Note: Descriptions are shown in the official language in which they were submitted.
~ cnld
10/15/81
~:Z02~.~98
CELL-SEEDING INTO FIBROUS
LATTICES BY MEANS OF
CENTRIFUGATION
Government Support
The invention describe~ herein was supported in
whole or in part by grants from the National Institutes
of Health.
Technical Fields
This invention is in ~he fields of medicine,
surgery, biology, biochemistry and polymers.
BACKGROUND ART
Damage to or loss of the skin can be a very
serious injury, resulting in extreme pain, disfigurement,
mutilation, and frequently death. Medical and surgical
treatment of severely burned people is very time-
consuming and expensive, and requires elaborate equip-
ment and highly-trained personnel. Extensive scarring
and wound contracture can lead to physiological,
emotional and societal impairment.
Skin loss or injury is most commonly caused by
fire or scalding, by mechanical or chemical injury,
or by skin lesions. Since burns are the most common
cause of skin injury, burn injury is referred to
herein; however, it should be understood that, with
possîble minor exceptions known to those who are
skilled in the art, the treatment of skin loss or
damage caused by any type of injury or disease
may be conducted according to the methods of this
invention.
A person or animal that suffers from a burn,
injury, disease, or removal of all or a portion
of the skin or an organ is referred to herein
120Z~8
as a "patient." An area of the body where tissue
has been lost, damaged, diseased, or surgically
removed is referred to herein as a "wound" or a
"woundbed. n An area of intact skin or organ from
which cells are harvested, or a cell bank or tissue
culture from which cells are harvested or otherwise taken,
is referred to hereih as a "donor site." Tissue that is
regenerated by cell growth upon a wound is referred to
herein as "neodermal," "neoepidermal," or "neo-organ"
tissue.
Skin Structure
Normal, undamaged skin is composed of several
layers [1]. The outer layer, usually called the
epidermis, is composed of several types of epithelial
cells. It contains nerve fibrils, but not blood
vessels. The outermost layer of epidermis, usually
called the stratum corneum, comprises squamous (i.e.,
relatively flat) cells that typically have low rates of
reproduction; these cells are gradually sloughed
off by abrasion, and are replaced by cells from the
lower layers. The next lower layer is usually
called the stratum lucidum, which may be absent in
certain areas. The lowest layer of squamous cells
is usually called the stratum granulosum. Below it
are two layers of nonsquamous cells: the stratum
malpighii (also called the rete mucosum) and the
stratum germinativum.
Cells that are at or near the base of the
epidermis (near the dermis) are often called basal
cells. These have relatively high rates of repro-
duction, and may give rise to other basal cells, orto maturing epidermal cells which do not subsequently
reproduce. Epidermal cells produce keratin, a
protein which may ~e secreted or contained in cell
walls. This protein is relatively hard, and imparts
toughness and strength to the skin.
:~02~
--3--
Below the epidermis is a layer of cells and
connective tissue called the dermis. This layer
comprises mesenchymal cells, which includes fibroblast
cells and cells of blood and lymph vessels. Hair fol-
licles, sebaceous glands, and sweat glands extendfrom the dermis to the surface of the sXin; such glands
and follicles are lined by epithelial cells.
Mesenchymal cells produce and secrete collagen,
a fibrous protein. Such collagen forms a structural
matrix that surrounds and contains the cells, which
increases the strength of the tissue.
The interface between the aermis and epidermis
is folded and papillary, rather than flat or level.
Therefore, a burn that is relatively level may remove
all of the epidermis and part of the dermis at
numerous locations, while leaving clusters of intact
epidermal cells interspersed in the damaged area.
A burn that is somewhat deeper may remove all of the
surface layers of epidermal cells (and some dermal
cells as well), without destroying the epithelial cells
that line hair follicles and sebaceous and sweat
glands. If this occurs, the epithelial cells from
the follicles and glands may proliferate and migrate
over the wound, creating a shallow layer of epidermis.
~5 Such a layer is often irreyular and thin, and it
may impede proper healing of the wound. Either of
the burns discussed above is classified as a second
degree burn. A burn that destroys the epidermis and
the full thickness of the dermis, including the
epithelial cells that line follicles and giands, is
classified as a "full thickness" or third degree
burn 12].
~2C~2~1B
Burn Treatment
A patient who has suffered extensive skin loss
or injury is ir~ediately threatened by infection and
by excessive loss of fluids. To meet both of these
needs, a severe skin wound must be closed promptly by
some type of membrane. A variety of attempts have been
made to meet this need. References to papyrus or
animal skin date back to about 1500 B.C. Specially
prepared pigskin is often used by surgeons today because
of its ready commercial availability. These xenografts
~ (i e., membranes of non-human origin) can cover a wound
for about three to five days. Howe~er, they are
rejected by the patient, leaving behind an open
woundO Therefore, they must usually be removed or
changed after a few days, and serve essentially as a
stopgap while the patient's skin slowly heals [3].
Homografts, also called allografts, can be
obtained from human cadavers. However, they are in
short supply and, like xenografts, are commonly
rejected after a brief period. Immunosuppxessive
drugs may be used to delay or reduce the rejection
of xenografts or homografts, thereby extending the
period that ~hey may cover a woundbed. However,
the use of immunosuppressive drugs increases the
vulnerability of the patient to infection l4].
Autografts are partial-thickness sections of
skin which are removed ("harvested") from an un-
damaged area of the patient or possibly from the
patient's identical twin, and transplanted onto a
wounded area. Vnlike xenografts or homografts,
autografts are not rejected by the patient; instead,
they become permanently attached to the wound and
proliferate, thereby providing a new layer of
epidermis and dermis which closes the wound l5].
~20ZP,~8
-5-
The harvesting operation is usually performed
with an instrument called a dermatome, which contains
an oscillating blade and adjusting devices that
control the depth and the width of the cut [6]. Since
cells in the stratum corneum normally do not reproduce
rapidly, virtually all harvesting operations remove
cells from the stratum granulosum. Because of
the papillary nature of skin, most harvesting
operations also remove cells from the stratum
malpighii and the stratum germinativum, as well
as significant amounts of dermis.
The harvesting operation is a painful, invasive
process, which causes scarring. It should therefore
be kept to a minimum. In addition, a badly in~ured
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.
In one important modification of the autograft-
ing process, a pattern of slits is cut into a piece
of harvested skin. This allows the skin to be
stretched into a mesh, thereby increasing the wound
area that can be covered by that piece of skin [7].
After grafting, the autologous cells migrate and
proliferate, closing up the gaps caused by the slits.
