U.S. patent application number 10/024360 was filed with the patent office on 2002-06-27 for artificial skin.
This patent application is currently assigned to IsoTis N.V.. Invention is credited to Ponec, M., Riesle, J. U., van Blitterswijk, C. A., van Dorp, Annette G. M..
Application Number | 20020082692 10/024360 |
Document ID | / |
Family ID | 26150928 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082692 |
Kind Code |
A1 |
van Blitterswijk, C. A. ; et
al. |
June 27, 2002 |
Artificial skin
Abstract
The invention relates to an artificial skin based on a copolymer
of a polyalkylene glycol and an aromatic polyester, which skin has
a thickness between 50 and 2000 .mu.m, and which skin has an upper
and a lower side, both having a macroporosity between 10% and
95%.
Inventors: |
van Blitterswijk, C. A.;
(Hekendorp, NL) ; van Dorp, Annette G. M.; (Alphen
a/d Rijn, NL) ; Ponec, M.; (Leiderdorp, NL) ;
Riesle, J. U.; (Amsterdam, NL) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
28 STATE STREET
28th FLOOR
BOSTON
MA
02109
US
|
Assignee: |
IsoTis N.V.
Bilthoven
NL
|
Family ID: |
26150928 |
Appl. No.: |
10/024360 |
Filed: |
December 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10024360 |
Dec 13, 2001 |
|
|
|
09451520 |
Nov 30, 1999 |
|
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Current U.S.
Class: |
623/15.12 ;
602/42; 604/304; 623/23.72 |
Current CPC
Class: |
A61L 27/56 20130101;
A61L 27/18 20130101; A61L 27/60 20130101; A61L 27/18 20130101; A61F
2/105 20130101; C08L 71/02 20130101; C08L 67/02 20130101; A61L
27/18 20130101 |
Class at
Publication: |
623/15.12 ;
602/42; 623/23.72; 604/304 |
International
Class: |
A61F 002/10; A61F
013/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 30, 1998 |
EP |
98204031.3 |
Dec 11, 1998 |
EP |
98204203.8 |
Claims
What is claimed is:
1. Artificial skin comprising a copolymer of a polyalkylene glycol
and an aromatic polyester, said artificial skin having a thickness
between 50 and 2000 .mu.m and having an upper and a lower side
comprising pores with the upper and the lower side each having a
macroporosity between 10% and 95%.
2. Artificial skin according to claim 1 further comprising a single
layer.
3. Artificial skin according to claim 1 wherein the upper and the
lower side each has a macroporosity of at least 15%.
4. Artificial skin according to claim 3 wherein the upper and the
lower side each has a macroporosity of at least 20%.
5. Artificial skin according to claim 1 comprising pores having a
diameter between 20 and 500 .mu.m.
6. Artificial skin according to claim 5 comprising pores having a
diameter between 50 and 250 .mu.m.
7. Artificial skin according to claim 1 wherein the pores at the
upper side have a diameter smaller than the diameter of the pores
at the lower side.
8. Artificial skin according to claim 8 wherein the pores at the
upper side have a diameter of between 0.2 and 5 .mu.m.
9. Artificial skin according to claim 7 wherein the pores at the
lower side have a diameter of between 20 and 500 .mu.m.
10. Artificial skin according to claim 1 wherein the aromatic
polyester comprises an alkylene glycol having from 2 to 8 carbon
atoms and an aromatic dicarboxylic acid.
11. Artificial skin according to claim 1 wherein the polyalkylene
glycol comprises one or more of polyethylene glycol, polypropylene
glycol, or polybutylene glycol.
12. Artificial skin according to claim 10 wherein the aromatic
polyester is one or more of poly(ethyleneterephtalate),
poly(propyleneterephtalate)- , and poly(butyleneterephtalate).
13. Artificial skin according to claim 1 wherein the copolymer is a
polyethylene glycol/poly(butyleneterephtalate) copolymer.
14. Artificial skin according to claim 1 wherein the copolymer
comprises 40-80 wt. % of the polyalkyleneglycol.
15. Artificial skin according to claim 1 wherein the copolymer
comprises 50-70 wt. % of the polyalkyleneglycol.
16. Artificial skin according to claim 1 comprising autologous
cells.
17. Artificial skin according to claim 16, wherein the autologous
cells comprise epithelial cells or fibroblasts.
18. Artificial skin according to claim 1 comprising a calcium
phosphate coating.
19. Artificial skin according to claim 1 compatible with skin
glands, hair follicles or cells from which skin glands or hair
follicles may develop.
20. Artificial skin according to claim 1 comprising skin glands,
hair follicles or cells which may develop into skin glands or hair
follicles.
21. Artificial skin according to claim 1 comprising a biologically
active agent.
22. Artificial skin according to claim 1 comprising
keratinocytes.
23. A method of using an artificial skin according to claim 1
comprising contacting the artificial skin to a wound of an animal
or human.
24. A method according to claim 23 wherein the artificial skin
comprises one or more of autologous epithelial cells or
fibroblasts.
Description
[0001] The invention relates to artificial skin which is suitable
for wound covering, and which can be used externally on various
types of wounds.
[0002] Human skin forms a barrier against adverse external
influences such as infections. If part of the skin is damaged, as
for example in the case of a burn, complications usually arise.
