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FIELD OF THE INVENTION
This invention relates to artificial skin, its preparation, and its use.
BACKGROUND OF THE INVENTION
For some time, there has been a move to develop an artificial skin that can
be used (1) for wound healing and the repair of ulcerated, burned or
lacerated skin, and (2) as a model to test substances for irritation,
toxicity, inflammation and pharmacology so as to reduce the number of
tests using live animals. This latter application for artificial skin is
commonly referred to as an in vitro alternative to animal testing.
Berg and colleagues (U.S. Pat. Nos. 4,703,108 and 4,970,298, the
disclosures of which are herein incorporated by reference) have developed
a biocompatible, chemically crosslinked, three-dimensional collagen matrix
as a substitute for the dermal layer of the skin where it promotes
fibroblast ingrowth and proliferation when implanted into animals
(Doillon, et al., "Fibroblast-collagen sponge interactions and the spatial
deposition of newly synthesized collagen fibers in vitro and in vivo," J.
Scanning Electron Microscopy, III:1313-1320 (1984). It has also been used
to treat human Decubitus ulcers where it promotes healing (Silver, F. H.,
et al., "Collagenous materials enhance healing of chronic skin ulcers,"
Biomedical Materials and Devices Research Society, 110:371-376 (1989)). In
vitro studies, this matrix has been used as a model for examining the role
of various matrix components on fibroblast ingrowth (Doillion, et al.,
"Fibroblast growth on a porous collagen sponge containing hyaluronic acid
and fibronectin," Biomaterials 8:195-200 (1987).
In this invention, this matrix is used as a support for human keratinocyte
growth and differentiation. The matrix described herein, containing
keratinocytes and fibroblasts, is referred to herein as a "skin model
system" or "SMS." Dollion, et al., in "Behavior of fibroblasts and
epidermal cells cultivated on analogues of extracellular matrix,"
Biomaterials, 9:91-96 (1988), report on efforts to use the Berg collagen
matrix in attempts to manufacture artificial skin, but such attempts were
not successful. Epidermal cells on the surface of the matrix were neither
differentiated nor in stratified layers.
An alternative collagen-based system has been developed by Bell et al.
(Bell, E., et al., "The reconstitution of living skin," Journal of
Investigative Dermatology, 81:2s-10s (1983); Bell, E., et al., "Recipes
for reconstituting skin," J. Biomechan. Eng., 113:113-119 (1991)) as a
dermal replacement called "living skin equivalent" or "LSE". The LSE is
manufactured by mixing living human fibroblasts with soluble rat tail
collagen under conditions where the collagen forms a gel (See, U.S. Pat.
Nos. 4,485,096, 4,604,346, 4,8356,102, and Bell, E., et al., "Production
of a tissue-like structure by contraction of collagen lattices by human
fibroblasts of different proliferative potential in vitro," Proc. Natl.
Acad. Sci. USA, 76:1274-1278 (1979)). During the five days of culture, the
gel containing fibroblasts undergoes a contraction process where the
collagen volume is reduced to a small disc approximately 10% to 20% of the
original volume depending on the concentration of collagen, the cell
number and the composition of the growth medium. This contracted collagen
matrix is then used to support human keratinocyte growth. Although the
Bell LSE is an advance over other previously known artificial skin
systems, it does suffer from disadvantages. Since the manufacture or the
collagen matrix requires living fibroblasts, it is expensive to
manufacture and the matrix is not easily stored. The Bell collagen matrix
is not cross-linked and contracts with the addition of the fibroblasts, so
it is difficult to manufacture the matrix in a desired shape and size. It
is difficult to make large sizes of LSE; the matrix contracts
substantially and large numbers of living cells are required. The Bell
collagen matrix utilizes soluble collagen, which is more difficult and
expensive to extract than insoluble collagen. Still further, since the
Bell collagen matrix requires living cells, the manipulations to which it
can be exposed are limited, e.g., it cannot be exposed to toxic conditions
which might manipulate or favorably alter the matrix structure but which
would kill the cells. In view of cheese limitations, there remains a need
for improved artificial skin systems.
