Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02286548 1999-10-14
University Hospital der Albert-Ludwigs-Universitat Freiburg
Hugstetter Strafe
79106 Freiburg
Composition for treating wounds which comprises
fibroblasts harboring a foreign gene
The present invention relates to a composition
for treating skin damage which can only be treated with
difficulty. In the case of burns covering a large area,
it can represent a substantial problem to restore the
regions of the skin which have been destroyed by the
1.0 damage,~in particular when major areas of the skin have
been severely damaged. It is frequently also quite a
substantial problem to restore the damaged skin regions
in the case of other diseases such as neoplastic
diseases or chronic skin lesions.
Gene-transfected fibroblasts per se are already
known from the state of the art. International Patent
Application WO 95/07105 describes a method for inhibit-
ing or preventing the growth of tumor cells in the
central nervous system of a patient in which the immune
response of the patient is stimulated by immunization
with either gene-transfected tumor cells or unmodified
tumor cells and gene-transfected, autologous fibro-
blasts. The cytokines which are used are first and
foremost those which stimulate the immune system.
2.5 DE-OS 44 06 073, which was also filed in the
name of the Albert Ludwigs University Hospital complex,
Freiburg, relates to a general method for preparing
human, clonogenic fibroblasts and to a method for gene-
transfecting fibroblasts. This application does not
disclose topical application or gene transfection
specifically with genes which promote wound healing.
WO 92/15676 describes the use of gene-
transfected fibroblasts for somatic gene therapy. In
this case, the transfected fibroblasts are fixed in an
extracellular collagen matrix and implanted under the
skin. The object of this application is aimed at
CA 02286548 1999-10-14
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remedying genetic defects in the human genome. In this
context, a functionally active "replacement" gene is
introduced into a somatic cell and used to compensate
for the damage arising as a result of the defective
gene. This literature reference does not address the
treatment of wounds or cytokines which promote wound
healing, such as TGF-a, EGF or b-FGF. This citation
mentions, in particular, cytokines which possess other
biological properties, such as GM-CSF, TNF or EPO. The
fibroblast growth factor which is mentioned in claim 5
is used as a blood vessel-forming substance.
Various growth factors which have an effect on
keratinocytes are also known. WO 90/08771 relates to
the recombinant preparation of growth factor proteins
which affect epithelial cells. However, the human
keratinocyte growth factor is used as an essentially
pure protein in this application.
US Patent Specification 5,302,701 describes the
development of an artificial polypeptide which is cell
adhesive and also possesses cell growth-promoting
activity. The polypeptide in this case is a combination
of fibronectin and fibroblast growth factor (FGF).
While US Patent 5,196,196 relates to a wound
dressing which also comprises a support matrix, there
is an important difference from the present invention
with regard to the principle by which the method works
and also the active agent. The US patent uses purified
protease nexin I (PN-I) in the wound dressing. However,
this protease is not a growth factor but an enzyme
having defined properties. Protease nexin I is a serine
protease inhibitor; i.e. a member of the serine
protease superfamily which is synthesized and secreted
by human fibroblasts in culture. The US patent uses the
protein in purified form and not in the form of a gene
which is expressed by fibroblasts into which this gene
has been incorporated.
Furthermore, the use of cultured, autologous
keratinocytes, which are suspended in fibrin adhesive,
for treating extensive burn wounds is known from the
_ _ __ ___
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state of the art [Stark et al., Eur. J. Plast. Surg.
(1995) 18, pp. 267-271]. An attempt has also already
been made to use keratinocytes which have been trans-
fected in situ by means of particle DNA transfer in
wound healing in a pig model [Andree et al., Proc.
Natl. Acad. Sci. USA, (1994) 91, pp. 12188-12192].
Andreatta-van Leyen et al. (J. Biomedical
Materials Res., Vol. 27 (1993), pp. 1201-1208] describe
a wound bandage which comprises gene-transfected
keratinocytes which produce bovine growth hormone
(bGH) .