These complications are due to the protective function of the skin
being lost, as a result of which microbial invasion may occur, and
to a substantial loss of moisture that may take place at the place
of the wound.
[0003] Many studies have been carried out in order to provide an
artificial skin which is able to take over all, or a large part of
the functions of natural skin during the period that the wound is
not covered by an epidermis and dermis. To this end, the artificial
skin preferably comprises autologous cells provided on a scaffold.
Alternatively, the artificial skin itself may serve as carrier
material for cultured autologous keratinocytes and/or fibroblasts,
which have a favorable influence on the recovery of the epidermis
and/or dermis. Either way, it is desired that an artificial skin,
or that a scaffold material comprised by the artificial skin, has
suitable properties to serve as carrier for said cells.
[0004] An artificial skin of this type must, however, satisfy
various requirements. On the one hand, it should provide a barrier,
so that the wound is closed to bacteria and the like, and so that
substantial moisture loss is avoided. On the other hand, it must be
possible for adequate water vapor transport to take place through
the artificial skin. During this transport, nutrients from the
underlying tissue may reach the recovering skin in sufficient
quantity. Another important requirement is that the artificial skin
adheres to the underlying wound bed immediately after its
application to the wound. Furthermore, a permanent adhesion must be
formed as a result of ingrowth of tissue.
[0005] In U.S. Pat. No. 5,147,401, an artificial skin is disclosed,
of which the outer surface (the surface facing away from a wound to
which the skin is to be applied) is virtually closed. This is
achieved, in one embodiment, by providing a bi-layer system
comprising an upper layer, which is dense and non-porous, on top of
a lower layer, which is porous. In a different embodiment, this is
achieved by providing a single-layer system of a segmented
material, so that one of the sides of the skin is virtually closed,
and the other side is fairly open.
[0006] It has now been found that when the outer surface of the
artificial skin is virtually closed, a poor adherence of the skin
to a wound may be observed. Under certain conditions, the
artificial skin shows more or less a `curling-up effect`, in that
the edges of the skin are forced away from the wound, leading to a
poor adherence.
[0007] Of course, when the adherence of the artificial skin to the
wound is unsatisfactory, the protection of the wound by the
artificial skin is equally unsatisfactory. It is therefore an
object of the present invention to provide an artificial skin which
shows an improved adherence.
[0008] Surprisingly, the desired improved adherence may be obtained
by the provision of an artificial skin comprising an upper and a
lower side, both of which are porous. Thus, the invention relates
to an artificial skin based on a copolymer of a polyalkylene glycol
and an aromatic polyester, which skin has a thickness between 50
and 2000 .mu.m, and which skin has an upper and a lower side, both
having a macroporosity between 10% and 95%.
[0009] An artificial skin according to the invention adheres very
well to a wound when applied thereto. Under many circumstances,
adherence is achieved in a period of a few minutes after
application. The so-called `curling-up effect` that has been
observed with the prior art artificial skins has not been found to
occur with the present skin. Furthermore, the artificial skin of
the invention provides a highly suitable carrier for autologous
cells, thus enabling tissue repair.
[0010] An artificial skin according to the invention is based on a
specific copolymer, which is biodegradable. Advantageously, the
biodegradability (the rate of degradation under certain conditions)
may be controlled, depending on the envisaged site of application
of the artificial skin.
[0011] The specific copolymer on which the present skin is based,
is a copolymer of a polyalkylene glycol and an aromatic polyester.
In a preferred embodiment, an artificial skin according to the
invention is a single-layer system composed of the specific
copolymer.
[0012] Preferably, the copolymer comprises 40-80 wt. %, more
preferably 50-70 wt. % of the polyalkylene glycol, and 60-20 wt. %,
more preferably 50-30 wt. % of the aromatic polyester. A preferred
type of copolymers according to the invention is formed by the
group of block copolymers.
[0013] Preferably, the polyalkylene glycol has a weight average
molecular weight of from 150 to 4000, more preferably of 200 to
1500. The aromatic polyester preferably has a weight average
molecular weight of from 200 to 5000, more preferably of from 250
to 4000. The weight average molecular weight of the copolymer
preferably lies between 20,000 and 200,000, more preferably between
50,000 and 120,000. The weight average molecular weight may
suitably be determined by gel permeation chromatography (GPC). This
technique, which is known per se, may for instance be performed
using tetrahydrofuran as a solvent and polystyrene as external
standard.
[0014] In a preferred embodiment, the polyalkylene glycol component
has units of the formula -OLO-CO-Q-CO-, wherein O represents
oxygen, C represents carbon, L is a divalent organic radical
remaining after removal of terminal hydroxyl groups from a
poly(oxyalkylene)glycol, and Q is a divalent organic radical.
[0015] Preferred polyalkylene glycols are chosen from the group of
polyethylene glycol, polypropylene glycol, and polybutylene glycol
and copolymers thereof, such as poloxamers. A highly preferred
polyalkylene glycol is polyethylene glycol.
[0016] The terms alkylene and polyalkylene generally refer to any
isomeric structure, i.e. propylene comprises both 1,2-propylene and
1,3-propylene, butylene comprises 1,2-butylene, 1,3-butylene,
2,3-butylene, 1,2-isobutylene, 1,3-isobutylene and 1,4-isobutylene
(tetramethylene) and similarly for higher alkylene homologues. The
polyalkylene glycol component is preferably terminated with a
dicarboxylic acid residue -CO-Q-CO-, if necessary to provide a
coupling to the polyester component. Group Q may be an aromatic
group having the same definition as R, or may be an aliphatic group
such as ethylene, propylene, butylene and the like.