~2~Z~
-- 6 --
Eventually, with the aid of autografts, the entire
wound area is covered by a layer of regenerated skin
which is subject to various problems such as hyper-
trophic scarring, discomfort, and disabling contracture.
The Bilayer Me~brane
The Applicants are co-inventors (along with
certain other persons) of a synthetic multilayer mem-
brane (herein referred to as a bilayer membrane) that
is useful in treating skin loss or injury. See U.S.
Patent 4,060,081 (Yannas et al, 1977~ and U.S. Patent
4,280,954 (~annas et al, 1981). Briefly, the top layer
of this membrane comprises a polymer such as a silicone
elastomer. This layer imparts several desired physical
properties to the membrane, iincluding tensile strength,
suturability, control of moisture flux, and impermea-
bility to bacteria~and viruses. The bottom layer
comprises a highly porous lattice made of collagen and
glycosaminoglycan (GAG, also referred to as mucopoly-
saccharide). Various forms of GAG which may be suitable
for use in this material include chondroitin 6-sulfate,
chondroitin 4~sulfate, heparin, heparan sulfate, keratan
sulfate, dermatan sulfate, chitin, and chitosan.
The collagen/GAG lattice effectively serves
as a supporting or "scaffolding" structure in or on
which epithelial and mesenchymal cells from the burn
victim can grow and proliferate. Its composition
and structure are controlled so that it does not provoke
a substantial immune response by the graft recipient,
and it is slowly biodegraded into non-toxic substances
that are utilized or eliminated by the body.
It is possible to control several parameters
(primarily crosslinking density, porosity and GAG
content) of the collagen/GAG lattice to control the
~OZP~)8
rate at which the lattice is biodegraded by collagenase
and other enzymes. Lattices that are biodegraded
too quickly will disappear before sufficient healing
occurs, while lattices that are biodegraded too
slowly tend to impede cell migration and to cause
the formation of a fibrotic layer of cells surrounding
the lattice. It is believed that a lattice that
is biodegraded after about thirty days is preferable
for use on burn patients.
ln When a bur~ pat~-ent ls ad~itted to a hospl-tal,
areas of skin that have been entirely destroyed or
severely damaged often contain dead or damaged skin,
called "eschar." The eschar usually is suryically
removed to prevent it from interfering with the
healing process. The entire area of damaged and
dead skin is excised, so that intact epithelial
cells are present at the perimeter of the wound.
The bilayer membrane discussed above is carefully
draped across the wound surface to avoid the entrap-
2n ment of air pockets between the wound and themembrane. The membrane normally is sutured to the
intact skin using conventional techniques. The
grafted area'is then covered with-a bandage.
The collagen/GAG lattice has been observed by
the Applicants to reduce wound contraction. In general,
wound contraction comprises horizontal motion by exist-
ing cells in, and the periphery of, the wound. It
results in substantial distortion and scarring, so
its prevention or reduction is very desirable.
Within a period of several days, healthy cells
from the woundbed begin to migrate into and proliferate
within the collagen/GAG lattice of the membrane.
Mesenchyma;l cells and minute blood vessels migrate
in a direction that is perpendicular to the plane
of the membrane. Since the skin surface is regarded
herein as horizontal, the direction of mesenchymal
skin growth is referred to herein as vertical.
~202~
--8--
Epithelial cells migrate across the surface of the
lattice in a horizontal direction (i.e., along the
plane of the membrane). Since bllrns and other skin
wounds tend to be relatively shallow, mesenchymal
cells need not migrate very far in order to create
-5 a neodermis. However, epithelial cells may be
required to migrate great distances to create a
neoepidermis and close the wound.
Within thirty days, epithelial cells are capable
of migrating and proliferating horizontally a distance
of about 0.75 cm. Therefore, a wound with a
horizontal minor dimension no larger than about
1.5 cm could be closed by epithelial migration
within about thirty days, assuming that epithelial
cells are closing the wound from all sides. However,
extensive burns often exceed 1.5 cm in every direction.
For example, it is not unusual for a badly burned
patient to lose virtually all of the skin below
the shoulders. Therefore, epithelial cells cannot
reach the interior areas of a large wound by normal
migration for periods exceeding hundreds of days.
When used with very large wounds, a collagen/GAG
lattice with a biodegradation rate of about 30
days would be entirely biodegraded long before
the epithelial cells could close the wound.
Preparation of an Aqueous Suspension of Cells
There are several known techniques for dis-
sociating a cohesive piece of skin into a suspension
of living, reproducing cells in a liquid solution [8].
One common technique involves treating a piece of
harvested skin with trypsin, collagenase, or other
enzymes that cause cells to become detached from
other cells or from solid surfaces. After a piece
of skin is treated with one or more enzymes, a layer
~02F)'~8
of epidermis is removed and discarded. The remaining
tissue, which contains basal cells that reproauce at
relatively high rates, is agitated with enough
force to separate the cells without damaging them.
For example, stirring at low speeds, vortexing,
pipetting, and other forms of mi~ing can be used for
this purpose. The cells are usually suspended in
an aqueous solution that contains various salts
that resemble the substances found in body fluids;
this type of solution is often called physiological
saline. It may be buffered by phosphate or other
non-toxic substances, in order to maintain the pH
at approximately physiological levels, and it may
be supplemented by animal or human blood serum
or other sources of protein or other nutrition.
The density of the liquid-may be controlled so that
it is less than the density of the cells.
1~02.~,~8
--10--
DISCLOSURE OF THE
INVENTION
This invention comprises the use of centrifugal
force to introduce viable cells into a fibrous
lattice, as well as fibrous lattices that are seeded
with cells by the use of centrifugal force. A
variety of fibrous lattices may be seeded by the
methods of this invention, such as a highly porous
lattice comprising collagen fibers crosslinked with
glycosaminoglycan. Before the centrifugation, a
piece of intact tissue is harvested from a donor site.
It is treated with one or more substances, such
as trypsin or collagenase, to dissociate cells
from the tissue. The cells are then mixed with
an aqueous solution to create an aqueous suspension
of cells. A piece of fibrous lattice is placed
within a container, referred to herein as a "bucket,"
that is suitable for rotation by a centrifuge.
The aqueous suspension of cells is placed within the
bucket, in contact with the lattice. The centrifuge
is then rotated. Centrifugal force causes the
cells, which are denser than the aqueous solution,
to be forced into the lattice in a relatively
uniform distribution. By controlling various
parameters, cells can be seeded into any desired
location within a lattice. Lattices that are seeded
by centrifugal metho~s may be used to promote the
growth of cells or the generation of tissue at a
wound.