Some of the proposed solutions known from the
state of the art rely partly upon keratinocyte
preparations (e. g. so-called keratinocyte sheets) which
are very difficult to handle and can often only be used
when three-dimensional structures have formed during
the cell culture. There is the risk that allogenic
preparations, in particular those which come from
different donors and have not been characterized, are
contaminated with viruses (HIV, HCV or viruses which
have not yet been characterized) and that the patient
who is to be treated can be infected by these viruses.
The present invention therefore relates to
compositions for treating wounds, which compositions
comprise fibroblasts, which harbor at least one foreign
gene encoding a wound healing-promoting cytokine, and
at least one additional wound healing-promoting com-
ponent. In addition to this, the composition according
to the invention can comprise further components which
are customarily used in such compositions.
The additional wound healing-promoting com-
ponent can be a so-called fibrin adhesive. Such fibrin
adhesives are commercially available and are used in
various areas of medicine, in particular in surgery.
In principle, fibrin adhesion makes use of the
last phase of blood coagulation. The fibrin adhesive
contains fibrinogen, which is converted by thrombin
into monomeric fibrin. This in turn forms aggregated
fibrin by means of end-to-end or side-to-side addition.
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Furthermore, in a preferred embodiment, the fibrin
adhesive contains fibronectin. At the same time,
thrombin activates factor XIII, which is normally
present in adequate quantity in the fibrin adhesive. As
a further component, the fibrin adhesive contains a
thrombin solution which is frequently added, together
with calcium chloride, as a separate component.
In addition to this, the fibrin adhesive can
also contain sufficient quantities of albumin or
plasminogen.
In a preferred embodiment, the additional wound
healing-promoting component can also be a solid con-
stituent which serves as the support matrix. Supports
of this nature for "artificial skin" are commercially
available (for example Laserskin° or Biobrane~). The
solid components can preferably be a matrix composed of
a derivative of hyaluronic acid, preferably a
hyaluronic acid ester. Hyaluronic acid is an endogenous
constituent of the connective tissue which is subject
to an extraordinary high turnover rate. Therefore, when
the matrix is composed of hyaluronic acid, it is very
rapidly degraded in the wound by the endogenous
hyaluronidase. For this reason, preference is given,
according to the invention, to using those derivatives
of hyaluronic acid which are degraded somewhat more
slowly in the body.
A particular advantage which can be achieved by
using a support matrix is that cells which have still
not formed a confluent association of cells can be
applied to the wound. When keratinocytes are also
employed in the composition according to the invention,
these cells can, with the help of the support matrix,
be used in a subconfluent state. At this time, they are
in their phase of optimal growth and are still dividing
very frequently and react particularly well to the
cytokines which are produced by the gene-transfected
fibroblasts.
In a preferred embodiment, the composition
according to the invention can also comprise
CA 02286548 1999-10-14
keratinocytes, preferably autologous keratinocytes. The
keratinocytes are particularly involved in forming the
outer layer of the skin; the so-called epidermis.
Keratinocyte culturf~s can be cultured using a routine
technique which is known per se [e . g. Rheinwald et al .
Nature 265 (1977) pp. 421-424]. In this technique, a
small piece of skin is normally removed by biopsy and
the epidermis and dermis are then separated from each
other. A cell suspension is prepared from the
epidermis, preferably by treating the latter with a
proteolytic enzyme. The individual cells which result
are then propagated under sterile conditions in culture
vessels (flasks or dishes) and detached from the
culture vessels at a suitable point in time.
The composition according to the invention
comprises genetically altered fibroblasts. Fibroblasts
are mesenchyme-derived cells which have a large cell
body and a somewhat flattened nucleus. The fibroblasts
are particularly involved in forming the intercellular
substance of the connective tissue. According to the
invention, either autologous fibroblasts can be used or
else, in a preferred form, use is made of allogenic
fibroblasts which are obtained from another individual,
that is not from the patient to be treated. Very par-
ticularly preferably, use is made of fibroblasts which
have been clonally selected, i.e, which derive from one
clone.