[0017] The polyester component preferably has units
-O-E-O-CO-R-CO-, wherein O represents oxygen, C represents carbon,
E is a substituted or unsubstituted alkylene or oxydialkylene
radical having from 2 to 8 carbon atoms, and R is a substituted or
unsubstituted divalent aromatic radical.
[0018] In a preferred embodiment, the polyester is chosen from the
group of polyethylene terephtalate, polypropylene terephtalate, and
polybutylene terephtalate. A highly preferred polyester is
polybutylene terephtalate.
[0019] The preparation of the copolymer will now be explained by
way of example for a polyethylene glycol/polybutylene terephtalate
copolymer. Based on this description, the skilled person will be
able to prepare any desired copolymer within the above described
class. An alternative manner for preparing polyalkylene
glycol/polyester copolymers is disclosed in U.S. Pat. No.
3,908,201.
[0020] A polyethylene glycol/polybutylene terephtalate copolymer
may be synthesized from a mixture of dimethyl terephtalate,
butanediol (in excess), polyethylene glycol, an antioxidant and a
catalyst. The mixture is placed in a reaction vessel and heated to
about 180.degree. C., and methanol is distilled as
transesterification proceeds. During the transesterification, the
ester bond with methyl is replaced with an ester bond with
butylene. In this step the polyethyene glycol substantially does
not react. After transesterification, the temperature is raised
slowly to about 245.degree. C., and a vacuum (finally less than 0.1
mbar) is achieved. The excess butanediol is distilled and a
prepolymer of butanediol terephtalate condenses with the
polyethylene glycol to form a polyethylene/polybutylene
terephtalate copolymer. A terephtalate moiety connects the
polyethylene glycol units to the polybutylene terephtalate units of
the copolymer and thus such copolymer also is sometimes referred to
as a polyethylene glycol terephtalate/polybutylene terephtalate
copolymer (PEGT/PBT copolymer).
[0021] The thickness of an artificial skin according to the
invention will depend on the envisaged site of application of the
skin. Generally, the thickness will be within a range of 50-2000
.mu.m. The skilled person will be able to select a suitable
thickness, given a certain site of application of the skin.
[0022] An important aspect of an artificial skin according to the
invention is that it has a macroporosity between 10 and 95%, on
both sides of the skin. In other words, both the side of the skin
facing the wound to which the skin is to be applied, and the side
of the skin facing away from the wound have said macroporositiy.
Preferably, the macroporosity is at least 15%, more preferably at
least 20%. It has been found that a skin having this specific
macroporosity adheres very well to a wound.
[0023] The size of the pores in an artificial skin according to the
invention preferably lies between 20 and 500 .mu.m, more preferably
between 50 and 250 .mu.m.
[0024] In a preferred embodiment, particularly when before or after
implantation of the artificial skin keratinocytes are applied on
top of the skin, the pore size varies within the skin. Preferably,
the pores at the upper side of the skin, which faces away from a
wound to which the skin is to be applied, are smaller than the
pores at the lower side of the skin. According to this embodiment,
the chance of microorganisms finding their way through the skin
into a wound, and of moisture finding its way through the skin out
of the wound are very small. Moreover, the ingrowth of
keratinocytes into the artificial skin is substantially prevented,
while a suitable porosity of the artificial skin is still present,
providing a favorable diffusion profile for nutrients and waste
products.
[0025] Preferably, in accordance with this embodiment, the diameter
of the pores at the upper side of the skin lies between 0.2 and 40
.mu.m, preferably between 0.4 and 2 .mu.m. The porosity at the
upper side of the skin preferably is higher than 15%. The diameter
of the pores at the lower side of the skin preferably lies between
20 and 300 .mu.m, more preferably between 50 and 250 .mu.m.
[0026] An important advantage of an artificial skin according to
the invention is that it forms a highly suitable substrate for
culturing epithelial cells, such as keratinocytes. It has also been
found to be feasible to culture fibroblasts on or within the
artificial skin. Due to the porous character of the present skin,
the fibroblasts can grow both on the upper and on the lower side of
the skin, as well as within the skin. The keratinocytes will
normally be provided on top of the skin.
[0027] If necessary, a possible delay in the initial cell growth on
the present skin may be overcome by improving the cell adhesion
using, for instance, laminin, collagen (type IV), proteoglycans or
fibronectin, which may be applied by (pre)coating to the surface of
the material which is to be covered. Improvement of the initial
cell growth may further be attained by modification of the surface
of the artificial skin. This modification can, inter alia, be
carried out by a plasma or glow discharge treatment, radiation,
monomer grafting or hydrolytic etching (e.g. with sulfuric
acid).
[0028] Accordingly, the invention also relates to the therapeutic
use of an artificial skin as described herein above. An important
example of said use concerns the use of the skin as wound-covering
material, particularly in the case of deep or large wounds, such as
burns.