BRIEF DESCRIPTION OF
DRAWINGS
The' drawing is a flow chart indicating a sequence of
steps comprising the invention.
~02~.~8
!~ Best Mode of Carrying Out the Invention
In one preferred embodiment of this invent;on, a
piece of epidermis containing healthy, reproducing
epithelial cell~ is harvested from a donor site on
a burn patient. This piece of epidermis is dissociated
into an aqueous suspension of cells by treating it
with trypsin, collagenase, or other suitable enzymeS.
A piece of bilayer membrane, described in U.S. Patent
4,060,081 (Yannas et al, 1977) is placed on the bot-
tom of a "swinging"-type bucket that is suitable for
rotation by a centrifuge, or within a specimen holder
that is suitable for placement within such a bucket.
The membrane is arranged within the bucket so that the
silicone layer is pressed against a wall of the bucket
or the specimen holder, and the collagen/GAG lattice
is exposed to the interior of the bucket. The cellular
suspension is introduced into the bucket, so that the
solution and the cells within it contact the collagen/
GAG lattice. The bucket is then rotated to generate
centrifugal force upon the lattice and suspension.
The cells in the suspension, which have greater
density than the liquid, are forced toward the
- walls of the bucket, and thereby become embedded
in the collagen/GAG lattice.
The speed and duration of rotation of the
bucket may be controlled to embed the epithelial
cells into a lattice at a desired depth or range
of depths. For example, if the bucket is rotated
at sufficiently high speed for a sufficiently
long period of time, a substantial number of cells
will be forced through the entire thickness of
the collagen/GAG lattice and will come to rest against
the silcone layer. If the container is rotated
at lower speed or for a shorter period of time,
a substantial number of cells may be embedded at
various depths within the collagen lattice.
Centrifugation may be performed in stages. For
example, a collagen/GAG lattice may be fitted into
(
~oz~
a bucket, and a cellular suspension added to the
bucket. The bucket may be rotated for a predetermined
period of time, and then stopped. A second volume
of cellular suspension may be placed in the bucket,
and the bucket may be rotated again. This two-stage
centrifugation may be used to seed cells into a thick
lattice with more uniformity than a single-stage
centrifugation. The process may be repeated any
number of times.
The seeding density of cells within a lattice
may be controlled, primarily with respect to the
horizontal area but also with respect to the thick-
ness of the lattice, by controlling the concentration
of cells within the suspension, i.e., the number
of cells within a given volume of solution or by
controlling the quantity of suspension placed
within.
After a membrane that has been seeded
centrifugally is grafted onto a woundbed, surviving
cells will reproduce and form numerous colonies
of cells. Each colony will grow radially until
it meets a neighboring colony. In this way, the
seeded cells grow to confluence and close the
wound.
A wound may be closed more quickly if cells
are seeded within the membrane in a relatively
dense areal pattern, i.e., if the seeded cells
are closer together in the plane of the membrane,
~eferred to herein as horizontal. The appropriate
areal density will normally depend upon numerous
specific factors involving the wound and the
patient's condition. In general, the closure
time for a wound of a given size is inversely
related to the seeding density and to the size
of the harvested piece of skin.
It is possible to embed more than one type
of cell into a lattice using the centrifugation
(
~02~98
-13-
technique. Under the present state of cell culturing
techniques, it is believed that epithelial cells
tend to reproduce more rapidly when they are in
intercellular communication with fibroblast or
other mesenchymal cells [~]. A aelay of several
days normally is necessary after grafting an unseeded
bilayer membrane onto a wound, before removing the
silicone layer and suturing a layer of autologous
cells onto the lattice. One of t~e-purposes of this
delay is to allow mesenchymal cells and blood vessels
to grow vertically into the lattice from the wound-
bed. However, this delay may be avoided or reduced
if mesenchymal cells are harvested from the patient
and centrifuged into the lattice prior to being
grafted onto the wound. In one embodiment of this
invention, epithelial and-mesenchymal cells may
be mixed within the same liquid suspension and
centrifuged simultaneously into a lattice. In
an alternate embodiment of this invention, epithelial
cells may be centrifuged into a collagen lattice,
embedding them near the silicone layer, and mesenchymal
cells may then be centrifuged into the lattice.
In either embodiment, at least-some of the mesenchymal
cells will be below (i.e., closer to the wound su~face)
the epithelial cells when the membrane is grafted
z5 onto the wound, and epithelial cell reproduction may
begin very quickly. In another alternate embodiment,
mesenchymal and/or endothelial cells may be centrifuged
into the lattice in order to reduce the delay that
is required be~ore epidermis is autografted onto
the lattice.
It is possible to commence the harvesting,
dissociation, and centrifuging operation as soon `
as a patient is admitted to a hospital. All three
of these procedures can be completed within the
/
~02~,~8
-14-
space of a few hours. Therefore, it is possible
to prepare and graft a fully-seeded membrane onto a
patient while the patient is still under general
anesthesia during the admittance operation, while
eschar is being removed from the wound. In this
way, a single operation may be sufficient to clean
the wound and replace it with a cell-seeded synthetic
membrane that is capable of promoting full closure
of the wound~ This may eliminate the need for a
long and painful series of operations to place
xenografts or allografts on a wound, remove them
before they are rejected, and eventually replace
them as autografts become available.
The centrifuging procedure also reduces or
eliminates difficulties that might arise in removing
lS the silicone layer from the collagen lattice of the
bilayer membrane. Over a period of several weeks
or months ~which can be varied by controlling
certain parameters of the collagen lattice~ the
collagen lattice is eventually biodegraded. It is
replaced by collagen which is produced and secreted
by cells growing within the lattice. This collagen
is produced and secreted under wet conditions, and
it does not become affixed to the silicone layer.
The silicone layer, which was initially attached
to a lattice of dry collagen, spontaneously peels
off of the collagen lattice when epidermal cells
grow between the collagen lattice and the silicone
layer. This eliminates the need for surigcal -
removal or peeling of the silicone layer.
An important advantage of centrifugal seeding
is that it can be used to greatly expand the area
or volume of a wound that can be closed quickly
by multiplication of a limited number of cells.
This provides for two distinct advantages. First,
if a very limited amount of intact tissue is
~02~,~8
-15-
available on a seriously burned patient, then the
centrifugation method may be used to greatly increase
the area or volume of a lattice that may be seeded
with the limited number of available cells. Second,
if a given area or volume of a latti~e needs to
be seeded with cells, then the amount of intact
tissue that needs to be harvested from a donor site
may be greatly reduced. The optimal seeding
densities for specific applications may be determined
through routine experimentation by people skilled
- 10 in the art.