In a particularly preferred embodiment, the
gene-transfected fibroblasts have been treated with a
dose of ionizing radiation such that they are no longer
able to replicate and die after a given period of time.
An advantage of this irradiation is that the gene-
transfected cells die in the body after a relatively
short and clear-cut period of time (< 3 weeks). An
additional advantage of using irradiated fibroblasts is
that the cells which have been treated in this way
sti_11 continue to express the cytokine satisfactorily.
In a particularly preferred embodiment, use is
made, according to the invention, of cells from
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immortalized fibroblast cell lines, in association with
which a cell line which has been given the designation
KMST-6 has proved to be particularly satisfactory.
Immortalized fibroblast cell lines are advantageous
since they can be cultured continuously and can be used
in a precisely characterized manner from the biological
point of view. However, it is also possible to use
other suitable immortalized fibroblast cell lines
without any difficulty.
A foreign gene which encodes a suitable cyto-
kine is introduced into the fibroblasts which are used
in accordance with the invention. This foreign gene is
preferably a gene which encodes a cytokine such as EGF,
TGF-a or KGF.
The epidermal growth factor is termed EGF for
short. This factor is a globular protein of about
6.2 kDa which possesses 53 amino acids. According to
the invention, it is not absolutely necessary for the
complete gene to be introduced into the transfected
fibroblasts; it is sufficient for that part which
possesses the biological activity to be inserted. In
order to enable the biologically active peptide to be
secreted, the appropriate cDNA moiety is preferably
fused in-frame to the secretory signal sequence of
human G-CSF. According to the invention, preference is
also given to using EGF-like proteins, such as
transforming growth factor a (TGF-a), which exhibits a
high degree of homology with EGF. The biological acti-
vities of EGF and TGF-a are comparable. Both cytokines
exert an effect on epidermal development and cell
differentiation.
NGF (nerve growth factor) may also be used in a
preferred manner. This cytokine is in particular
responsible for the survival, differentiation and
functional activity of sensory and sympathetic neurons
in the peripheral nervous system. The accelerating
effect on wound healing is probably due to the ability
of NGF to increase the survival and functional
activities of a, variety of immunocompetent cells such
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as granulocytes, mast cells, macrophages and
lymphocytes.
Another cytokine which is preferably employed
is keratinocyte growth factor (KGF), which in particu
lar stimulates the division of keratinocytes which are
able to divide. According to the invention, variants of
the genes can also be employed. Such variants can
exhibit deletions or additions and the sequence can be
altered in a controlled manner. It is also perfectly
possible to employ cytokine variants which have a
higher biological activity than do the naturally
occurring cytokines, such as, in particular, constructs
obtained by fusing two or more cytokines.
The fibroblasts which are employed in accor
dance with the invention harbor a foreign gene which
encodes a cytokine which promotes wound healing. While
this foreign gene can preferably be derived from
humans, it can also be derived from an animal such as a
mouse or a cow. However, a prerequisite is that the
cytokine is not species-specific when the foreign gene
comes from another species.
The foreign gene has to be introduced into the
fibroblasts. This can be brought about by the desired
gene being incorporated into a suitable vector and then
being transfected into the fibroblasts. Suitable
vectors are viral vectors or plasmid vectors, with
particular preference being given to plasmid vectors
since, when viral vectors are used, additional investi-
gations have to be carried out in order to exclude the
possibility of contamination with replication-competent
viruses.
The compositions according to the invention
exhibit a variety of advantages as compared with the
solutions known from the state of the art.