SUMMARY OF THE INVENTION
It has now been found that a cross-linked matrix of insoluble collagen can
be used as the matrix for preparing a skin model system mimicking human
skin. This invention relates to such a skin model system, comprising a
three-dimensional, cross-linked matrix of insoluble collagen containing
fibroblast cells therein, and stratified layers of differentiated
epidermal cells supported thereon.
This invention further relates to a method for preparing such a skin model
system comprising (a) providing a three-dimensional, cross-linked collagen
matrix, (b) seeding said matrix with fibroblasts and culturing the seeded
matrix under conditions to allow ingrowth and proliferation of said
fibroblasts, (c) seeding the surface of said matrix with epidermal cells
in a manner to deter ingrowth of said epidermal cells, (d) culturing the
seeded matrix for a first period of time under conditions to allow said
epidermal cells to attach to said matrix and proliferate to form a
monolayer and (e) culturing the seeded matrix for a second period of time
under conditions to allow said epidermal cells to differentiate.
Still further, this invention relates to methods for using the skin model
system of this invention to determine the effect of an agent on human skin
comprising contacting the skin model system with said agent and measuring
the interaction of the skin model system and said agent.
Still further, this invention relates to methods for using the skin model
system of this invention to treat wounds comprising transplanting said
skin model system as a graft at the wound.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a scanning electron micrograph (SEM, of a cross-section of
unmodified, cross-linked collagen matrix of insoluble collagen. A 1000.mu.
bar is shown.
FIG. 2 is a graph of the streaming potential versus flow rate for matrices
of this invention not containing polylysine (UMDNJ-DHT), matrices of this
invention containing polylysine (UMDNJ-DHT-PL), and a commercially
available collagen hemostatic sponge (INSTAT).
FIG. 3 is a graph of optical density of cell cultures on several different
matrices, evidencing the ability of dermal fibroblasts to attach to these
matrices. PLASTIC is polylysine-coated plastic tissue culture dish; INSTAT
is a commercially available collagen sponge; INSTAT-C is a commercially
available collagen sponge crosslinked with cyanamide; UMDNJ-C is a
collagen matrix of this invention crosslinked with cyanamide; UMDNJ-DHT is
a collagen matrix of this invention crosslinked by dehydrothermal
treatment; UMDNJ-DHT-PL is a collagen matrix of this invention crosslinked
by dehydrothermal treatment and incorporating polylysine; UMDNJ-DHT-PA is
a collagen matrix of this invention crosslinked by dehydrothermal
treatment and incorporating polyaspartic acid. Cell density was determined
by the MTT assay as described in the examples.
FIG. 4 is a graph illustrating the ability of cells to grow on plastic
tissue culture dishes (PLASTIC) compared to their ability to grow on a
commercially available collagen sponge (INSTAT), a cyanamide crosslinked
version of the same matrix (INSTAT-CYANAMIDE), and a cyanamide crosslinked
version of the matrix described here (UMDNJ-CYANAMIDE).
FIG. 5 is a graph showing the effect of added polyamino acids on cell
growth in collagen matrices. 3.times. cells were incubated either on
tissue culture dishes (PLASTIC) or with one of several collagen matrices:
the matrix described here crosslinked by a dethydrothermal technique
(UMDNJ-DHT), the same matrix with polylysine (UMDNJ-DHT-PL), or
polyaspartic acid (UMDNJ-DHT-PA) incorporated during its manufacture. Cell
density was determined by the MTT assay described in the examples.
FIGS. 6 are photomicrographs of cross-sections of human foreskin or of
epidermal cell cultures grown on a collagen matrix. Magnification is
400.times., except were noted. FIG. 6A shows human foreskin. FIG. 6B shows
the Bell skin equivalent after ten days of culturing; FIG. 6C shows the
same after 21 days of culture. FIG. 6D shows keratinocytes seeded on the
"air side" of a cross-linked matrix of insoluble collagen (no polylysine
or polyaspartic acid). FIGS. 6E and 6F show the skin model system of this
invention in which the matrix has been modified with polylysine.
