a 24 hour dehydrothermal treatment at 105.degree. 
C. and 30 milliTorr. The now sterile material is coated with a thin 
(approximately 0.3 mm thick) layer of silicone, which is cured in 0.05M 
acetic acid at room temperature for 24 hours. The matrix is further 
cross-linked by a 24 hour treatment with a 0.25% (by volume) 
glutaraldehyde solution in 0.05M acetic acid. The ECM analog is then 
exhaustively dialyzed in sterile, de-ionized water and stored in sterile 
70% isopropanol until use. Before grafting, the matrix is rinsed in 
phosphate buffered saline (PBS) to remove the alcohol. 
RESULTS 
TABLE 
______________________________________ 
Gross Histologic 
Treatment Take (%) Take (%) 
______________________________________ 
Type I CEA on CG 100 (n = 16) 
97 .+-. 3 
(n = 2) 
Type II 
CEA on fascia 
0 (n = 4) 
0 (n = 1) 
Type III 
CEA on fat 0 (n = 8) 
8 (n = 1) 
Type IV 
CEA on 77 .+-. 10 
(n = 8) 
granulating 
wounds 
______________________________________ 
n = number of wounds. 
Gross observations showed complete take of CEA on CG substrate. 
Histological measurements from 2 sites confirmed that the CEA was 97 
.+-.3% adherent to the CG substrate, by 7 days after grafting. By 
contrast, gross observation indicated no take of CEA on fascia or fat. 
This was confirmed by an observed histologic take on fat and fascia of 8% 
and 0%, respectively, by 7 days after grafting. Histological observation 
indicates that CEA take on granulating wounds (77% .+-.10) was better than 
with CEA on freshly excised wounds, but still inferior to CEA take on CG 
matrix. The ability of CEA to take when applied directly to granulating 
wounds has little relevance clinically. A patient suffering from burns or 
wounds that cover a large portion of the patient's body surface is faced 
with an immediate threat of a loss of body fluids and infection. 
Consequently, such a patient cannot afford to wait seven days for a wound 
to granulate before closing the wound. Therefore, the improvement 
represented by the present method over existing methods in a clinically 
relevant setting is determined by comparing the take rates of CEA on CG 
matrix with the take rates of CEA on freshly excised wounds. These results 
indicate that the claimed invention represents a clear improvement over 
existing methods of regenerating skin at wound sites. 
Growth, maturation and differentiation of the CEA on CG were histologically 
similar to that of normal epidermis. CEA grafted on CG matrix, as compared 
to on full-thickness wounds, appeared less fragile and seems more 
resistant to shearing. As early as 7 days after grafting CEA onto CG 
matrix, the resulting tissue was pink, soft and supple. 
EQUIVALENTS 
Those skilled in the art will know, or be able to ascertain using no more 
than routine experimentation, many equivalents to the specific embodiments 
of the invention described herein. These and all other equivalents are 
intended to be encompassed by the following claims.