The genetically altered fibroblasts which, in a
preferred embodiment, have been irradiated lethally,
can express the desired cytokine(s) far a predetermined
time and secrete it(them) into the tissue. Because the
cytokine is being produced continuously, it is possible
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to circumvent the problems which are due to the short
half-life of cytokines when they are applied externally
in a local manner. In the compositions according to the
invention, different cell types (keratinocytes/fibro-
blasts) can be combined with each other with it also
br::ing possible for the gene-transfected fibroblasts to
contain different cytokines. This makes it possible to
introduce different growth factors into the wound
region in a deliberate manner. If autologous keratino-
cytes are used, it is then possible to avoid the
histoincompatibility rejection reaction.
It is also conceivable to additionally intro-
duce genes for growth factors, such as G-CSF or GM-CSF,
which act on white blood corpuscles for the purpose of
stimulating the endogenous immune defense in the wound
regions. This can boost the body's own defense against
infections, something which is particularly relevant in
the case of burn wounds.
It is also perfectly possible to use the
composition according to the invention without any
keratinocytes. In this case, the genetically altered
fibroblasts release the cytokine into the environment
and the treated patient's keratinocytes which are
located, for example, in the marginal regions of the
wound are stimulated.
The compositions according to the invention are
preferably in the form of pharmaceutically acceptable
formulations. These formulations can either be suspen-
sa.ons of fibrin adhesive and gene-transfected fibro-
blasts which can be applied to the wound as solutions,
suspensions or ointments or in the form of gels. When
the composition comprises a solid component, that is a
support matrix, they are then preferably present in the
form of sterile units which can be preserved in a
suitable manner. Such preparations can, for example, be
in the form of deep-frozen, sterile dressings.
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Description of the figures
Figure 1 shows the structure of the vector for
expressing human EGF. Part (A) shows the plasmid
construct containing the chimeric EGF gene. Part (B)
shows the sequence of the in-frame fusion between the
human G-CSF signal sequence (underlined) and the
mature, human EGF-encoding region (Asn Ser Asp ...).
Figure 2 shows the secretion of EGF by fibro
blasts which have been transfected with the plasmid
pCMV-EGF-IRES-TKNeo. The fibroblasts are fibroblasts of
the KMST-6 cell line which have been irradiated
(100 Gy). In the experiment, 2 x 105 irradiated cells
were sown on six-well culture plates and the concentra-
tion of EGF was determined in the culture supernatants
after 24 hours. The values are mean values.
Figure 3 shows the bioactivity of chimeric EGF
polypeptides which were obtained from clones #3 and #6
of gene-transfected KMST-6 fibroblasts.
Part (A) shows the bioactivity on BALB/MK
strain mouse keratinocytes.
Part (B) shows the results which were obtained
using human primary keratinocytes. The values indicate
the average results from eight measurements, together
with the standard deviation. The dotted bars represent
the calibration curve which was obtained using recombi-
nant EGF. The empty bars represent the control values
which were obtained using fibroblasts of the KMST-6
cell line. The hatched and gray bars represent the
results which were obtained using culture supernatants
from the two clones 3 and 6, respectively.
Figure 4 shows the proliferation of primary
human keratinocytes in a culture test using irradiated
fibroblasts of the KMST-6 cell line which had been
gene-transfected.
Part (A) shows the results after four days of
coculture.
Part (B) shows the results after 10 days of
coculture.
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The present invention is clarified further by
the examples which are listed below:
Example 1
Constructing a plasmid which contains a gene for EGF,
and transfection
In most human cells, EGF is synthesized as a
130 kDa transmembrane glycoprotein precursor molecule
which is proteolytically cleaved into the biologically
active 6.2 kDa EGF peptide containing 53 amino acids.
In accordance with the invention, therefore, a chimeric
construct was prepared which encodes an in-frame fusion
of the mature EGF peptide and the human G-CSF secretory
signal sequence. The structure is depicted diagrammati-
cally in Figure 1 (A). The DNA fusion fragment which
resulted was placed under the transcriptional control
of the human CMV (cytomegalovirus) promoter and
incorporated into a dicistronic vector in order to bind
the expression of the transgene to that of the neomycin
phosphotransferase selection marker.
