U.S. patent application number 13/392222 was filed with the patent office on 2012-08-16 for tissue dressing kit.
This patent application is currently assigned to Medvoent GmbH. Invention is credited to Thomas Freier, Rivelino Montenegro.
Application Number | 20120209320 13/392222 |
Document ID | / |
Family ID | 42115621 |
Filed Date | 2012-08-16 |
United States Patent
Application |
20120209320 |
Kind Code |
A1 |
Montenegro; Rivelino ; et
al. |
August 16, 2012 |
TISSUE DRESSING KIT
Abstract
A kit comprising a tissue dressing material for being applied in
contact with the tissue of a patient and a detachment solvent for
removing the tissue dressing material from the tissue. A method of
treating a tissue of a patient, the method comprising the steps of:
applying a water-soluble tissue dressing material in contact with
the patient's tissue; and applying an acidic detachment solvent to
the tissue dressing material for removing the tissue dressing
material from the tissue. A method of treating a tissue of a
patient, the method comprising the steps of: applying a liquid
tissue dressing material in contact with the patient's tissue; and
applying a detachment solvent to the tissue dressing material for
removing the tissue dressing material from the tissue. A method of
treating a tissue of a patient, the method comprising the steps of:
applying a water-soluble tissue dressing material in contact with
the patient's tissue; and allowing the water-soluble tissue
dressing material to convert into a form in which it is insoluble
in water at neutral pH. And a solid material comprising a polymer
salt and glycerol, the glycerol content being at least 10% of the
polymer salt content by weight, for use in a method of treating a
human or animal tissue.
Inventors: |
Montenegro; Rivelino;
(Mainz, DE) ; Freier; Thomas; (Mainz, DE) |
Assignee: |
Medvoent GmbH
Mainz
DE
|
Family ID: |
42115621 |
Appl. No.: |
13/392222 |
Filed: |
September 1, 2010 |
PCT Filed: |
September 1, 2010 |
PCT NO: |
PCT/EP2010/062822 |
371 Date: |
May 3, 2012 |
Current U.S.
Class: |
606/213 |
Current CPC
Class: |
A61P 17/02 20180101;
A61L 15/28 20130101; A61P 31/04 20180101; A61P 31/06 20180101; A61P
31/10 20180101; A61L 26/0023 20130101; A61L 2300/232 20130101; A61P
31/12 20180101; A61P 7/04 20180101; A61P 31/02 20180101; A61L
2300/404 20130101; A61P 31/00 20180101; A61L 26/0066 20130101; A61L
15/28 20130101; C08L 5/08 20130101; A61L 26/0023 20130101; C08L
5/08 20130101 |
Class at
Publication: |
606/213 |
International
Class: |
A61B 17/03 20060101
A61B017/03 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 1, 2009 |
EP |
PCT/EP2009/006323 |
Claims
1. A kit comprising: a tissue dressing material for being applied
in contact with the tissue of a patient; and a detachment solvent
for removing the tissue dressing material from the tissue.
2. The kit according to any one of the previous claims,
characterized in that the detachment solvent for removing the
tissue dressing from the tissue is an aqueous detachment
solvent.
3. The kit of claim 2, characterized in that the detachment solvent
for removing the tissue dressing material from the tissue is
acidic.
4. The kit according to any one of the previous claims,
characterized in that the tissue dressing material for being
applied in contact with the tissue of the patient is solid or
gel-like.
5. The kit according to claim 4, characterized in that the tissue
dressing material for being applied in contact with the tissue of
the patient is water-soluble.
6. The kit according to claim 4 or 5, characterized in that the
main component of the tissue dressing material for being applied in
contact with the tissue of the patient is a polymer salt.
7. The kit according to any one of claims 1 to 3, characterized in
that the tissue dressing material for being applied in contact with
the tissue of the patient is liquid.
8. The kit according to any one of claims 4 to 7, characterized in
that the kit in addition to the solid or gel-like tissue dressing
material and the detachment solvent comprises a liquid tissue
dressing material.
9. The kit according to any one of claim 7 or 8, characterized in
that the liquid tissue dressing material is an aqueous
solution.
10. The kit according to any one of claims 7 to 9, characterized in
that it further comprises a spraying apparatus for spraying the
liquid tissue dressing material on the tissue of the patient.
11. The kit according to any one of the previous claims,
characterized in that the tissue dressing material comprises a
pH-sensitive dye for visually indicating the pH at the site of the
tissue where the tissue dressing in applied.
12. The kit according to any one of the previous claims,
characterized in that the tissue dressing material when applied to
the tissue in transparent.