A piece of fibrous lattice or bilayer membrane
may be placed directly into a centrifugal bucket, or
into a specimen holder that is fitted into a cen-
trifugal bucket. Specimen holders may be fabric~ted
from polycarbonate, aluminum, or other materials
which can be conveniently sterilized by autoclaving
or other methods. Typically, a specimen holder will
contain one or more depressions or "wells" into
which a piece of lattice or membrane may be fitted.
- A potential problem exists regarding gaps
between the edge of a lattice and the wall of a
specimen holder or centrifugaI bucket. When driven
by centrifugal force, cells in an aqueous suspension
will travel to the lowest or outermost accessible
area. If a large gap exists between the edge of a
lattice and the wall of a specimen holder, a large
number of cells will collect in the gap rather than
be properly seeded in the lattice. This potential
problem can be avoided or mitigated in a variety
of ways, including the foliowing.
First, a piece of membrane or lattice may be
placed into a well that is the same size. A variety
of specimen holders with different size wells could
be kept on hand to accommodate a variety of membrane
or lattice sizes. After a membrane or lattice has
OZ~98
-15.1-
been centrifugally seeded, it may be trimmed to the
proper size to inlay into a woundbed. Most of the
cells seeded into the unused areas may be recovered
if desired, by techniques such as wringing or
centrifuging. To centrifugally remove cells from
a seeded bilayer membrane, the membrane could be
placed in a specimen holder or centrifugal bucket
with the moisture transmission control layer oriented
toward the axis of rotation.-- -
Alternately, gaps that surround a lattice could
be filled with impermeable material that is as thickas, or somewhat thicker than, the lattice that is
to be seeded. There are several ways to accomplIsh
this. For example, a set of impermeable sheets of
plastic, the same size as the well in a specimen
holder, may be kept in stock. A piece of lattice
can be trimmed by a surgeon to inlay into a woundbed.
The trimmed lattice may then be placed on top of
a sheet of impermeable plastic. An incision through
the plastic may be made along the perimeter of the
trimmed lattice, allowing a piece of plastic identical
in size to be remoued from the~sheet of plastic and
discarded. The surrounding piece of plastic and
the trimmed lattice may then be placed into the
specimen holder or centrifugal bucket.
A variety of centrifugation techniques may be
used in conjunction with this invention. For
example, a quantity or a continuous flow of cellular
suspension may be administered to or removed from
a lattice while the lattice is being rotated.
!
3~Z02~98
-~6-
ALTERNATE MOD~S OF CARRYING OUT THE INVENTION
Cells can be seeded by the methods of this
invention into a porous lattice of virtually any
chemical composition. Although the collagen/GAG
5 lattices disclosed in U.S. Patènt 4,060,081 (Yannas
et al, 1977) and U.S. Patent 4,280,954 (Yannas et al,
1981) contain a relatively small weight percentage
of glycosaminoglycan (GAG) to improve the biocompatibility
and physical properties of the ccllagen, the presence
10 of GAG or any other substance within a collagen
lattice is not necessary for the purpose of this
invention.
Although the research that led to this invention
involved collagen, the cell seeding me~hods of this
15 invention are not limited to methods for seeding
collagen. Subsequent research may reveal that other
fibrous proteins or other polymeric molecules may
also be suitable for prosthetic or other medical
purposes. If such other molecules are formed into
20 porous lattices that are seeded by the methods of
this invention, then such seeding processes, and such
seeded ~attic`es, are within the scope of this invention.
The term "lattice" is used broadly herein to
include any material which is in the form of a highly
porous and permeable structure in which cells can
migrate and proliferate.
"Fibrous lattices" should be construed broadly
to include all lattices which include material that
is fibrous at the macroscopic, microscopic, or
molecular le~el. For example, many polymeric
foams comprise long organic molecules, which may
have numerous side chains or extensive crosslinking.
Alternativèly sintered ceramic materials comprise
numerous particles which may be regarded as fibrous
1~02~J98
- 17 -
in shape or nature. Any such material, if formed
as a lattice that is seeded with cells by the methods
of this invention, is within the scope of this
invention.
It is possible to seed cells into lattices of
any shape or configuration. For example, it may be
possible to create molded bilayer membranes in the
shape of a face, a hand, or another irregular surface.
Such lattices may be seeded with cells by the methods
of this invention, and are within the scope of this
invention.
The methods of this invention may be used in
combination with other methods for seeding cells
into a fibrous lattice. For example, when a burn
patient is first admitted to a hospital, autologous
cells may be harvested from ~he patient, dissociated
into an aqueous suspension, and centrifugally seeded
into a bilayer membrane that is grafted onto the
patient during the initial operation. If an insuffi-
cient number of healthy epithelial cells is available.or if some of the centrifuged cells fail to generate
colonies *ox any reason, then areas of the wound might
not be closed by neoepidermis generated from the
centrifugally seeded cells. Areas of unclosed wounds
can be identified by visually monitoring the transparent
silicone layer of the membrane. These areas can be
reseeded by one or more other methods which are the
subject of U.S Patent No. 4,458,678. For example,
large voids in the epithelial coverage may be seeded
by removing an area of the silicone layer and spraying
or spreading a quantity of a suspension of cells onto
the exposed collagen lattice. Small gaps in the
epithelial coverage may be seeded by syringe emplace-
ment of cellular suspension.
~oz~
-18-
Autologous cells, as aescribed previously
herein, were restricted to cells taken from the
patient, or from the patient's iaentical twin.
This is a reflection of the current status of
5 grafting techniques Using the current techniques,
non-autologous cells tend to be rejected by a wound.
~owever, subsequent advances in cell typing and
matching, cell treating to remove or inactivate
surface or secreted antigens or other molecules,
immunosuppressive agents, and other techniques may
reduce or eliminate this problem, thereby rendering
non-autologous cells suitable to reconstitute lost
tissue, bone, or organ. Any such cells which are
matched or treated in such a manner would be suitable
15 for seeaing into a fibrous lattice by the m~thoa~ of
this invention. Such cells are within the scope of
this invention.
Various types of fibrous lattices may be suitable
for use as prosthetic devices within most regions
of the body, including s~in, blood vessels, bones,
connective tissue, contractile tissue, and organs.
Such lattices provide a structural system in which
virtually any type of cell may grow, migrate, and
proliferate. They can be surgically emplaced within
virtually any region of the body, and if properly
seeded with the appropriate type(s) of cells, may
allow f~r the regeneration of new tissue. For example,
if a patient suffers damage to or disease of an organ,
a portion of the organ may need to be removed. A
fibrouslatticemay be emplaced in the location
created by removal of part of the organ. If a
sufficient number of healthy cells from another part
of that organ, or from a compatible donor, is
seeded into the lattice by the methods of this
invention, it may be possible to greatly promote
~()2P.~98
--19--
the recovery and regeneration of the organ. Such use
falls within the scope of this invention. CentrifuqOl
force may be very useful to seed cells throughout such
lattices, which may be severaI centimeters thick in
5 all directions.