DETAILED DESCRIPTION OF THE INVENTION
Skin is composed of both a dermal layer, consisting primarily of types I
and III collagen, proteoglycans, elastin and other matrix macromolecules,
and epidermal layers, consisting of epidermal cells containing keratin
filaments undergoing progressive differentiation from a basal
proliferating layer to a surface consisting of terminally differentiated,
epidermal cells that protect the skin from the environment. The skin model
system of this invention mimics the composition of normal skin. Fibroblast
cells are grown within a three-dimensional matrix formed of cross-linked,
insoluble collagen to form a dermal-type layer which supports stratified
layers of differentiated epidermal cells.
The collagen matrix utilized in this invention is based on insoluble
collagen. "Insoluble collagen" refers to collagen which cannot be
dissolved in aqueous acidic or alkaline or in any inorganic salt solution
without chemical modification. Preferred sources of the insoluble collagen
include hides, splits and other mammalian or reptilian coverings. More
preferably, the collagen is derived from the corium, the intermediate
layer of a bovine hide between the grain and flesh sides. More generally,
however, the collagen can be derived from the following typical sources:
type I collagen: bovine, chicken and fish skin, bovine and chicken tendons
and bovine and chicken bones including fetal tissues; type II collagens:
bovine articular cartilage, nasal septum, sternal cartilage; and type III
collagen, bovine and human aorta and skin.
The insoluble collagen is dispersed and swollen in a suitable liquid media,
such as dilute hydrochloric acid, acetic acid or the like, having a Ph
between about 3.0 and 4.0. Generally, a weight ratio of insoluble collagen
to dispersion agent of from about 1 to 15 is used to form the dispersion.
This dispersion is poured into molds (generally, plastic or metal trays)
of the desired shape and size. For skin model systems, it is generally
preferred that the thickness of the matrix be within about 1 to 5 mm,
preferably about 2-3 mm, so the size and shape of the mold can be
determined accordingly. The dispersion is solidified by freezing and is
then lyophilized to form a three-dimensional, porous matrix.
Prior work has demonstrated that the pore size of the matrix is important
to achieve optimal cell ingrowth. See U.S. Pat. No. 4,970,298, the
disclosure of which is herein incorporated by reference. Generally, a
matrix having a pore size of 50 to 250 microns, preferably in the range of
100.+-.50 microns, containing channels is an ideal structure for a
collagen-based material for cell ingrowth. (Pore size may be measured from
a photomicrograph using a ruler, averaging two measurements of a pore
taken at 90.degree. angles from one another, and averaging such
measurements over a representative number of pores.) The optimum
conditions for forming a matrix having these characteristics are: (1) to
avoid excessive blending and obtain a well dispersed mixture of large
collagen fibers, (2) to disperse the collagen in a liquid media having a
Ph of about 3.0 to about 4.0, (3) to freeze the collagen dispersion to
from about -30.degree. C. to about -50.degree. C. in an ethanol bath, and
(4) to keep the ethanol bath in direct contact with the plastic or metal
tray to avoid any air gap. After freezing, lyophilization is generally
carried out under conditions of a sample temperature of about 0.degree. to
20.degree. C. and a vacuum below about 200 mTorr Hg.
As noted in U.S. Pat. No. 4,970,298, the upper surface of the sponge in
direct contact with the atmosphere during the freeze-drying process,
called the "air side", is found to have a collapsed form or a sheet-like
structure in almost all cases. The other side of the sponge, in direct
contact with the tray, called the "pan side", is found to have a more
open, delicate structure. The average pore size on the pan side tends to
be significantly smaller (e.g., generally at least about 100.mu. smaller)
than that of the air side. Not reported in U.S. Pat. No. 4,970,298, but
described hereinbelow, is the importance of seeding the epidermal cells on
the "pan side" of the sponge rather than the "air side" to achieve the
optimal skin model system.