13. A method of treating a tissue of a patient, the method
comprising the steps of: applying a water-soluble tissue dressing
material in contact with the patient's tissue; and applying an
acidic detachment solvent to the tissue dressing material for
removing the tissue dressing material from the tissue.
14. A method of treating a tissue of a patient, the method
comprising the steps of: applying a liquid tissue dressing material
in contact with the patient's tissue; and applying a detachment
solvent to the tissue dressing material for removing the tissue
dressing material from the tissue.
15. The method of claim 13 or 15, characterized in that the tissue
dressing comprises a water-soluble tissue dressing material before
application of the detachment solvent, the water-soluble tissue
dressing material is allowed to convert into a form in which it is
insoluble in water at neutral pH.
16. A method of treating a tissue of a patient, the method
comprising the steps of: applying a water-soluble tissue dressing
material in contact with the patient's tissue; and allowing the
water-soluble tissue dressing material to convert into a form in
which it is insoluble in water at neutral pH.
17. A solid material comprising a polymer salt and glycerol, the
glycerol content being at least 10% of the polymer salt content by
weight, for use in a method of treating a human or animal tissue.
Description
FIELD OF THE INVENTION
[0001] The invention relates to kits comprising a tissue dressing
material for being applied in contact with the tissue of a patient.
It further relates to methods of treating a tissue of a patient, in
which methods a tissue dressing material is applied in contact with
the patient's tissue. The invention also relates to a solid
material for use in a method of treating a human or animal
tissue.
BACKGROUND OF THE INVENTION
[0002] The polysaccharide chitosan is the at least partially
N-deacetylated derivative of chitin. Chitin can be found widely in
the exoskeletons of arthropods, gels, crustaceans and the cuticles
of insects. It is usually derived from such natural sources.
Chitosan in general is synthetically prepared by hydrolysis of
chitin, although it can also be naturally derived directly, e.g.
from certain fungi in which it occurs. The different solubilities
of chitin and chitosan in dilute acids are commonly used to
distinguish between the two polysaccharides. Chitosan, the soluble
form, can have a degree of acetylation (DA) between 0% and about
60%, the upper limit depending on parameters such as processing
conditions, molecular weight, and solvent characteristics. While
soluble in acidic aqueous media, chitosan precipitates at a pH of
above 6.3.
[0003] Both chitin and chitosan are promising polymers for
biomedical applications because of their biocompatibility,
biodegradability and structural similarity to the
glycosaminoglycans. For comprehensive reviews of potential
applications of chitin and chitosan see, e.g., Shigemasa and
Minami, "Applications of chitin and chitosan for biomaterials",
Biotech. Genetic. Eng. Rev. 1996, 13, 383; Kumar, "A review of
chitin and chitosan applications", React. Funct. Polym. 2000,
46(1), 1; and Singh and Ray, "Biomedical applications of chitin,
chitosan and their derivatives", J. Macromol. Sci. 2000, C40(1),
69.
[0004] Chitin and chitosan have been held to be of particular
promise in wound healing applications, early scientific reports on
this subject dating back to 1970 when Prudden et. al. in "The
discovery of a potent pure chemical wound-healing accelerator", Am.
J. Surg. 1970, 119, 560 described the successful application of
chitin powder on human wounds. The primary factor in the
acceleration of wound healing was reported to be the presence of
N-acetyl-D-glucosamine (in contrast to D-glucosamine) which is
released from chitin due to enzymatic degradation by lysozyme,
which is abundantly available in fresh and healing wounds.
[0005] The use of poly(N-acetyl-D-glucosamine), i.e. chitin, as a
wound healing accelerator is disclosed in the U.S. Pat. No.
3,632,754. U.S. Pat. No. 4,532,134 discloses the application of
chitosan solutions, powders, films, and mats to wounds. The claimed
method asks for chitosan being between 42 to 100% deacetylated.
Animal experiments using 78 to 92% deacetylated chitosan are
disclosed that show acceptable results when the material is applied
to wounds of dogs, while interference with early wound healing is
observed when the material is used to cover wounds of rats.
[0006] In the U.S. Pat. No. 5,902,798 and the US patent application
US 2001/0056079 degrees of acetylation of less than 25% are asked
for. In experiments applying 16% acetylated chitosan, inferior
stimulation of cell proliferation and wound healing were found in
an in vitro model using human skin compared to chitosan/heparin
materials.