It is possible ~o culture cells in vitro after
they have been harvested, before they are seeded into
a fibrous lattice. This would allow for several
distinct advantages. For example, it can be used to
10 increase the number of cells that are available for
seeding, thereby reducing the amount of tissue that
must be harvested to cover a wound. In addition,
this allows for the use of cell "banks." For example,
people who work in high-risk occupations could donate
15 cells that can be cultured in vitro and available
for seeding into a fibrous lattice if an accident
or injury occurs. The seeding of preserved or cultured
cells into fibrous lattices by the methods disclosed
herein are within the scope of this invention. Cell
2n banks and tissue cultures from which cells of a desired
variety are taken for seeding are within the term
"donor sites" for the purposes of this invention.
A variety of techniques are known for contacting
cells with various substances that increase the
25 reproductive rate of certain types of cells. For
example, it is known that epidermal growth factor 110],
fibronectin lll], cyclic nucleotides [12], choleratoxin
1131, platelet derived growth factor [14], tissue
angiogenesis factor [15], and various other substances
30 116] are capable of increasing the rate of proliferation
and/or surface adherence of one or more types of cells.
Prior to seeding cells into a fibrous lattice by
the methods of this invention, it is possible to
~ ~Q
~iC~V~
--19 . 1--
contact such cells with any substance that is known
or hereafter discovered to increase the rate of
reproduction of such cells. Such pre-seeding treatment
may be usea to increase the number of cells that are
available for seeding, or to induce the cells to
reproduce more rapidly after they have been seeaed.
Such pre-seeding treatment or neodermal surface treat-
ment is within the scope of this invention.
~.~02F..~,~18
- 20 -
Characteristics of Seeded Oollaqen Lattices
The invention described herein comprises a
method of centrifugally seeding cells into or onto
fibrous lattices. It also comprises a composition of
matter which is a fibrous lattice that is seeded with
cells by the method of this invention. In order to
further define that composition of matter, the
following information is provided regarding the
collagen/GAG lattice that is further described in
10 U.S. Patent 4,060,081 (Yannas et al, 1977) and U.S.
Patent 4,280,954 (Yannas et al, 1981).
The physiological response of a wound to a
grafted collagenilattice depends upon a combination
of characteristics of the lattice, rather than upon
any single characteristic acting as an isolated factor.
Therefore, it is preferable not to specify an optimal
numerical value of any single characteristic. Instead,
a range of values can be specified for most charac-
teristics, which assumes that all other characteristics
are simultaneously within suitable ranges. It must
also be noted that the correlations mentioned between
parameters and characteristics are not exhaustive;
instead, only the most direct correlations are mentioned.
, ~
2~.
-21-
1. Controllable Biodegradation. A collagen
lattice, when in biochemical communication with
a wound surface, eventually is biodegraded by
collagenase and other natural enzymes into
non-toxic substances that are digested, utilized,
or eliminated by normal bodily processes. The
lattice must retain its structural integrity
until an ade~uate number of cells have re~
produced within the lattice to regenerate
the lost or removed tissue. If the lattice
is biodegraded more quickly than this, it
will be liquified and renaered useless ~efore
the wound has healed. On the other hand,
research by the Applicants indicates that if
the lattice is biodegraded too slowly, it
tends to promote the formation of a dense
fibrotic sac surrounding the lattice. This
sac impedes the healing of the wound and
tends to exacerbate scarring.
Research with the bilayer membrane indicates
that the ideal biodegradation rate should be
roughly egual to approximtely 25 to 30 days.
This does not mean that the entire lattice
should be biodegraded within 30 days. Instead,
it indicates that a significant amoung of
biodegradation should comrnence within about
30 days, although remnants of the lattice
may persist for several months or more.
Routine experimentation by persons skilled in
the art might indicate that this biodegradation
rate should be modified somewhat for lattices
that are seeded with cells, or for lattices
that are used for purposes other than
synthetic skin.
The biodegradation rate of a collagen lattice
may be decreased ~i.e., the lattice will endure
~Z02~8
-22-
for a longer period of time after grafting onto
a wound~ by increasing the collagen cross-
linking density, by increasing the content
of GAG that is crosslinked with collagen, or
by decreasing the porosity of the lattice.
The s;licone layer of the bilayer
membrane is not biodegradable. However, this
is satisfactory and even preferable, since
this layer is spontaneously ejected
(without requiring surgical invasion or
removal) after neoepidermal tissue has been
regenerated below it.
2. Non-antigenic and non-inflammatory.
Xenografts, allografts, and transplanted organs
normally contain cells that are recognized
as foreign by the immune system of the patient.
In a typical immune response, antibodies
and certain types of cells such as lymphocytes
identify and take part in the attack on
foreign cells unless i~munosuppxessive drugs
are usea to suppress the formation of antibodies
or defensive cells. However, the use of
such drugs renders the patient more vulnerable
to infection. The use of such drugs can
be rendered unnecessary if the grafted substance
does not have antigenic or inflammatory
properties.
The collagen/GAG lattice that has been co-
invented by the Applican~s may be manufactured
so that it does nol possess antigenic or
inflammatory properties, by adjusting the
chemical content and crosslinked structural
arrangement of the collagen and GAG molecules.
If properly prepared, it is readily accepted
by wound surfaces without provoking rejection
by the patient.
1~Z02~9~
-23-
3. Affinity for a Wound Surface. A collagen
lattice must possess sufficient affinity for
a wound surface to efficiently wet the surface
and maintain contact with it. This affinity
is usually expressed as surface tension or
surface energy of an interface, measured in
terms of force per area. The surface energy
of an interface between a wound and a
collagen lattice should be lower than the
surface energy of an interface between the
wound and the atmosphere. This criterion
i5 satisfied by the collagen/GAG lattice co-
invented by the Applicants.
4. Tensile Strength. A synthetic membrane
or prosthetic device should be sufficiently
tough and strong to withstand suturing
without tearing, and to prevent or limit
tearing if subjected to accidental stresses
caused by bandaging or medical operations
or by patient movement. The two most
important indices of strength of a lattice
are tensile strength (which measures how
much force is required to pull apart a specimen
with a known cross-sectional area) and
fracture energy (which measures how much work
is required to create a tear of a given size).