[0029] When the artificial skin is used per se, that is without
providing it with cells prior to implantation in a patient, it may
be advantageous to cover the wound to which the artificial skin has
been applied with a semi-permeable membrane. This membrane ensures
a suitable barrier against microbial invasion and loss of moisture
until cells of the patient surrounding the wound have taken over
the barrier function of said membrane, or until the membrane may be
replaced by an epidermal graft or a cultivated cornified
keratinocyte sheet, at which time the membrane may be removed. For
a general discussion of how artificial skin may be applied to a
patient, reference is made to Morgan et al., Science &
Medicine, July/August 1997, pp. 6-15, Yannas, Biomedical
Engineering Handbook, CRC Press, 1995, pp. 2025-2038, and Wood et
al., in Essays in Biochemistry, Ed. Apps and Tipton, Portland
Press, Vol. 29, 1995, pp. 65-85.
[0030] Preferably, the artificial skin is provided with autologous
epithelial cells and/or fibroblasts when it is applied to a wound.
These cells may be obtained from a biopsy taken from the human or
animal to be treated. According to the invention, it is possible to
culture these autologous cells on the artificial skin both in-vitro
and in-vivo. In case the artificial skin is provided with a
stratified corneum prior to implantation, the use of a
semi-permeable membrane will generally not be necessary.
[0031] In case the cells are cultured on the skin in-vitro, it has
been found highly advantageous when the artificial skin comprises a
calcium phosphate coating. This coating facilitates the adhesion of
the cells to the artificial skin. The calcium phosphate may be
applied to the artificial skin by soaking the skin into a highly
concentrated calcifying solution at low temperature. The calcifying
solution is preferably composed of at least calcium and phosphate
ions, and optionally of magnesium, carbonate, sodium and chloride
ions, which are dissolved into water by bubbling carbon dioxide
gas. During the natural release of carbon dioxide gas or its
exchange with air, the pH of the calcifying solution is increased
and the saturation is raised until the nucleation of carbonated
calcium phosphate crystals on the surface of the artificial skin.
The process of bubbling and/or releasing CO.sub.2 gas through or
from the calcifying solution can be repeated until a sufficient
thickness of the coating has been reached.
[0032] In a preferred embodiment, the artificial skin is designed
to accommodate skin glands (sebaceous, apocrine or eccrine glands),
hair follicles or cells from which skin glands or hair follicles
may develop. The skin glands or hair follicles may be obtained, for
example, from a suitable donor, such as a cadaver, preferably
having the same blood type. It is preferred, however, that
autologous hair follicles are used, which may be obtained in a
biopsy from the patient undergoing a skin transplantation. Cells
which may be used having the ability to form or develop into hair
follicles are, among others, stem cells, hair follicle dermal
papilla, fibroblasts, keratinocytes, germinative matrix cells, and
melanocytes. It will be understood that the invention also
encompasses an artificial skin provided with skin glands, hair
follicles or cells which may develop into hair follicles or skin
glands.
[0033] In accordance with this embodiment, the artificial skin may
for instance be provided with orifices in which hair follicles, or
cells which may develop into hair follicles, may be located. These
orifices may be created by drilling, possibly by laser drilling,
or, and this is preferred, by punching with a, preferably hollow,
needle having the desired diameter. It is also possible to form the
artificial skin in a mold provided with protuberances having the
form of the desired orifices. The amount of orifices per surface
unit of the artificial skin will typically depend on the density of
the hair growth on the skin of the patient which is to be treated
with the artificial skin. In general, there will be between 1 and
50 orifices per square centimeter. It will be clear that it is
aimed at to achieve as natural an appearance of the patient's skin
after implantation of the present artificial skin as possible. The
diameter of the orifices may vary, dependent on whether hair
follicles or cells from which hair follicles may develop are to be
provided in the artificial skin. Typically, the diameter will lie
between 10 .mu.m and 10 mm. The depth of the orifices will,
depending on the thickness of the artificial skin, vary within a
range of 5 to 2000 .mu.m, preferably of 5 to 1500 .mu.m.
[0034] An artificial skin designed to accommodate skin glands or
hair follicles according to the invention may be provided with a
dense top layer, preferably a semi-permeable membrane as mentioned
above or an epidermal equivalent provided with keratinocytes, which
may be put on top thereof, and can be kept in place by gluing it to
the artificial skin using any biocompatible glue. Of course, it is
also possible that the dense layer forms an integral part of the
artificial skin. In fact, in accordance with this embodiment it is
possible that the entire artificial skin is dense or substantially
dense.
[0035] The dense top layer contributes to the provision of space
and configuration for hair growth. Further, it may assist in
keeping the hair in place. When the artificial skin is provided
with cells which may form or develop into skin glands or hair
follicles, it is also possible to apply the dense top layer on top
of an artificial skin not containing orifices. In that case, a
substance improving cell adhesion as discussed above, such as
integrin, CD2, CD48, laminin or fibronectin, may be applied at the
orifices in the artificial skin, thus defining the locations for
the skin glands or hair follicles to develop. Through the orifices,
cells may be seeded onto the artificial skin, which will adhere to
the substance improving cell adhesion. The dense top layer may be
removed before or after seeding the cells. It is another advantage
of the present artificial skin that it has such an optimal
degradation profile that it remains intact long enough to keep a
hair, a hair follicle or a skin gland in place, while on the other
hand it degrades fast enough to meet all requirements of a tissue
engineered artificial skin. In addition, in case the hair follicle,
the skin gland or the cells which may develop into a hair follicle
or a skin gland is applied to the artificial skin when said skin is
in a dry, non-swollen state, the swelling behavior of the
artificial skin when in contact with an aqueous environment will
greatly contribute to the keeping the hair follicle or skin gland
in place.