[0007] The UK patent GB 2358354 B teaches a flexible polymeric film
comprising at least 80% by weight of chitosan with a degree of
acetylation between 12 and 30%. A slightly higher rate of wound
healing compared to non-treated wounds was found. The relatively
weak mechanical properties which necessitate the use of an
epichlorhydrine cross-linker or silicon coating may constitute a
disadvantage of this prior art material. The document also suggests
washing off the film in saline solution after healing of the
wound.
[0008] Azad et. al., "Chitosan membranes as a wound-healing
dressing: Characterization and clinical application", J. Biomed.
Mater. Res. 2004, 69B, 216, discloses the use of 25% acetylated
chitosan for the fabrication of films and meshes (perforated
films). The authors found that chitosan films cause an impaired
wound healing in patients undergoing skin grafting as a result of
blood clot formation underneath the film, while the use of meshes
led to a more efficient removal of blood, resulting in faster
healing with good epithelialization and without scar formation.
[0009] In the U.S. Pat. No. 7,482,503 a chitosan acetate foam is
described for use as a hemorrhage control wound dressing for severe
bleeding. The chitosan is required to be at least 70% deacetylated
and in the examples, degrees of deacetylation between 85 and 93%
are used.
[0010] The US patent application US 2005/042265 A1 discloses a
hydrogel for skin repair, the hydrogel containing a maximum of 5%
chitosan. The chitosan's degree of acetylation is required to be no
greater than 40%, in particular between 2% and 6%. Finally, the
international patent application WO 2008/128567 A1 discloses
medical articles, including wound dressings, at least partially
made of chitosan. The lowest degree of acetylation disclosed is
3%.
[0011] The German patent application DE 10 2007 038 125 A1
discloses an adhesive composition for gluing or fixing biological
tissue. The composition comprises a 50 to 98% deacetylated
polysaccharide, e.g. chitosan. Another component of the composition
is a functionalized oligolacton. The two components are provided
into separate chambers of a spraying apparatus.
[0012] The International patent application WO 2008/1128567
discloses the biodissolution of least a part of a medical device in
a dilute acid. The part of the device is made of N-acetylchitosan
with a degree of acetylation of more than 3% and less than 25%. The
biodissolution of the part of the device is controlled by adjusting
the pH of the aqueous medium in contact with the N-acetylchitosan
part of the device to a value of equal or less than 6.0.
PROBLEM TO BE SOLVED BY THE INVENTION
[0013] It is an object of the present invention to provide a kit
for improving the treatment of the tissue of a patient, the kit
comprising a tissue dressing material to be applied in contact with
the tissue of the patient. The invention further aims to provide
improved methods for treating a tissue of a patient. Moreover, the
invention seeks to provide a new material for use in a method of
treating a human or animal tissue.
SOLUTION ACCORDING TO THE INVENTION
[0014] According to the invention, the problem is solved by
providing a kit comprising: a tissue dressing material for being
applied in contact with the tissue of a patient; and a detachment
solvent for removing the tissue dressing material from the
tissue.
[0015] The problem is also solved by a method of treating a tissue
of a patient, the method comprising the steps of: applying a
water-soluble tissue dressing material in contact with the
patient's tissue; and applying an acidic detachment solvent to the
tissue dressing material for removing the tissue dressing material
from the tissue. The problem is further solved by a method of
treating a tissue of a patient, the method comprising the steps of:
applying a liquid tissue dressing material in contact with the
patient's tissue; and applying a detachment solvent to the tissue
dressing material for removing the tissue dressing material from
the tissue. Moreover, the problem is solved by a method of treating
a tissue of a patient, the method comprising the steps of: applying
a water-soluble tissue dressing material in contact with the
patient's tissue; and allowing the water-soluble tissue dressing
material to convert into a form in which it is insoluble in water
at neutral pH. Finally, the problem is solved by providing a solid
material comprising a polymer salt and glycerol, the glycerol
content being at least 10% of the polymer salt content by weight,
for use in a method of treating a human or animal tissue.
[0016] In the context of the present invention, a "detachment
solvent" is a liquid that can be applied to the tissue dressing
material when it is in a solid or gel-like state and that can
facilitate detachment of the tissue dressing material from the
tissue, preferably by at least partly dissolving and/or swelling
the tissue dressing material. The preferred detachment solvent can
reduce the adherence of the tissue dressing material to the tissue.
Thus, with the detachment solvent it can be avoided that the tissue
is damaged during removal of the tissue dressing material, and in
particular it can be avoided that when the tissue dressing material
is removed, parts of the tissue beneath it that adhere to the
tissue dressing material are torn away. Amongst other cases, this
can be of great advantage where the tissue dressing material is
applied to a wound as a wound dressing material, because wound
tissue can be very sensitive to mechanical stress. With the
invention, therefore, irritation or damage of the regenerating
tissue due to adhesions to the wound dressing material being
removed can be avoided.