Prior to solidifying the collagen dispersion other agents may be
incorporated into the dispersion. For example, polycationic polymers such
as polylysine or polyaspartic acid may be incorporated to improve cell
growth. Carrier compounds such as collagen types IV and V, fibronectin,
laminin, hyaluronic acid, proteoglycans, epidermal growth factor, platelet
derived growth factor, angiogenesis factor, antibiotic, antifungal agent,
spermicidal agent, hormone, enzyme and enzyme inhibitor.
Following lyophilization, the collagen matrix is cross-linked. Preferably,
cross-linking is carried out by a dehydrothermal treatment. Suitable
conditions include subjecting the matrix to temperatures of from about
50.degree. C. to about 200.degree. C. at a vacuum of 50 millitorr or less
for a period of time from about 2 to 96 hours. However, the collagen may
also be cross-linked by chemical agents (or a combination of
dehydrothermal treatment and chemical agents) such as carbodiimide or
succinimydyl ester/carbodiimide.
Examples of the carbodiimides include cyanamid and
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. Examples of
bifunctional succinimidyl active esters include bifunctional
N-hydroxysuccinimide, 3,3'-dithio(sulfosuccin-imidyl)proprionate and
bis(sulfosuccinimidyl)suberate. When using a carbodiimide cross-linking
agent, the collagen-based matrix is immersed in a carbodiimide solution at
a concentration of from about 0.1 to 10% (W.V) maintained at a temperature
of from about 2.degree. C. to 40.degree. C. and at a Ph of between 2 to 11
for a period of time of from about 2 to 96 hours. When using a
succinimidyl active ester crosslinking agent, the collagen-based sponge or
sheet is immersed in a solution thereof at a concentration of from about
0.1 to about 15.0% (W/V) maintained at a temperature of from about
2.degree. C. to 40.degree. C. for a period of time of from about 2 to 96
hours. The collagen-based sheet is placed in a solution containing 0.1 to
about 15% (W/V) of N-hydroxysuccinimide and carbodiimide at a Ph between 2
to 11 for a period of time between 2 to 96 hours at a temperature of from
about 2.degree. C. to 40.degree. C. The thus-treated intermediate
collagen-based matrix is exhaustively washed to remove excess
cross-linking agent.
Unlike prior art collagen matrices prepared from collagen and living cells,
the collagen matrices of this invention can be stored for periods of time
in a dry state. For example, experience has suggested that sterilized
matrices can be stored under dry and sealed conditions in excess of two
years. Prior to storage or use, it is generally preferred to sterilize the
matrices, e.g., with gamma irradiation. Prior to seeding the matrices with
living cells, the matrices are preferably treated (e.g., soaked overnight
in DMEM supplemented with 10% calf serum to remove any residual acid.
The next step in preparing the skin model system described herein is
seeding the collagen matrix with fibroblasts. Fibroblasts will be
dispersed throughout the collagen matrix to simulate the dermal layer of
humans and other mammals. Although the type of fibroblast utilized is not
critical, it is preferred to use dermal fibroblasts as they will deposit
the appropriate types of collagen and other dermal components. The
fibroblasts may be of human or animal origin. They may be commercially
obtained or may be cultured from a patient biopsy. Fibroblast-like cells
may also be used. Other cells may also be cultured in the collagen matrix,
including but not limited to endothelial cells, pericytes, macrophages,
monocytes, lymphocytes, plasma cells and adipocytes.
The fibroblasts are generally inoculated onto the matrix at a density of
about 0.2 to 1.times.10.sup.6 cells per 2.times.2 centimeter of matrix.