[0036] An orifice in the artificial skin forms a chamber in which
the skin gland or hair follicle may be located. Through the orifice
in the dense top layer (the semi-permeable membrane), a hair may
grow. Also, the dense top layer may assist in keeping the hair in
place. It is one of the advantages of the artificial skin according
to the invention, that nutrients will be able to reach the skin
gland or hair follicle by diffusion. Alternatively, the diffusion
may be amplified by providing a channel below the chamber, which is
smaller in diameter than the chamber itself, and through which
nutrient and waste product transport may take place.
[0037] In another preferred embodiment, the artificial skin is
provided with biologically active agents, which may be released
after implantation of the artificial skin in a patient. Preferred
biologically active agents to be incorporated into the artificial
skin are vitamins, such as vitamin C or E, antibiotics, such as
gentamycin, and growth factors, such as FGF, EGF, TGF-.alpha., and
IGF-1. The provision of the artificial skin with biologically
active agents may be accomplished as described in EP-A-0 830 859,
which is incorporated herein by reference.
[0038] Above it has been mentioned, that an artificial skin
according to the invention can be applied to a wound with or
without cells seeded thereon. If a cell-seeded approach is taken,
the cells used will preferably be autologous epithelial cells,
fibroblasts, or cells that can develop into epithelial cells or
fibroblasts. Whether cells are seeded before application or whether
a cell-free approach is taken, in accordance with the invention it
has been found feasible to apply keratinocytes onto the artificial
skin. The provision of the keratinocytes onto the skin may
advantageously be carried out in one of three different
manners.
[0039] In accordance with a first manner, a differentiated
(stratified) keratinocyte sheet may be obtained by applying
keratinocytes onto a suitable carrier which allows keratinocyte
attachment (e.g. of the above described copolymer of a polyalkylene
glycol and an aromatic ester) having pores which allow for nutrient
and waste material transport. The pores are preferably smaller than
1 .mu.m. The keratinocytes on the carrier are preferably first
cultivated submerged in a suitable culture medium, followed by
cultivation at the air-liquid interphase until stratification
occurs. Subsequently, the keratinocyte sheet may be detached from
the carrier, e.g. using by a protease such as dispase. The thus
obtained keratinocyte sheet is preferably applied to an artificial
skin according to the invention with its basal side facing the
artificial skin. This may be carried out either in vitro or in
vivo, wherein in the latter case the artificial skin is applied
onto a skin wound.
[0040] In accordance with a second manner, keratinocytes are seeded
on a dense polymer film which has been provided with holes or pores
for nutrient and waste material transport. The film preferably has
a thickness of 5-100 .mu.m, more preferably of 10-80 .mu.m, and is
preferably of the above described copolymer of a polyalkylene
glycol and an aromatic ester, more preferably of a copolymer of
polyethylene glycol and polybutylene terephtalate. The holes or
pores preferably have a diameter of less than 1 .mu.m to prevent
migration of keratinocytes across the polymer film. Preferably, the
holes or pores are provided in the dense film by laser
drilling.
[0041] Onto this polymer film, keratinocytes may be seeded to reach
a subconfluent or confluent state, at the side which, after
application on an artificial skin according to the invention, faces
away from the wound. Thus, the polymer film will be provided
between the keratinocytes and the artificial skin. Nutrient supply
to the keratinocytes may be ensured either via diffusion through
the polymer film or through the holes or pores.
[0042] In accordance with a third manner, the polymer film as
described in the discussion of the second manner is seeded with
keratinocytes at its side which is to face an artificial skin
according to the invention to which it may be applied. Thus, the
keratinocytes are provided between the polymer film and the
artificial skin. In one embodiment, the polymer film may be removed
soon, preferably within 24 hours, after the film with the
keratinocytes has been applied to the artificial skin. The
keratinocytes will remain behind, attached to the artificial skin.
In another embodiment, the keratinocytes are allowed to form a
differentiated cornified epidermis, which leads to a detachment of
the (differentiated) keratinocytes from the polymer film, which may
then conveniently be removed. The formation of the cornified
epidermis will usually take place after the artificial skin with
keratinocytes has been applied to a wound. It is preferred that
accumulating wound fluid is soaked up by an absorbent material (a
dressing) which is placed on the polymer film.
[0043] The invention will now be elucidated by the following,
non-restrictive example.
EXAMPLE
Materials and Methods
[0044] Manufacture and Composition of Polyactive.TM. Substrate
[0045] The material used was an elastomeric Poly(Ethylene Glycol
Terephtalate) and Poly(Butylene Terephtalate) (PEGT/PBT) copolymer
with a PEGT/PBT ratio of 55/45 and PEG weight average molecular
weight (M.sub.w) of 300 Dalton [HC Implants BV., Leiden, The
Netherlands]. In the literature, this type of polymer is often
referred to as PEO/PBT or PEG/PBT copolymer.