[0017] The terms "dissolve" and "dissolution" in context with a
polymer is meant to refer to a process of mass loss of a solid or
gel-like polymer form without molecular weight decrease (i.e.,
without decrease in polymer chain length) due to solubility in an
aqueous environment. This is to be distinguished from
"degradation", which is the process of molecular weight decrease
due to depolymerization of a polymer. Advantageously dissolution
and/or swelling can facilitate the removal of the tissue dressing
material. It can be achieved with the invention that the tissue
dressing material is partly or even entirely dissolvable.
[0018] The inventors have found that providing the tissue dressing
material together with the detachment solvent in a kit can
considerably improve compliance in the sense that the patient is
less likely to attempt to separate the tissue dressing material
from the tissue without previous application of the detachment
solvent. The kit according to the invention can also prevent the
user from applying another, unsuitable or possibly even harmful
solvent.
[0019] The term "water-soluble" in the context of the present
invention refers to a state of the tissue dressing material in
which it is soluble in water at neutral pH. In one aspect, the
invention exploits the inventor's finding that some liquid tissue
dressing materials, such as chitosan solutions, as well as some
water-soluble but solid or gel-like tissue dressing materials, such
as chitosan salts, upon application to the patient's tissue can
transform into a solid or gel-like state, in which they are only
soluble in an acid liquid solvent. This transformation may, for
example, occur due to evaporation of a constituent of the tissue
dressing material upon contact of the tissue dressing material with
air. It may also be a result of an interaction of the tissue
dressing material with a body fluid and/or the tissue itself; for
example the relatively high pH of blood and/or the attachment of
proteins present in the blood to the tissue dressing material may
induce the transformation. Alternatively or additionally,
transformation may be achieved by applying a transformation medium,
e.g. an aqueous alkaline solution, to the tissue dressing material.
Advantageously, it can be achieved that after transformation the
tissue dressing material remains in place under normal condition,
e.g. when the tissue is cleaned under tap water (neutral pH) or
when soap (alkaline) is applied, and detaches only upon application
of the detachment solvent. Moreover, as a result of the
transformation the adhesion of the tissue dressing to the tissue
can be reduced, facilitating later removal with the detachments
solvent. Also, the water uptake capacity of the tissue dressing
material can be reduced as a result of the transformation, which is
desirable in certain applications.
[0020] The inventors have found that the presence of glycerol in
the solid material for tissue treatment can accelerate the
transformation from a water-soluble state into a state in which the
material is only soluble in an acid liquid solvent. For example, in
the case of a native chitosan salt as a polymer salt,
transformation can be accelerated from approximately one month to a
mere week. Without limiting the invention to a specific theory, the
inventors believe that the acceleration may be due to the
glycerol's effect of disrupting the crystalline structure of the
polymer salt. Advantageously, the faster transformation allows the
beneficial effects of the transformation (i.e. for example that
after transformation the tissue dressing material remains in place
when the tissue is cleaned under tap water) to set in earlier.
[0021] The kit, the methods and the solid material according to the
invention advantageously can be used for locally confined
antibacterial treatment of a patient's tissue. Thereby, it can be
exploited that the site where the tissue dressing material is
applied and thus the antibiotic activity takes place can be well
controlled in order to achieve only a local presence of the
antibiotic activity.
[0022] The kit, the methods and the solid material according to the
invention advantageously can be used for the treatment of acute
wounds, chronic wounds, and burn wounds or other types of wounds.
It can also be used to treat tissue affected by dermatosis, for
example athlete's food disease and psoriasis. The tissue dressing
material of the kit and methods according to the invention can be
applied in wound coverings, for example band aids, gauzes, films
and foams, and in support aids, for example bandages, support
tights and plaster casts. The invention may advantageously be used
to treat cuts and abrasions, nose bleeding, severely bleeding
wounds, and external and internal wounds in general. Thus, the
invention can be of use when surgery is performed on a patient. The
invention can also be applied advantageously to treat acne, razor
burn and insect bites as well as in cosmetic application such as
face masks and peelings. Preferably, the tissue dressing material
not only contacts the tissue to be treated but also tissue
surrounding the tissue to be treated.