Preferably, the fibroblasts are inoculated onto the "air side" of the
matrix as the greater pore size on this side encourage ingrowth of the
fibroblasts into the matrix. However, it should also be possible to seed
the "pan side" of the matrix. As mentioned above, pore sizes in the range
of about 100.+-.50.mu. have been found to be ideal for cell ingrowth. The
seeded matrix is cultured for a period of time and under conditions to
allow the cells to grow and modify the collagen matrix so that it will
support the growth of epidermal cells. During this period, the fibroblasts
substantially fill the spaces in the collagen matrix, generally, a period
of from about one to fifteen days, preferably about five days. Suitable
culture conditions would be known to those skilled in the art and would be
conditions conducive to the proliferation of the fibroblasts. Such
conditions include immersing the seeded matrix in DMEM (Dulbeco's Modified
Eagle's Medium) supplemented with 5% FBS (fetal bovine serum) or 10%
bovine serum for five to seven days.
The next step involves seeding the matrix with epidermal cells. These
epidermal cells must be keratinocytes, although other cells may be used in
conjunction with the keratinocytes (e.g., melanocytes). Epidermal cells
are generally inoculated onto the matrix at a density of about 100,000 to
about 500,000 cells per cm.sup.2, although lesser or greater amounts could
be used. The epidermal cells must be seeded onto the "pan side" of the
matrix. It has been found that if the cells are seeded onto the "air
side", with its open, large pore structure, the cells migrate into the
matrix to produce swirls and pockets of epidermal cells. Applying
epidermal cells to the pan side, that side with the smallest pore
structure, improved epidermal cell growth and proliferation by minimizing
ingrowth of the epidermal cells into the matrix.
The matrix seeded with epidermal cells is cultured for a first period of
time and under conditions suitable to allow the epidermal cells to attach
and form a monolayer. Generally, this may be accomplished by submerging
the seeded matrix in an epidermal growth media for approximately one week
at 37.degree. C. and 7% CO.sub.2. A suitable epidermal growth media
("E-media") contains: 3:1 high glucose DMEM supplemented with Ham's F-12
Nutrient Mixture (Gibco), 5.times.10.sup.-10 M cholera toxin (Schwarz-Mann
Biotech), 2 .mu.g/ml hydrocortisone, 25 .mu.g/ml insulin, 25 .mu.g/ml
transferrin, and 1.times.10.sup.-10 M triiodothyronine (all from Sigma
Chemical Company).
Once the epidermal cells have proliferated to form a monolayer, the matrix
is cultured for a second period of time under conditions to allow the
epidermal cells to differentiate. The culture or matrix is raised so that
the surface seeded with epidermal cells is exposed to the atmosphere,
e.g., by raising the matrix to the liquid/air interface in the culture.
The matrix continues to be fed with the epidermal growth media and held
until the epidermal cells have differentiated as desired. A period of
twenty-one days has been found to be sufficient, although shorter or
longer periods may be utilized.
Tests indicate that the epidermal cells grown on the collagen matrix as
described above form stratified layers similar to skin with the epidermal
cells differentiating as they approach the surface. Those skilled in the
art would know how to determine whether a stratified layer of cells is
present. Stratified layers of epidermal cells are found where several
layers of cells are piled on top of one another as opposed to being in a
single monolayer (or to simply filling in holes in the collagen matrix).
Generally, the stratified layers formed in this invention will comprise a
layer of three or more, preferably five or more, cell thicknesses.
Those skilled in the art would also know how to determine whether epidermal
cells have differentiated. Cells that have differentiated show a change in
morphology (shape and appearance) and often secrete certain proteins.
Changes in morphology include (1) the presence of granules in the granular
layer (or a layer in the SMS approximating the granular layer), (2) the
flattening of the cells and (3) the loss of cell nuclei. The presence of
any one or more of these changes in morphology indicates that the cells
have differentiated or are differentiating.
When epidermal cells differentiate, they also start to make certain
proteins. For the purpose of this invention, the presence of any one or
more of those proteins in the epidermal cells is evidence that the cells
have differentiated or are differentiating. Those proteins include
involucrin, filaggrin, keratin K1 and loricrin.