[0046] The material was subjected to a solvent casting procedure,
wherein a substrate was formed by liquefying the material in
chloroform containing sodium citrate particles having a particle
size of 75-212 .mu.m, to acquire the desired pore structure. The
salt/copolymer solution was then casted on a glass plate using a
substrate-casting apparatus fixed at a height of approximately 250
.mu.m. The salt particles were allowed to sink, and a substrate was
formed wherein the pores gradually changed from one surface to the
opposite one (BISKIN-M).
[0047] A second substrate was casted analogous to the manner
described above. This second substrate was provided with a dense
top layer, which was prepared by casting a layer as described
above, except that no salt particles were used. This dense top
layer was applied on top of the second substrate to obtain the
substrate BISKIN.
[0048] Standard techniques were used to remove the salt from all
substrates (Beumer et al., Clin. Mater., 1993, Vol. 14, pp.
21-26).
[0049] Fibroblast Isolation and (Sub) Culture
[0050] A split-thickness skin obtained from the rump of a Yucatan
miniature pig using a mechanical dermatome was used for
establishment of fibroblast culture. The skin biopsies were first
extensively washed in phosphate buffered saline (PBS) supplemented
with Penicillin (1,000 IU/ml) and Streptomycin (1,000 .mu.g/ml)
[ICN Biomedicals, Inc.], subsequently cut into 0.5 cm.sup.2 pieces
and finally incubated overnight at 40.degree. C. in trypsin
solution (0.3% trypsin, 0.15 M NaCl, 0.04% KCl, 0.1% glucose, pH
7.6). Thereafter, the skin specimens were incubated for 60 min at
37.degree. C. after which epidermal cells were mechanically
separated from the dermis by means of a forceps. Next, the dermis
was digested with a collagenase solution (0.35% collagenase type 1A
[Sigma] in a Dulbecco's Modification of Eagle's Essential Medium
(DMEM [ICN Biomedicals, Inc], pH 7.6) for 60 min at 370.degree. C.
Released fibroblasts were grown in DMEM that was supplemented with
5% Fetal Calf Serum (FCS [Gibco]) and Penicillin (500
U/ml)/Streptomycin (500 .mu.g/ml) [ICN Biomedicals, Inc.]. The
medium was refreshed three times a week.
[0051] This method usually rendered confluent cultures of
fibroblasts within one week, which then were trypsinized (0.5%
trypsin in PBS, supplemented with 0.05% EDTA and 0.1% glucose, pH
7.6) and subcultured. Passages one to five were used for the
experiments.
[0052] Fibroblast Culture on PolyactiveT.TM. [300PEG55PBT45]
[0053] Gamma irradiated sterile polyactive.TM. substrates (BISKIN
and BISKIN-M, size 5.times.5 and 6.times.6 cm) were immersed in
complete culture medium and incubated overnight at 37.degree. C. on
polycarbonate filters (0.4 .mu.m [Costar]). Thereafter, the
substrates were kept immersed by gluing them on the filter with
Histoacryl.TM. [Melsungen, Germany].
[0054] 1.5 ml of suspension autologous or allogeneic dermal
fibroblasts (200,000-300,000 cells/cm.sup.2) were seeded onto the
porous layer of the substrates. Cells were allowed to attach for 5
hours after which 14 ml of culture medium were added. The
fibroblasts were cultured for three weeks in culture medium
supplemented with Epidermal Growth Factor (EGF, 5.0 ng/ml [Sigma])
and ascorbic acid (100 .mu.g/ml [Sigma]). The medium was changed
every other day.
[0055] Prior to transplantation, the cell-free or
fibroblast-populated substrates were washed three times in
serum-free medium and directly transported to the operating
theatre.
[0056] Animal Operation/Transplantation
[0057] This study was approved by the Animal Use Committee from the
University of Leiden. Five Yucatan miniature pigs weighing 17-23 kg
were used. The animals were fed a basal swine diet and housed in
animal facilities with controlled temperature (19-21.degree. C.)
and light (12 h light/12 h darkness). Immediately prior to the
operation the pigs were sedated with a mixture of Stressnil
injected intramuscularly, and general anaesthesia was maintained
with mask inhalation of isoflurane, oxygen, and nitrous oxide. The
backs were shaved, cleansed with chlorhexidine, and covered with
Ophraflex.TM.. A maximum of five square wounds (5.times.5 cm) were
marked on the Ophraflex.TM. on each flank of the animal and
subsequently full-thickness slices of skin, subcutaneous fat and
panniculus carnosus were excised, exposing the muscle fascia of the
external intercostal muscles. Cell-free or cell-seeded substrates
(seeded with allogeneic or autologous fibroblasts) were placed into
the wounds (Table 1). All wounds were covered with a non-adherent
polyamide mesh [Surfasoft, Mediprof, The Netherlands]; fixed with
skin staplers [Johnson & Johnson Medical, The Netherlands] or
with paraffin impregnated gauzes [Unitulle, Roussel B. V.; The
Netherlands]. Finally, the wounds were protected against mechanical
trauma by gauze layers, and fixed with adhesive tape [Fixomull.TM.
stretch, Beiersdorf, Hamburg] and elastic stockings [Tubigrip.TM.,
Seton Healthcare Group plc., England]. For control, wounds were
treated with the bandage as described above, without a dermal
substrate.