[0023] While the invention preferably is used for external wounds,
it may also be applied to internal wounds. In a preferred method,
the invention is used internally as a hemostatic agent to control
bleedings during surgeries, or it is applied in the treatment of
injuries or diseases that cause internal bleedings. In another
preferred method, the invention is used internally to prevent or
limit infections by exploiting the potential of the deacetylated
native chitosan as polymeric antibiotic. In another preferred
method, the wound dressing material of the kit and methods
according to the invention is used in regions of the body that are
difficult to reach or treat, such as orifices, the genital area, or
parts of the body where wound healing can be delayed, due to
limited exposure to air. In a particularly preferred method, the
wound dressing material is applied in areas that are sensitive to
the application of foreign-body materials and conventional wound
dressings, such as mucosa. The material may be applied into or onto
the wound. A patient in the context of the present invention can be
a human or an animal.
PREFERRED EMBODIMENTS OF THE INVENTION
[0024] Preferred features of the invention which may be applied
alone or in combination are discussed in the dependent claims and
in the following description.
[0025] Preferably, the detachment solvent for removing the tissue
dressing material from the tissue is an aqueous detachment solvent.
Preferred detachment solvents include distilled water, aqueous
solutions of ionic compounds, such as an aqueous sodium chloride
solution, buffered solutions, such as an acetic acid/acetate
buffered solution, as well as aqueous solutions of non-ionic
compounds, such as an aqueous glucose solution. Advantageously,
water as a solvent is less irritating to the skin than many organic
solvents. While in principle, the aqueous detachment solvent
according to the invention may in addition to water comprise one or
more co-solvents other than water, e.g. an organic co-solvent such
as isopropanol or another alcohol, the preferred detachment solvent
is free of organic solvents, including alcohols, esters, alkanes,
halogenated solvents, amines, amides. It may, however, frequently
contain an organic acid; organic acids are not considered organic
solvents in the context of the present invention.
[0026] The detachment solvent for removing the tissue dressing
material from the tissue, in particular if it shall be removed by
dissolution, preferably is acidic. This embodiment of the invention
exploits the fact that the solubility of the tissue dressing
material, at least in a particular state of the tissue dressing
material, can be pH-dependent. Thus, advantageously the pH of the
detachment solvent can be selected from a range in which the tissue
dressing material is dissolvable to detach the tissue dressing
material from the tissue. The preferred pH of the detachment
solvent is below 7, more preferably below 6.5, more preferably
below 6.3. Advantageously, at a pH below 6.3 the base-form of
native chitosan, one of the preferred wound dressing materials, is
dissolvable in an aqueous medium. More preferably, the pH of the
detachment solvent is below 6, more preferably below 5.5, more
preferably below 5. The pH of the detachment solvent preferably is
above 3.5. Thereby, advantageously, irritation of the tissue due to
high acidity of the detachment solvent can be avoided. More
preferably, the pH of the detachment solvent is above 4, more
preferably above 4.5.
[0027] A preferred detachment solvent comprises a surfactant, e.g.
a polysorbate such as Tween. Alternatively or in addition in may
comprise substituted or unsubstituted polyalkyleneoxides, such as
polyethylene glycol or polyethylene glycol esters. It has been
found that the presence of such additives can considerably
facilitate detachment of the solid, gel-like or solidified liquid
tissue dressing material.
[0028] The amount of detachment solvent provided in the kit is at
least 5 times per weight, more preferably at least 50 times per
weight of the amount of the constituent(s) of the tissue dressing
material other than water or the chitosan provided in the kit. By
providing a sufficient amount of detachment solvent, it can be
avoided that the pH of the tissue dressing material solution falls
under a certain threshold. For application, the detachment solvent
may be sprayed or brushed or applied by means of a sponge, a
spatula, a pipette or gauze. Accordingly, a preferred kit contains
a sponge, a brush, a spatula, a pipette or gauze for applying the
detachment solvent. The detachment solvent may for example be
provided in a sealed bottle or a disposable pipette, or by means of
gauze, a sponge or a gel soaked with the detachment solvent. It may
also be provided in a spraying apparatus. The preferred spraying
apparatus comprises a container for storing the detachment solvent.
It may also comprise pressurised gas for expelling the detachment
solvent.
[0029] If in relation to preferred formulations, properties and
features of tissue dressing materials described below it is not
specified whether they concern a solid, gel-like or liquid tissue
dressing materials, it shall be assumed that they may apply equally
to any of such materials. The preferred tissue dressing material is
a polymer or a co-polymer, preferably comprising or consisting of a
polysaccharide, for example a chitosan such as native chitosan.