Involucrin is a cell envelope protein present in all but the basal layers
of normal skin and is one of the earlier proteins produced for the
formation of the corneocyte envelope in human skin. The corneocyte
envelope is a rigid alkaline-resistant structure produced only by
differentiating keratinocytes and not by basal keratinocytes.
Filaggrin is a protein produced in the suprabasal layer as profilaggrin
which is processed to filaggrin. In human skin, it is found in the upper
stratum spinosum and granulosum. Filaggrin is generally not found in the
stratum corneum, possibly because it is degraded to amino acids in that
layer. Filaggrin is involved in the preparation of keratohyalin granules
formed in differentiating cells of the epidermis. Only differentiating
cells would produce filaggrin.
Keratins are a family of proteins whose members are expressed as a function
of the differentiation state of the epidermal cells. The keratin K1
(having a molecular weight of 67 kDa), is the keratin present in the most
highly differentiated keratinocytes. In normal skin, K1 is localized to
the upper stratum spinosum and granular layers.
Loricrin is a major cornified envelope precursor, which is one of the
latest envelope proteins to be expressed in normal skin. When using an
antibody specific to the epitopes on the N-terminal portion of loricrin,
normal foreskin has been found to stain specifically from the upper
stratum spinosum to the stratum corneum.
The presence of these proteins in the skin model system can be determined
by standard immunohistochemical techniques described in more detail in the
Examples. Tests show that the amounts and distribution of each of these
proteins in the skin model system of this invention mimic their
distribution in normal neonatal skin. The only exception to this pattern
was that reduced amounts of loricrin were determined in the skin model
system matrix described herein compared to normal skin.
The skin model system described herein should have a variety of uses.
Chemicals such as drugs, cosmetics, pesticides and food additives must
often be tested for skin irritation, for toxicity and/or for efficacy.
Currently, much of this testing is carried out on laboratory animals. The
skin model system of this invention, however, closely resembles human skin
and can be used not only to spare laboratory animals but also to more
accurately gauge the effect of a chemical on human skin. One aspect of
this invention, therefore, relates to a method of determining the effect
of a chemical or other agent on human skin comprising contacting a skin
model system of this invention with said chemical or agent and measuring
the interaction of the skin model system and said chemical or agent. The
term "agent" is intended to encompass not only substances but conditions
such as light, heat, etc. Such interactions could include, but are not
limited to, the release of one or more substances by the skin model
system, an effect on metabolism or cell proliferation or differentiation
of the cells, or the reorganization of the cells of the system. The extent
to which the chemical or agent is likely to affect human skin is
determined by the extent of any such interaction with the skin model
system. In this way, the potential toxicity or potential for irritation of
a chemical or other agent may be determined, as may the potential
pharmacological efficacy of a chemical or other agent be determined.
Other utilities for the skin model system of this invention include its use
as a model for studying skin diseases and developing new treatments for
skin ailments. For example, one could form the skin model system of this
invention using cell lines from patients with a certain disease to learn
more about that disease and to study and evaluate the efficacy of
treatments for it. More specifically, one aspect of this invention relates
to a method for determining the efficacy of a treatment for a skin
condition comprising (a) providing the skin model system of this invention
wherein diseased cells typical of said condition have been used to produce
said system, (b) exposing said skin model system to said treatment, and
(c) monitoring any change in said skin model system.
The skin model system of this invention may also be used for treating
patients suffering from a wound to the skin, for example, burn patients.
The skin model system may be applied to the wound, for example, by
transplantation or grafting.
This invention is further illustrated by the following examples, which are
provided for purposes of illustration only. These examples are not
intended to limit the scope of this invention.
EXAMPLE 1
Collagen Matrix Formation
Three-dimensional collagenous matrices were prepared from collagen (Devro,
Inc.; microcut) which was isolated from the corium layer of the bovine
hide by shredding and washing in various solvents in order to obtain a
slurry of insoluble collagen particles in water.