In the cloning, the sequence encoding the
mature human EGF peptide was amplified by PCR tech-
nology and the resulting product was subcloned into the
pBluescript (Stratagene) vector and subsequently
sequenced. The human G-CSF signal sequence was likewise
amplified from human G-CSF cDNA by means of PCR
technology, with an improved Kozak consensus sequence
at the 5' end and a single NheI restriction cleavage
site being created at the same time. The relevant
sequence is depicted in Figure 1 (B).
The plasmid which had been produced in this way
was transfected into human fibroblasts of the KMST-6
cell line, and all the neomycin-resistant clones
secreted human EGF, as was demonstrated by means of an
ELISA test. The control showed that untransfected human
fibroblasts of the KMST-6 cell line did not secrete any
detectable quantities of hEGF either before or after
irradiation.
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Clones #3 and #6, which secreted 37 and 8 ng of
EGF/106 cells and 24 hours, respectively, were used for
the subsequent investigations.
The secretion of human EGF into the supernatant
by transfected fibroblasts was determined using the
ELISA technique (Quantikine, R & D Systems). The super
natants from irradiated or non-irradiated starting
cells or EGF gene-transfected cells were removed after
24 hours and investigated for EGF production after the
number of viable cells had been determined.
An irradiation was carried out at room tempera-
ture using a 13'Cs radiation source at a dose rate of
3 Gy/minute.
Example 2
Effect of lethal irradiation on the expression of
chimeric EGF protein by gene-transfected fibroblasts
In order to achieve optimal wound-healing
results, it is necessary for EGF to be available during
the first days of the treatment. On the other hand, the
uncontrolled in vivo growth of genetically modified
cells can be prevented by lethal irradiation. The
effect of lethal irradiation on the expression of EGF
was therefore investigated using KMST-6 fibroblasts
(clone #3, which were transfected with the plasmid
pCMV-EGF-IRES-TKNeo). It was found that, although the
secretion of EGF slowly declined after irradiation with
100 Gy, EGF was detectable in the supernatant in vitro
over a period of at least seven days. The results are
summarized in Figure 2.
Example 3
Biological activity of the chimeric EGF polypeptide
In order to demonstrate that the EGF protein
which is secreted by the transfected fibroblasts also
actually does exhibit biological activity, the super
natants of the media from the EGF gene-transfected
clones were examined for their mitogenic activity on
permanent mouse keratinocyte cell lines and on primary
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human keratinocytes. Different concentrations of recom-
binant human EGF were used as controls. The two cell
types were stimulated in a dose-dependent manner both
by the recombinant protein and by the culture
supernatants from clones #3 and #6. Optimal stimulation
of the mouse keratinocytes was observed at a concentra-
tion of recombinant hEGF of between 2 and 20 ng/ml.
This corresponds to the stimulation which can be
achieved by a 1:5 dilution of the culture supernatant.
The results are depicted in Figure 3 (A).
When primary human keratinocytes were used, the
effective dose was marginally lower and optimum pro-
liferation was already observed at 0.2 ng of rEGF/ml
and at a 1:50 dilution of the culture supernatant from
the pCMV-EGF-IRES-TKNeo/KMST6#3 clone. The results are
depicted in Figure 3 (B). In both tests, it was
observed that there was a tendency for cell growth to
be inhibited at the higher concentrations. Culture
supernatants from untransfected fibroblasts of the
KMST-6 cell line did not show any stimulation of
proliferation~when compared with cell culture super-
natants without any added growth factor. In order to
investigate the therapeutic activity in wound healing
in more detail, lethally irradiated EGF gene-
transfected fibroblasts were cocultured in vitro with
primary human keratinocytes. In the experiments, it was
found that, after incubating for four days with
different concentrations of irradiated, EGF-secreting
fibroblasts, it was possible to obtain a dose-dependant
stimulation of keratinocyte proliferation which was
similar to that which was induced by recombinant growth
factor. This is depicted in Figure 4, with Figure 4 (A)
showing the value after four days of coculture and
Figure 4 (B) showing the values after 10 days of
coculture.