[0030] The term "native chitosan", in the context of the present
invention refers to the defined chemical entity chitosan, which is
a poly(N-acetyl-D-glucosamine-co-D-glucosamine) copolymer or a
poly(D-glucosamine) homopolymer. Any cross-linked or otherwise
chemically modified chitosan is considered a chitosan derivative,
having different properties than native chitosan. In the context of
the present invention the term "native chitosan" includes both the
chitosan base and chitosan in the form of a chitosan salt,
dissolved or un-dissolved. When in the context of the present
invention it is referred to "chitosan" in general, this can be any
form, salt or base, of native chitosan or any derivative of a
poly(N-acetyl-D-glucosamine-co-D-glucosamine) copolymer or a
poly(D-glucosamine) homopolymer, cross-linked and/or otherwise
modified. The preferred chitosan is native chitosan. One of the
advantages of native chitosan is its high biocompatibility and
bioactivity. The preferred tissue dressing material can be
essentially non-degradable, as is for example the case for
deacetylated chitosan as defined below, or it can be partly or
entirely degradable, for example biodegradable.
[0031] The preferred tissue dressing material has an antibacterial
property. Thereby, advantageously, a localized antibacterial
treatment can be achieved, thereby avoiding a systemic
antibacterial activity, i.e. an antibacterial treatment of regions
of the patient's body where such treatment is not required and/or
not desirable. The invention can thus reduce side effects and
contribute to the swift recovery of the patient. A suitable tissue
dressing material with antibacterial properties is chitosan, in
particular native chitosan.
[0032] Advantageously, with chitosan as the tissue dressing
material or a constituent of the tissue dressing material, a tissue
dressing material can be provided that is essentially free of toxic
compounds. The invention can reduce the risk of wound infections,
by exploiting the antibiotic nature of chitosan, in particular
native chitosan, as a natural polymeric antibiotic with no systemic
but only topical activity. Preferably, the tissue dressing material
comprises no additional preservative. The inventors have found that
the antibacterial properties of chitosan are sufficient to provide
for a satisfactory shelf life of the product. This is particularly
advantageous in liquid tissue dressings, as many liquid tissue
dressing available on the market contain preservatives which may
have toxic effects and may induce tissue irritations or allergic
reactions.
[0033] The preferred chitosan has a degree of acetylation (DA) of
40% or less, preferably 20% or less, preferably 10% or less.
Preferably, the chitosan is deacetylated. Preferably, this is the
only chitosan component of the tissue dressing material. In the
context of the present invention the term "deacetylated chitosan"
means that the chitosan's DA is less than 2.5%. This embodiment of
the invention exploits the inventors' discovery that a
significantly accelerated rate of wound healing can be achieved by
applying a native chitosan material that is deacetylated, i.e.
essentially free of N-acetyl-D-glucosamine subunits. This finding
is surprising when considering the importance attributed to the
N-acetylated form of D-glucosamine in wound healing applications,
e.g. as described in the U.S. Pat. No. 3,632,754, supra.
Furthermore, it has been suggested, e.g., in Izume et. al., "A
novel cell culture matrix composed of chitosan and collagen
complex", in: Chitin and chitosan, Amsterdam 1989, 653, that
chitosan of a very low degree of acetylation may rather have
cytostatic properties, as it inhibits cell proliferation due to an
extremely high cell adhesion.
[0034] The DA can be obtained by means of .sup.1H NMR spectroscopy
as, e.g., disclosed in Layertu et al., "A validated .sup.1H NMR
method for the determination of the degree of deacetylation of
chitosan", J. Pharm. Biomed. Anal. 2003, 32, 1149. "Deacetylated
native chitosan" in the context of the present invention refers to
chitosan that is both native and deacetylated according to the
above definitions. In a preferred tissue dressing material
according to the invention, the deacetylated chitosan's or the
deacetylated native chitosan's DA is 2% or less, preferably 1.5% or
less, more preferably 1% or less, more preferably 0.5% or less.
Advantageously, such extremely low degrees of acetylation can
further improve the wound healing properties of the invention.
Also, biodegradation can be further inhibited, avoiding tissue
ingrowths and excessive adhesion of the tissue dressing material.
Moreover, by virtue of the low DA of the deacetylated native
chitosan, the tissue dressing material can be applied in
practically non-lysozyme biodegradable form, which can contribute
to preventing tissue ingrowths and undesired adhesion of the
polymeric matrix to growing tissue.