The collagen/GAG membrane has a tensile
strength range of approximately 50 to 1,000
psi, and a fracture energy that ranges from
approximately 1 x 105 to about 5 x 106 ergs/cm3.
~'~02~8
-24-
The strength of the lattice may be increased
by increasing the crosslinking density or by
decreasing the porosity of the lattice.
5. Morphology. In general, "morphology"
relates to the size and spatial arrangement
of the fibers wit,hin a lattice. As such,
it may be regarded as the converse of "porosity,"
which relates to the size, shape, and spatial
arrangement of the open spaces between the
fibers within a lattice.
A synthetic col:Lagen lattice that serves as
a prosthetic device should resemble the collagen
matrix that exists naturally within the type
of tissue that is to be regenerated. This
spatial arrangement will promote the growth
of cells in orderly patterns that resemble
undamaged tissue, thereby reducing scarring
and promoting proper functioning of the
regenerated tissue.
Significant morphological characteristics
of a porous collagen lattice include:
~ a. Volume fraction of the fibers,
which ;s egual to the volume occupied by
the fibers, divided by the total volume of
the lattice. This fraction is the converse
of porosity, which is discussed below.
b. Mean aspect ratio, which is the ratio
of the average length of the fibers to the
average width. A lattice composed of long
and thin fibers woula have a high mean aspect
ratio.
c. Mean orientation of fiber axes, which
indicates whether the fibers are randomly
oriented in all directions, or whether sub-
Z~.~8
stantial numbers o fibers are oriented in
roughly parallel directions along one or
more axes within the lattice.
d. Mean distance between fiber axes,
which indicates how far apart adjacent
fibers are. This characteristic is directly
related to pore size.
It is believed ~hat the porous collagen/GAG
lattice that has been coinventea by the Applicants
has morphological characteristics that resemble
the collagen matrixes that exist normally in
mammalian skin, corneas, and tendons. There-
fore, the aforementioned lattice is very suitable
as a prosthetic device to promote the regeneration
15; of lost or aamage~l skin, corneas and tendons.
Research may indicate that other types of tissue
also have similar morphological characteristics,
and thus may be well-suited to regeneration by
the collagen/GAG lattice described above. In
addition, research may indicate methods of
altering the morphological characteristics
of collagen lattices to resemble thQ collagen
matrixes that exist in other types of tissue.
Such lattices, if seeded with cells by the
methods of this invention, are within the scope
of this invention.
6. Porosity. Four interrelated aspects of
porosity affect the rate of cell migration
and reproduction within a collagen lattice:
a. Porosity, also called pore fraction~
which is a fraction that is equal to the
volume of the lattice. This fraction may
~'02~ 8
-26:
~-~ be multiplied by 100 to convert it to a
percentage. High porosity is desirable,
because it provides more space in which
cells can grow and multiple. Porosity may
be modified to control the rate of
biodegradation and the flexural rigidity
of a collagen lattice.
Research involving the bilayer membrane
indicates that porosity of at least about
ninety percent is aesirable to encourage
cell migration and reproduction within or
on the surface of the lattice. Additional
research by the Applicants indicates that
if porosity is at least about ninety-
five percent, epithelial cells tend to migrate
between the collagen/GAG lattice and the
top silicone layer of the membrane. This
is very desirable, since it allows the silicone
layer to be spontaneously ejected when neo-
epidermal skin is regenerated beneath it.
b. Pore shape and distribution, which
relates to the shape of the pores and the
orientation of the fibers.
c. Pore size, which indicates the
diameter of the average or mean pore. The
pores within a collagen lattice must be
large enough for cells to grow in and migrate
through. Research by the Applicants indicates
that average pore sizes of approximately
50 um tend to encoura~e satisfactory cell
migration and reproduction. Routine
experimentation by those skilled in the art
may indicate that average size, and
possibly the distribution of pore size
about the average, should be varied to
~LX02~9~
enhance cell migration and reproduction
for various uses of collagen lattices.
- d. Connectivity, also callea permeability
whether the pores are isolated or inter-
connected. A closed-cell foam does not
S allow fluid or other material to move
through it; each bubble is trapped. This
type of lattice would be unsuitable for
cell migxation. By contrast, a permeable
lattice contains pores that are interconnected;
this allows the movement of fluids or cells
between pores. The fibrous nature of
collagen, and the freeze-drying procedures
that is used to create the lattices that
are used in this invention, ensure that the
lS lattices are sufficiently permeable to
permit cell migration.
7. Reduction of Wound Contraction. Wound
contraction normally involves migration of cells
in and on the periphery of a wound. For example,
if a small piece of skin is lost or removed
from an animal or ~uman, the surrounding skin
will tend to move across the fascia to close
the wound. -This results in distortion and
scarring of the wounded area, and it is very
detrimental to accurate return to normal
function. Research by the Applicants indicates
that proper creation and emplacement of the
bilayer membrane tends to delay and reduce
wound contraction, which reduces scarring and
contracture deformity and promotes the re-
generation and proper functioning of neoepidermal
skin.
~L~Z02~ 8
-2~-
8. Flexural Rigidity. When placed in con-
tact with a wound surfa~e, a collagen lattice
should be sufficiently flexible to prevent
pockets of air from being trapped between
the woundbed and the lattice. Such pockets
of entrapped air, often called dead space,
become filled with fluid and often develop
into sites of bacterial proliferation and
infection, and therefore should be avoided.
Efficient wetting requires the use of a
lattice with relatively low rigidity.
Flexural rigiaity is a function of the shape
of the lattice and the modulus of elasticity
of the material. The rigidity of a membrane
used as artificial skin may be reauced by
reducing the thickness of the membrane;
however, an organ or bone prosthesis may be
constrained to a specific shape. The
modulus of elasticity (often called Young's
modulus) must be sufficiently low to reduce
the flexural rigidity of a collagen lattice
to acceptable levels, but suffîciently high
to withstand moderate compressive forces with-
out buckling. Materials with a Young'e modulus
between about 1 ana about 100 psi ~depending on
the thickness and shape of the lattice) are
preferred. The Young's modulus of a collagen
lattice may be increased by decreasing the
porosity or increasing the crosslinking density.
9. Moisture Flux. Moisture flux relates
to the amount of water or other liquid that
will permeate through a given area of a
membrane during a given period of time,ex-
pressed by gm/cm2/hr or similar terms.
~IZ~28~38
--29--
~, .