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Example 4
Demonstration of the in vivo production of EGF by
KMST-6 cells which are liposomally transfected in vitro
Experimental organization
Full-thickness skin wounds of 1.5 x 1.5 cm in
size were produced on the backs of 42 nude mice.
9.4 x 106 hEGF-transfected and lethally irradiated
KMST-6 cells were suspended in 2.8 ml of fibrin
adhesive and transplanted onto the full-thickness skin
wounds of 14 nude mice (300,000 cells/cniz, group I).
14 full-thickness skin wounds, which were
transplanted with untransfected KMST-6 cells (300,000
cells/cm2, group II), and 14 untreated full-thickness
skin wounds (group III), served as controls.
On days 1, 2, 3, 4, 5, 7 and 14, two animals
from each group were in each case necropsied and 0.8 g
of wound tissue was homogenized using a Triton-X/PBS
buffer. The homogenates were centrifuged and the con-
centration of EGF in the supernatants was evaluated
using an anti-human EGF ELISA.
Resul is
On day 1, 470 pg/ml were detectable, in vivo,
in group I (in accordance with the invention) as
compared with 18 pg/ml in group II and 1.3 pg/ml in
group III. While the concentrations of EGF in group I
declined on days 2-7, they were nevertheless signifi-
cantly higher than in the control groups. It was not
possible to detect any EGF in any of the three groups
on day 14.
In summary, these results demonstrate that it
was possible to successfully transplant fibroblasts
which had been transfected with EGF in vitro in a
fibrin adhesive suspension. It was possible to detect
the transgenic protein in vivo at least up to day 7.
The results obtained in the experiment are
summarized in Table I below.
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Table I
DayspCMV-EGF-IRES- KMST6 control Untreated
TKNeo/KMST6#3 (transplanted with full-thickness
in accordance withuntransfected KMST6skin wounds
the invention cells, group II) (group III)
1 470 18 1.3
2 393 58 1.6
3 330 28 2.3
4 150 8 6.5
180 8.5 8
7 140 8.3 2.6
14 0.4 0 0
Table I shows the release of hEGF, in pg/ml, in
wounds in vivo, by irradiated KMST6 fibroblasts which
5 were transfected with a chirneric hEGF gene.
Example 5
Wound healing-promoting effect following the transplan
tation of KMST-6 cells, which have been transfected
wi th EGF in vi tro, in combination wi th human keratino
cytes (mixed cell transplantation)
Experimental organization
Full-thickness skin wounds of 1.5 x 1.5 cm in
size were produced on the backs of 72 nude mice.
1.0125 x 106 EGF-transfected and lethally irradiated
KMST-6 cells were suspended, in combination with
2.025 x 106 human keratinocytes, in 3.6 ml of fibrin
adhesive and transplanted onto the full-thickness skin
wounds of 18 nude mice (ratio 1:2, 75,000 cells/cm2,
group I ) .
18 full-thickness skin wounds which were trans-
planted with EGF-transfected KMST-6 cells on their own
(25,000 cells/cm, group II), 18 full-thickness skin
wounds which were transplanted with human keratinocytes
on their own (50,000 cells/cm2, group III) and 18 full-
thickness skin wounds which were transplanted with
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untransfected KMST-6 cells in combination with human
keratinocytes (ratio 1:2, 75,000 cells/cm2, group IV)
served as controls. Table II shows the division into
groups.