[0035] Preferably, chitosan, more preferably native chitosan, is
the main component of the tissue dressing material. In the context
of the present invention, the expression "main component" with
regard to the tissue dressing material and a type of chitosan (such
as chitosan in general, deacetylated chitosan, native chitosan or
deacetylated native chitosan) means that the respective type of
chitosan makes up at least 50% by weight of the tissue dressing
material. Thus, if e.g. the tissue dressing material is provided as
a solid or gel-like film to be applied to the tissue, this film is
required to be made up of the respective type of chitosan by at
least 50% by weight. In the case of the liquid tissue dressing
material, the expression "main component" with regard to the
constituent(s) other than water in the aqueous mixture means that
at least 50% by weight of the combination of all constituents other
than water must be the respective type of chitosan. Also, as
discussed further below, the tissue dressing may comprise a first
layer, which layer is formed of the tissue dressing material, and
another layer formed of another material, this other layer acting
as a support. In such a case, according to the above definition, it
would be the first layer but not the support layer that is required
to be made up of the respective type of chitosan by at least 50% by
weight. Note that material which the tissue dressing material takes
up from a tissue, such as exudative fluid from a wound, is not
considered a component of the tissue dressing material.
[0036] In one embodiment of the invention, the tissue dressing
material is a solid or a gel preferably a hydrogel. Preferably, it
is present in the form of a film. The preferred film has a smooth
surface, preferably with an average roughness R.sub.a of 1 .mu.m
(micrometer) or less, more preferably 0.3 .mu.m or less, more
preferably 0.1 .mu.m or less. Advantageously, a smooth surface can
reduce the formation of mechanical anchoring to the tissue, thereby
further facilitating removal of the tissue dressing material.
Typically, the dried film is between 0.5 and 500 .mu.m thick,
preferably between 10 and 100 .mu.m. It has a surface area
sufficient to cover the tissue to be treated, such as a wound, and
preferably also some of the surrounding tissue. Preferably, at
least 70%, more preferably at least 90%, more preferably at least
95% by weight of the solid or gel-like tissue dressing material is
chitosan, preferably native chitosan.
[0037] The preferred solid or gel-like tissue dressing material for
being applied in contact with the tissue of the patient is at least
partly water-soluble. In other words, at the time it is provided
for being applied to the patent's tissue it can be dissolved at
least partly in water at neutral pH. The tissue dressing material
may for example be a polymer salt, e.g. the salt of a
polysaccharide such as a chitosan salt, e.g. the salt of native
chitosan or a chitosan derivative. It is an achievable advantage of
this embodiment of the invention that the tissue dressing material
adheres well to the tissue. Thereby, it can be avoided that the
tissue dressing material prematurely detaches from the tissue. This
embodiment of the invention advantageously exploits the fact that
chitosan salt is soluble in an aqueous solvent of neutral pH. Thus,
wet or pre-wetted tissue can liquefy the tissue dressing material's
surface, providing for a durable contact with the tissue. Preferred
salts are those derived from the dissolution of a polymer,
preferably chitosan such as native chitosan, in a inorganic acid,
such as hydrochloric acid, or an organic acid selected from the
group of monobasic or multibasic organic acids having 2 to 12
carbon atoms and a first pKa value between 1 and 5, such as acetic
acid, citric acid, lactic acid, malic acid, succinic acid, mandelic
acid, oxalic acid, tartaric acid, ascorbic acid, etc. In an
alternative embodiment of the invention the respective type of
polymer, is present in the form of the chitosan base.
[0038] A preferred solid tissue dressing material entirely consists
of chitosan, preferably native chitosan. Preferably, a polymer salt
such as a polysaccharide salt, preferably a chitosan salt,
preferably a salt of native chitosan makes up the main component of
the solid or gel-like tissue dressing material. More preferably, at
least 70%, more preferably at least 90%, more preferably at least
95% by weight of the solid or gel-like tissue dressing material is
a polymer salt such as a polysaccharide salt, preferably a chitosan
salt, preferably a salt of native chitosan. A preferred solid
tissue dressing material entirely consists of a polymer salt such
as a polysaccharide salt, preferably a salt of chitosan, preferably
a salt of native chitosan.
[0039] In a preferred embodiment, the tissue dressing material
comprises glycerol in addition to the polymer salt such as chitosan
salt, e.g. native chitosan salt. The glycerol content preferably
makes up least 10%, more preferably at least 15%, more preferably
at least 20% by weight of the solid tissue dressing material's
polymer salt content, more preferably chitosan salt content, by
weight. The glycerol preferably is present at a concentration of
more than 10%, more preferably more than 15%, more preferably more
than 20% by weight. The glycerol preferably is present at a
concentration of less than 60%, more preferably less than 45%, more
preferably less than 30% by weight.