See, for example, U.S. Pat. No. 4,703,108, U.S. Pat. No. 4,970,298 and
Jain, M. K., et al., "Material Properties of Living Soft Tissue
Composites," J. Biomed. Mat. Res., 22:311-326 (1988), the disclosures of
which are herein incorporated by reference. Since collagen swells when
exposed to acid, the slurry was prepared by adding 6 gm collagen to 100 ml
0.001N Hcl, Ph 3, and allowed to swell. The slurry was then blended to
produce a smooth dispersion. (In some preparations, 30 mg polylysine were
added per 100 ml of dispersion corresponding to 0.5% polylysine per
collagen wt/wt, as described below in Example 2.) The dispersion of
collagen in acid (250 ml) was then poured into molds of teflon lined pans
8" by 8" to a depth of 3-4 millimeters. The molds had flat bottoms and
were open to the air on top. The dispersions were frozen at -20.degree. C.
and lyophilized to form a matrix. The matrix was then crosslinked using a
dehydrothermal treatment (110.degree. C. in a vacuum of less than 10 m
torr). The collagen matrices were cut with a cork borer to a diameter of
1.5 cm or with a razor blade to squares of approximately 2 cm by 2 cm. The
matrices were sealed in plastic and sterilized with gamma irradiation
using 2.5 m rad of .sup.60 Co (Isomedix, Inc.) and stored in a dry state
(FIG. 1).
The pore structure of the matrices produced by freezing and lyophilizing
varied from approximately 50 to 200.mu.. The pore structure at the surface
of the matrix was more open for the air side of the matrix and more closed
for the side of the matrix in contact with the mold.
EXAMPLE 2
Preparation of Modified Collagen Matrices
In attempt to change the surface charge on the matrices to improve cell
attachment and growth, matrices were modified by the incorporation of 0.5%
wt per wt polylysine or polyaspartic acid per collagen. The polylysine or
polyaspartic acid was added to the collagen in the acid dispersion for
incorporation into the sponge prior to pouring into the mold and freeze
drying. In order to determine if the surface charge was changed, the
matrices were examined by measuring their streaming potential. Streaming
potentials of matrices were measured with a horizontal streaming potential
chamber. See Walsh, W. R., et al., "Electrokinetic Effects on Matrix
Fibroblast Interactions," BRAGS (1992). The electrical potentials in
0.145M, pH 7.3, sodium veronal buffer were measured using silver/silver
chloride electrodes and a Keithley 642 Electrometer and x-t recorder.
Collagen sponges were equilibrated in the streaming potential chamber for
30 minutes followed by 5 minutes at a flow rate of 1 ml/min. Streaming
potentials at several flow rates, corresponding to different pressures,
were determined after 30 seconds of flow at a given pressure when the
signal was stable as indicated on x-t recorder. The slope of the flow rate
versus streaming potential is inversely related to the zeta potential. The
zeta potential is defined as the average potential difference between the
bulk solution, considered to be zero potential, and the surface of shear
(near the matrix).
Results are presented in FIG. 2 as a graph of the screaming potential
versus flow rate for the matrix of this invention not containing
polylysine (UMDNJ-DHT), matrices of this invention containing polylysine
(UMDNJ-DHT-PL), and a commercially available collagen hemostatic sponge
(INSTAT) (INSTAT.TM. sponges are a product of Johnson & Johnson Co.) The
streaming potentials for all collagen matrices tested were negative at pH
7.3 in 0.145M buffer. Since the slope of the flow rate versus streaming
potential is inversely related to the zeta potential with the electrode
polarity used in the streaming potential chamber, the matrices all had
positive zeta potentials. The zeta potential was positive for the matrices
without polylysine and more positive for the polylysine matrices (FIG. 2).
The streaming potential increased as the flow rate increased (applied
pressure increased), as predicted by electrokinetic theory. (Lyklema, J.,
et al., J. Colloid Sci., 16:501-512 (1961).
EXAMPLE 3
Cell Attachment and Growth Curve Assays in Collagen Matrice | | |