Table II
Group I EGF-KMST-6 + keratinocytes, n = 18
Group II only EGF-KMST-6, n = 18
Group III only keratinocytes, n = 18
Group IV untransfected KMST-6 + keratinocytes, n = 18
On days 1, 3 , 5 , 7 , 10 and 12 , one animal f rom
each group was in each case necropsied and 0.8 g of
wound tissue was homogenized using a Triton-X/PBS
buffer. The homogenates were centrifuged and the con
centration of EGF in the supernatants was evaluated
using an anti-human EGF ELISA. The other half of the
biopsies was used for the histological investigations,
beginning with day 5.
On post-OP days 7, 10, 12, 14, 17 and 21,
biopsies from two further animals from each group were
in each case examined histologically.
Results
EGF was detectable in groups I and II but not
in groups III and IV.
As far as closure of the wound was concerned,
the results in group I continuously showed an almost
complete, pronounced epithelialization on days 7 to 12
and a complete epithelialization from day 14 onward.
The group I wounds also showed the best results with
regard to the quality of the reconstitution of the
epidermis (cell positioning in the epithelium). By
contrast, the control groups did not show pronounced
epithelialization until day 14 and still did not show
any high-grade epithelialization on day 21. The
epithelialization is depicted in tabular form in
Table III.
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Table III: Tabular representation of the epithelializa-
tion
Epithelialization/differentiation
EGF-KMST-6 EGF-KMST-6 Keratino- ut-KMST-6 +
+ cytes keratinocytes
keratinocytes (group II) (group III)(group IV)
(group I)
Day - - - -
Day ++ + - -
7
Day ++ + + +
Day ++ + + +
12
Day ~ +++ + ++ ++
14
Day +++ +++ ++ ++
17
Day +++ ++ ++ ++
21
5
- no epithelialization detectable
+ incipient epithelialization
++ pronounced but no complete epithelialization
+++ complete epithelialization
Example 6
Wound healing-promoting effect in a large animal model
following transplantation of KMST-6 cells which have
been transfected wi th EGF in vi tro
Experimental organization
In a large animal experiment, experiments were
carried out on a total of 3 pigs (P1, P2 and P3) using
a total of in each case 21 standardized burn wounds
(5 cm2 in size, grade 2a). In one pig (P1), 7 stan-
dardized burn wounds were transplanted with EGF-
transfected KMST-6 cells in fibrin adhesive (therapy
group). In this case, 7 untreated standardized burn
wounds (control group I).and 7 standardized burn wounds
which were treated with fibrin adhesive (control group
III) served as controls. In two further pigs (P2 and
P3), 7 standardized burn wounds were likewise in each
case transplanted with EGF-transfected KMST-6 cells in
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fibrin adhesive (therapy group). In each case 7
untreated standardized burn wounds (control group I)
and in each case 7 standardized burn wounds which were
transplanted with untransfected KMST-6 cells (control
group II) served as controls. Table IV shows the
division into groups.
Table IV
Therapy group EGF-KMST-6, n = 21
Control group I untreated burn wounds, n = 21
Control group II untransfected KMST-6, n = 14
Control ,groupIII only fibrin adhesive, n = 7
On days l, 3, 5, 7, 10, 21 and 35, one wound
from each group was biopsied in each case. 0.8 g of
wound tissue from the biopsies up to day 10 was
homogenized using a Triton-X/PBS buffer. The homo-
genates were centrifuged and the concentration of EGF
in the supernatants was evaluated using an anti-human
EGF ELISA. In addition, the biopsies were evaluated
histologically.
Results
Table V shows the EGF concentration values in
the groups in one wound in each case.
Table V: Tissue concentrations of EGF, in pg/ml, in the
large animal experiment
Therapy group Control Control Control
In accordance group I group II group III
with the inven-Untreated Transplanta- Treatment
tion, trans- burn tion of only with
plantation of wounds untransfected fibrin
EGF-transfected KMST-6 cells adhesive
KMST-6 cells
(P3) (P3) (P3) (P1)
Day 390 0 20 0
1
Day 200 0 20 0
3
Day 140 0 25 0
5
Day 0 0 0 0
7
Day 10 0 10 0
10