If the moisture flux of a membrane used as
synthetic skin is too high, too much fluid
will leave the wound, and the woundbea and
the membrane will dehydrate, causing shrinkage
ana curling of the membrane. On the other
- hand, if the moisture flux of the membrane is
too low, fluid will accumulate beneath the
membrane, disrupting the desired physiological
processies. Such fluid accumulation is
usually called exuaate or edema. To avoid
either extreme, the moisture flux of a
membrane used as synthetic skin should
approximate the moisture flux of normal skin.
The moisture flux of the collagen/GAG
membrane may be easily controlled by modifying
the thickness of the silicone layer. It
has been found that a silicone layer of
approximately 0.1 to 1.0 mm provides a
moisture flux that is in the appropriate
range.
-30-
EXAMPLES
Example 1: Preparation of Bilayer Membranes
Collagen from bovine hjae, prepared by the methods
- described by M. Komanowsky et al, J. ~ner. Leather
Chemists Assn. 69: #9, p. 410-422 (1974), was donated
by the Eastern Regional Research Center, U.S. Depart-
ment of Agriculture, Philadelphia, PA. It was ground
in a Wiley mill (A. H. Thomas Company, Philadelphia,
PA) using a 20-mesh screen, cooled with liquid
nitrogen. To prepare each membrane, 0.55 g (hydrated
weight) of milled collagen was added to 200 ml of 0.05 M
aqueous acetic acid. This solution was stirred for 60
minutes in an iced-jacketed blender (Eberbach Corp., Ann
Arbor, MI) on a 2-speed power unit (Waring Company,
Hartford, CT) set on high speed with the line voltage
reduced to 60 volts.
0.044 g of chondroitin 6-sulfate (hydrated
weight) obtained from shark cartilage (sodium salt
form, type C, Sigma Chemical, St. Louis, MO) was
dissolved in 40 ml of 0.05 M acetic acid. Over a
period of five minutes, the C6S solution was added
to the collagen dispersion during blending. The
mixture was blended for an additional 10 minutes,
then centrifuged at 1500 g for one hour in a refrigerated
centrifuge (Model CRU-5000, International Equipment,
Needham Heights, MA) maintained at 4C. The dispersion
was removed from the centrifuge, and 140 ml of
supernatant was decanted for each 240 ml of the dis-
persion which was centrifuged. The concentrated
dispersion was then blended for lS minutes in the
Eberbach blender at high speed setting, 60 volts.
The dispersion was then poured into freezing trays;
2 ml of dispersion were aJpplied to each square inch
- 31 -
of tray surface. The trays were placed on a pre-cooled
freezing shelf maintained at -45C (Model 10-MR-PC,
Virtis Company, Gardner, NY). The trays were allowed
to freeze and equilibrate with the shelf temperature
for about one hour. The pressure in the chamber was
then reduced to less than 100 mtorr, and the trays
were allowed to stand for an hour. The shelf tempe-
rature was increased to 0C. The samples were then
lyophilized for a period of 24 to 48 hours.
The resulting foams were removed, wrapped in
aluminum foil, and placed in~a vacuum oven maintained
at 105C and 50 mtorr for a period of about 24 hours.
After removal from the oven, the foams were either
stored in a dessicator, or cooled and coated with
silicone adhesive.
Silicone adhesive (medical grade, Dow
SILASTIC (trade mark) catalog No. 891, Dow Chemical
Company, Midland, MI? was coated over the entire surface
of the cooled foam. The silicone was coated over the
foam surface that was not in contact with the freezing
tray. The silicone was applied with a spatula to a
thickness of approximately 0.1 to 0.5 mm. The bilayer
membrane was placed silicone side down in 0.05 M acetic
acid at room temperature for 24 hours to allow the
silicone to cure. The membrane was then turned silicone
side up and allowed to rehydrate in 0.05 M acetic acid
for 24 hours at room temperature. The acetic acid was
removed and replaced with 0.05 M acetic acid which
contained 0.25% by volume glutaraldehyde (practical
grade, catalog ~o. 8-M752, J.T. Baker Chemical Co.,
Phillipsburg, N~). The glutaraldehyde cross-linking
treatment lasted for 24 hours at room temperature. The
glutaraldehyde solution was removed, and the material
o~
-32-
was rinsed twice in distilled, deionized water. The
foam was stored in water for 24 hours at room temperature,
then transferred to a storage container. It was
stored in a solution of 70% isopropanol in water at
4C until shortly before use.
Typical characteristics of membranes prepared by
these methods are indicated in Table 1.
1202~
-32.1-
TABLE.l
Characteristics of Bilayer Membranes
Prepared as Described in Example 1
Tensile strenyth
C/GAG lattice 2 to 5 x 10 newtons/m
Bilayer membrane 7 to 10 x 10 newtonsjm
Average pore diameter
C/GAG lattice before wetting 80 microns
Average porosity
C/GAG lattice before wetting 96%
Mois~ure flux
Bilayer membrane 1 to 10 mg/cm2/hr
Bending rigidity of 1 cm wide strip
C/GAG lattice 5 to 150 x 10 newton-m2
Bilayer membrane 10 to 500 x 10 newton-m2
Antigenicity Very low
Pyrogenicity . Not detectable
Significant biodegradation 25 to 30 days
Mean orientation of fiber axes Random
(~
~Z'02~98
-33-
Example 2: Preparation of Aqueous Cellular Suspensions
Autologous cells may be harvested from a guinea
pig back or from a human body using a dermatome, or
from the rim of a guinea pig's ear. Harvested cells
are placed in cold ~4C) phosphate-buffered saline
solution (PBS) without calcium or magnesium (catalog
~17-515B, M.A. Bioproducts, Walkersville, MD). Before
the skin is treated with ~rypsin, it is transferred
to warm PBS (about 30C). The skin is then incubated
at 37C for 40 minutes in a solution of 2.5~ trypsin
in Hanks' balanced salt solution without calcium or
magnesium (catalog $17-160H, N.A. Bioproducts), diluted
with PBS to 0.25% trypsin. Following incubation,
the epidermal layer is separated from the dermal layer
and discarded. The dermal layer, which contains
a relatively high number of reproductive basal cells,
is transferred to tissue culture medium (Dulbecco's
modified eagle medium without glutamine, catalog
~12-707B, M.A. Bioproducts, supplemented with 10%
fetal calf serum and L-glutamine shortly before use).
This solution is then vortexed for 1.5 minutes to
release basal cells from the tissue. The suspension
is then filtered through sterile gauze to remove
large tissue fragments.
Cell concentration is determined by using a cell
counting chamber or electronic particle counter. Cell
viability is determined by staining an aliquot of
cells with trypan blue (Grand Island Biological Company,
Grant Island, NY). The cell density is adjusted to
approximately 10 viable cells/ml by addition of tissue
culture medium.