[0058] Table 1 All experimental wounds with positions and
transplanted substrates as described in Materials and Methods.
1 Wounds Experiment 1 Experiment 2 position pig 1 pig 2 pig 3 pig 4
pig 5 Left flank 1 A D E F D 2 B E F D E 3 C F D E F 4 D G E F G 5
E A D G F Right flank 6 F B F D E 7 G C D E G 8 A D E F D 9 B E F D
E 10 C F G E F (A = cell-free BISKIN; B = BISKIN populated with
autologous fibroblasts; C = BISKIN populated with allogeneic
fibroblasts; D = cell-free BISKIN-M; E = BISKIN-M populated with
autologous fibroblasts; F = BISKIN-M populated with allogeneic
fibroblasts; and G = control)
[0059] Macroscopical Transplant Examination
[0060] Wounds were inspected, measured and photographed twice a
week. The extent of wound contraction was established using a
sterile marking gauge and calculating the decrease in surface area
of the wound until re-epithelialization occurred. Wound contraction
is defined as 1 Wound contraction = A 0 - A x A 0 * 100 %
[0061] wherein A.sub.0 is the wound area on day zero and A.sub.x is
the area on day x post-transplantation. For statistical analysis
the Student-t test was used.
[0062] Biopsies (5 mm in diameter) were collected under general
anesthesia after 17 days and 1, 2, 3, 6, 12 and 24 months
post-transplantation. Biopsies were taken from each individual
wound. Per wound a maximum of four biopsies were taken. Biopsies
were taken from different sites of the wounds.
[0063] If, however, in the postoperative period, wrinkles developed
in the material, the ridge of the wrinkle was debrided in the
operating-room in order to drain the fluid. The dermal substrate
was never removed prematurely.
[0064] Microscopical Examination
[0065] Preparation of Samples for Light and Electron Microscopy
[0066] Biopsies and ungrafted dermal substrates were rinsed in
saline, dissected, and fixed with a mixture of 2% formaldehyde
([Sigma], freshly prepared from paraformaldehyde), and 1.5%
glutaraldehyde [Polysciences, Inc.] in 0.2 M Cacodylate buffer, pH
7.4, and subsequently dehydrated in a graded ethanol series up to
100%.
[0067] For light microscopy (LM), specimens were embedded in
glycol-methyl-methacrylate [Merck]. Sections were cut on a Reichert
Jung Supercut 2050 at a thickness of 1-2 i m and stained with
Toluidine Blue. Polyactive.TM. was discriminated from the
surrounding tissue using polarized light.
[0068] For scanning electron microscopy (SEM), the specimens were
critical point dried [(Balzers CPD030], gold sputtercoated [Balzers
MED010] and examined with a Philips SEM 525M at 15 kV.
[0069] For transmission electron microscopy (TEM) and reflection
contrast microscopy (RCM), fixed specimens were briefly rinsed in
0.1M phosphate buffer (pH 7.4) and postfixed for 1 hour in 1.0%
OsO.sub.4 and 1.5% K.sub.4Fe(CN).sub.6. Finally, the specimens were
washed with phosphate buffer for 10 minutes followed by dehydration
through increasing concentrations of ethanol, which were
subsequently substituted by EPON resin. After embedding in EPON,
ultrathin sections (60-100 nm) were stained with uranyl acetate and
lead hydroxide and examined at 80 kV in a Philips EM 410 or were
stained with Toluidine Blue and examined in an Orthoplan light
microscope.
[0070] Immunohistochemistry
[0071] Biopsies were embedded in OCT compound (Tissue Tek Miles
Inc.), immediately frozen in liquid nitrogen, and stored at
-80.degree. C. until use. Five-micrometer thick cryostat sections
were cut at -25.degree. C., air-dried overnight, fixed in acetone
for 10 minutes, and immunolabelled at room temperature. Multiple
sections of each specimen were processed to assure representative
samples. The monoclonal antibodies used for this study were
directed against: type IV collagen (Dakopatts, Glostrup, Denmark;
1:200), vimentin and smooth muscle actin (Dakopatts, Glostrup,
Denmark; 1:200). The sections were incubated with the first
antibodies at room temperature for 60 minutes. Thereafter, the
sections were washed and further incubated respectively with
biotinylated second antibody [Dakopatts, Glostrup, Denmark] for 30
minutes at room temperature, followed by incubation with
streptavidin-biotinylated-horseradish-peroxid- ase complex
[Amersham; 1:100] at room temperature for 30 minutes. The sections
were soaked in a solution containing 0.005% of 3-amino-9-ethyl
carbazole, 0.03% of H.sub.2O.sub.2, and sodium acetate buffer, pH
5.0, for 1 to 5 minutes and counter-stained with Mayer's
Hematoxylin. As a control, parallel sections incubated with normal
serum were used.
RESULTS
[0072] Transplantation Material
[0073] It was observed using SEM, that all pores of the porous
substrate BISKIN-M were filled with fibroblasts and extracellular
matrix.
[0074] Macroscopical and Microscopical Evaluation
Post-transplantation
[0075] The general clinical observations concerning healing were
the lack of inflammation, contracture or rejection with maintenance
of elasticity and suppleness in the healed wound. These
observations were made at the various stages of healing, from week
one to twenty-four months.