~;zo~
-34-
Example 3: Centrifugal Seeding Methods and Results
A piece of bilayer membrane ~prepared as described
in Example 1) approximat:ely 1.5 by 3.15 cm in area,
about 1-2 mm thickness, was placed in a specimen
holder fabricated of milled polycarbonate. The holder
and membrane were then placed in a swinging centrifugal
bucket (International Equipment Model 353-S, Needham
Heights, MA). The silicone layer of the membrane was
placed against the bottom of the specimen holder, so that
the collagenlGAG lattice of the membrane was exposed.
About 1.3 ml of aqueous cellular suspension (prepared as
descrr~ed in Example 2) was placed on top of the lattice
by means of a pipette; this corresponds to a seeding
density of about 0.29 x 106 cells per cm2. The bucket
was placed in a regrigerated centrifuge (International
Eauipment Model CRV-5000, Needham Heights, MA~ main-
tained at about 4C, and rotated at about 50 g for about
15 minutes.
The seeded membrane was removed from the bucket.
A strip of mem~rane about 1.5 x 0.15 cm was removed
from each membrane, and subjected to biological analysis.
The remainder of the seeded membrane was sutured onto
a 1.5 x 3.0 cm wound on the back of a guinea pig.
This operation was performed on about 20 guinea pigs.
The operations succeeded-in seeding epithelial cells
into the membranes which reproduced into colonies of
cells. Most of the animals were sacrificed for histo-
logical sudies before the cell colonies fully closed the
wounds. However, on those animals that were not sacri-
ficed before wound closurle, the cell colonies grew to
confluence and created a ]permanent, functional layer of
neoepidermis. Although the neoepidermal areas tended
-35-
to lack hair follicles, sebaceous glands, or sweat
glands, the neoepiderma:L layers tended to be smoother,
less scarred, and less ibrotic than neoepidermis
generated by wound contraction, unaided healing, or
conventional autografting. Wound closure aided by a
properly seeded collagen/GAG lattice usually occurred
within about 7 to 14 days.
1202898
-35.1-
Example 4: Modifications of the Centrifugation Methods
Several modifications of the foregoing procedures
were perfo~med to assess the importance of several
parameters. In one such modification, the concentra-
tion of viable cells in the cellular suspension wasincreased to about 3 x 106 cells/ml. 1.3 ml of sus-
pension was applied to a 4.5 cm2 membrane, for a seeding
density of about 0.87 x Lo6 cells per cm2. However,
the rate of wound closure at the high seeding density
was not substantially improved by the increase in
seeding density, and the apparent condition of the
regenerated epidermis after 14 days was not markedly
improved.
In a second modification, the membrane and sus-
pension were placed in the centrifuge and rotated at
500g for 10 minutes. This figure was chosen based
upon published studies ir,dicating that cell populations
in te~t tubes were not adversely affected by forces
of such magnitude and duration. ~owever, wound closure
by membranes seeded by centrifugation at 500g for 10
minutes was substantially inferior to wound closure by
membranes seeded by centrifugation at 50g for 15 minutes.
In order to firmly establish the fact that the cell
colonies within the seeded membranes were generated
by seeded cells, rather than by migration or pro-
liferation of cells from the periphery of the wound,
several guinea pigs were fitted with "island grafts"
of 1 x 2 cm seeded membranes centered in 5 x 6 cm wounds.
The island grafts were 2 cm from the wound periphery.
Epidermal cell colonies grew in the is~and grafts,
generating neoepidermal tissue that was isolàted
from any other source of lepidermal cells.
~L21)2~39~
-36-
Industrial Applicability
This invention has industrial applicability in
the use of fibrous proteinous lattices to promote the
growth of cells and tissue.
Equivalents
Those skilled in the art will recognize, or be
able to ascertain using no more than routine experimenta-
tion, numerous equivalents to the specific procedures
and seeded lattices described herein. Such equivalents
are considered to be within the scope of this invention,
and are covered by the following claims.
lZ02~9~
-37-
REFERENCES
1. See~ e.g., R. H. Sims et al, An Introduction
to the Biology of thl_ Skin (F. A. Davis Co.,
Phila., 1970); W. Montagna et al, The Structure
and Function of Skin, 3rd edition (Academic
Press, New York,1974); H~ Gray, Anatomy,
Descriptive and Surgical, 15th edition, p.
1135 et seq. (Bounty Books, New York, 1977).
2. See, e.g., H. C. Polk Jr. et al, editors,
Contemporary Burn Management, p. 345 et seq.
(Little, Brown & Co., Boston MA, 1971).
3. See, e.g., Polk et al, supra note 2, p. 412 et
seq
4. See, e.g, J. F. Burke et al, Ann. Surg. 182(3):
p. 183-195 (1975~.
5. See, e.g., Polk et al, supra note 2, p. 362 et
seq.
6. See, e.g., Polk et al, supra note 2, p~ 385 et
seq.
7. See, e.g, Polk et al, supra note 2, p. 383 et
seq.
8. See, e.g., M. Prunieras, J. Investigative
Dermatology 67: p. 58 et seq. (Williams &
Wilkins, Baltimore, 1976).
12028~8
.,
-38-
9. See, e.g., R. Fleischmajer et al, Epithelial-
Mesenchymal Interaction (Williams and Wilkins,
Baltimore, 1968); R. H. Kahn et al, In Vitro 8:
451 (1973); R. H. Xahn et al, J. Nat'l Cancer
Inst. 53: 1471 (1974); M. Regnier, Acta Derma-
tovener (Stockhol~) 53:241 et seq. (1973r;
Rheinwald et al, CelL 6:317 tl975).
10. See, e.g., R. O. Grepp, Recent Progress in
~ormone Research 30:533 et seq. (Academic
Press, New York, 1974); R. H. Starkey et al,
Science 189:800 (1975).
11. See, e.g., L. B. Chen et ai, Science 197:776
(1977).
12. See, e.g, D. M. Prescott, editor, Reproduction
of Eukaryotic Cells, p. 107 et seq. (Academic
Press, New York, 1976).
13. See, e.g., A. W. Bernheimer, editor, Mechanisms in
Bacterial Toxicology p. 53-84 ~Wiley, New York,
1976); D. M. Gill, Adv. Cyclic Nucl. Res. 8:
85 et seq. (1977).
14. See H. N. Antoniades et al, Proc. Natl. Acad.
Sci. 76: 1809-1813 (1979).
15. See J. Folkman et al, J. Exp. Med. 133:275(1971~.
16. See,- e.g., H. Green, Cell 15: 801,805(1978).