[0076] I. Early Post-Transplantation Period (until one month
Post-Transplantation)
[0077] Adhesion to the Wound
[0078] After placement of the BISKIN grafts on the animal wounds it
was observed that all BISKIN-substrates showed little or no
adherence to the wound bed. Pre-swelling and transplantation site
were not of influence to adherence. In contrast to this, the
adherence of the BISKIN-M substrates to the underlying wound was,
however, achieved within a few minutes after application.
Therefore, in further evaluations BISKIN-M Polyactive.TM.
substrates are presented only.
[0079] There was no difference between wounds transplanted with
cell-free or cell-seeded BISKIN-M substrates in the incidence and
the appearance of wrinkles. There were no deaths, wound infections,
or acute graft failures in any of the animals.
[0080] Vascularization
[0081] Four days after grafting of cell-free or cell-seeded
BISKIN-M substrates, the grafted area showed red appearance due to
the ingrowth of capillary rich granulation tissue. Light and
electron microscopy revealed the presence of a thick layer of
highly vascularized granulation tissue surrounding the dermal
grafts in all experimental wounds. Cells from granulation tissue
(mainly fibroblasts, lymphocytes and macrophages) and capillary
vessels were invading the pores of the BISKIN-M substrates. There
was no difference between cell-free or cell-seeded BISKIN-M dermal
grafts, and the density of blood vessels in the subdermal
granulation tissue of BISKIN-M treated wounds was markedly higher
than in control wounds.
[0082] Wound Contraction
[0083] All wounds contracted, which was defined as the movement of
the wound edges towards the centre of the wound. There were
considerable differences, however, in the extent of contraction
during the post-transplantation time between substrates tested. The
extent of wound contraction was significantly lower in the
BISKIN-M-treated wounds as compared to non-treated wounds
(controls) (p<0.05). In control wounds (without substrate), 50%
wound contraction was observed within 14 days post-grafting,
whereas wounds treated with the BISKIN-M substrates showed
reduction of surface area with 5-15%. 30 days after transplantation
a reduction of surface area with cell-seeded and cell-free BISKIN-M
substrates treated wounds of 15-40% was observed, whereas control
wounds showed 75% reduction. Wounds treated with the
autologous-fibroblasts seeded BISKIN-M substrates contracted to a
lesser extent as compared to wounds treated with
allogeneic-fibroblasts seeded or with cell-free BISKIN-M
substrates. These differences, although undoubtedly present, were
not statistically significant. (p<0.15 when compared to
allogeneic-fibroblasts populated BISKIN-M substrates, and p<0.11
when compared to cell-free BISKIN-M substrates).
[0084] Formation of Granulation Tissue
[0085] In the first 2 weeks, macrophage phagocytic activity was
observed at most implantation sites. The level of this activity was
generally related to the degree of local trauma created by the
surgical procedure because it was seen in both control and
transplant-treated wounds. After two weeks lymphocytes were present
within and around the copolymer transplants. The presence of
condensed chromatin in the nucleus of these cells suggests that
they were not activated. Cellular debris but also some copolymer
fragments were incorporated in macrophages or multinucleated giant
cells.
[0086] Early Formation of Neo-dermis
[0087] In the first week post-operation all wounds were
characterised by the presence of a relatively high number of
myofibroblasts containing a-smooth muscle actin.
[0088] Degradation
[0089] Light and transmission electron microscopy showed a foreign
body reaction at the implantation site of all transplanted
substrates. Macrophages and multinucleated giant cells were present
during the first 4 weeks post-transplantation. At 8 weeks
post-transplantation the foreign body reaction was ceased. Four
weeks after transplantation, matrix degradation was observed.
Degradation started with fragmentation of the polymers into
particles that were phagocytized by macrophages and multinucleated
giant cells.
[0090] II. Late Post-Transplantation Period (from 2 up to 24 months
Post-Transplantation)
[0091] Formation of Neo-dermis
[0092] It was observed that, two months after grafting, newly
formed collagen in wounds treated with fibroblastpopulated
Polyactive substrates, was distributed in orthogonal arrays, or in
a "basket wave" pattern. The organisation of collagen bundles and
blood vessel formation was similar in wounds transplanted with
substrates seeded with allogeneic- or autologous-fibroblasts.
[0093] Degradation
[0094] Twenty-four months post-transplantation, the tissue
surrounding the Polyactive substrates consisted of a mature
connective tissue. At the macroscopic and at the light- and
electron microscopic level, the heart, spleen, liver, lung, glands
and kidneys did not show any signs of swelling, tissue
damage/necrosis or polymer fragments. The most important results on
the appearance of wounds after grafting are summarised in Table
2.
2TABLE 2 Characteristics of wounds transplanted with cell-free or
fibroblast-populated BISKIN-M matrices. treatment of the wounds
fibro blast- populated cell-free Poly- Parameters none Polyactive
active* adhesion time after 1-2 min 1-2 min application vasculari-
time after >14 days in 4 days in 4 days zation application wound
30 days >50% <50% <50% contraction after application
neo-dermis collagen thin, thin, less thick, formation appearance
less compact compact compact bundles bundles bundles collagen in in
in distribu- parallel parallel orthogo- tion arrays arrays nal
arrays degradation number of high low intracel- lularly located
fragments seeded with autologous or allogeneic fibroblasts
* * * * *