[0040] In another preferred embodiment of the present invention,
the tissue dressing material for being applied in contact with the
tissue of the patient is liquid. In general, after application, the
liquid tissue dressing material will solidify, i.e. it will turn
into a solid or a gel, for example a hydro-gel. In some embodiments
of the invention, removal, preferably evaporation, of the solvent
that was present in the liquid tissue dressing when it was applied
causes or at least contributes to the solidification. Additionally
or alternatively, solidification may be caused or contributed to by
other factors such as chemical or physical cross-linking of
polymeric components of the tissue dressing material.
[0041] Preferably, the liquid tissue dressing material is an
aqueous mixture, e.g. a dispersion or a suspension, more preferably
a solution, i.e. it comprises water as the mixture medium or
solvent, respectively. Moreover, it may comprise a co-solvent, for
example an alcohol such as isopropanol. This can have the advantage
of faster evaporation of the solvent which in turn leads to faster
solidification.
[0042] The solute or more generally the constituent(s) of the
mixture that remain once the mixture medium is removed upon
solidification of the liquid tissue dressing material preferably
comprises, more preferably consists of a salt, more preferably a
polymer salt, for example a chitosan salt such as the salt of
native chitosan or the salt of a chitosan derivative. Preferred
salts are those derived from the dissolution of a polymer,
preferably chitosan such as native chitosan, in an inorganic acid,
such as hydrochloric acid, or an organic acid selected from the
group of monobasic or multibasic organic acids having 2 to 12
carbon atoms and a first pKa value between 1 and 5, such as acetic
acid, citric acid, lactic acid, malic acid, succinic acid, mandelic
acid, oxalic acid, tartaric acid, ascorbic acid, etc.
[0043] Preferably, chitosan, more preferably native chitosan, is
the main component other than water of the liquid tissue dressing
material. Preferably at least 70% by weight of the constituent(s)
of the mixture other than water are a polymer, preferably a
polysaccharide such as chitosan, preferably native chitosan. A
particularly preferred mixture essentially only consists of a
polymer, preferably a polysaccharide such as chitosan, preferably
native chitosan, and water. The preferred mixture is acidic. The
concentration of the polymer preferably is less than 15%, more
preferably less than 10%, more preferably less than 5%, more
preferably less than 2% by weight.
[0044] For application, the liquid tissue dressing material
preferably is sprayed onto the tissue and the mixture medium or
solvent subsequently is allowed to evaporate to form a solid or
gel-like film. Typically the film is between 0.1 and 50 .mu.m
thick, preferably between 1 and 20 .mu.m. It has a surface area
sufficient to cover the tissue to be treated such as a wound, and,
preferably, also some of the surrounding tissue. Accordingly, the
preferred kit according to the invention that comprises a liquid
tissue dressing material further comprises a spraying apparatus for
spraying a liquid tissue dressing material onto the tissue of the
patient. The preferred spraying apparatus comprises a container for
storing the liquid tissue dressing material. It may also comprise
pressurised gas for expelling the liquid tissue dressing material.
The tissue dressing material can be provided in two or more liquid
components that are mixed shortly before or during application of
the liquid tissue dressing material to the tissue. In this case,
the spraying apparatus may comprise several containers and/or the
kit may comprise several spraying apparatus each containing one of
the liquid components. Alternatively, the liquid tissue dressing
material may be brushed onto the tissue or applied by means of a
sponge, a spatula, a pipette or gauze. Accordingly, a preferred kit
contains a sponge, a brush, a spatula, a pipette or gauze for
applying the liquid tissue dressing material or at least a
constituent of the liquid dressing material to the tissue.
[0045] After the liquid tissue dressing material or the solid or
gel-like water soluble tissue dressing material is brought into
contact with the tissue, and in the case of a liquid tissue
dressing during or after solidification, it preferably is allowed
to transform into a water-insoluble form, e.g. a chitosan base.
This may be achieved through exposure of the tissue dressing
material to air or by applying a transformation medium, e.g. an
aqueous alkaline solution. Thereby, advantageously, it can be
achieved that after transformation the tissue dressing material
remains in place under normal condition, e.g. when the tissue is
cleaned under tap water (neutral pH) or when soap (alkaline) is
applied, but detaches only upon application of the detachment
solvent. Subsequently, when the tissue dressing material shall be
removed, e.g. to be replaced or at the end of a therapy, the
detachment solvent is applied to facilitate detachment of the
tissue dressing material from the tissue. The preferred kit in
addition to the tissue dressing material and the detachment solvent
also comprises the transformation medium.
[0046] The preferred tissue dressing material, in particular the
preferred liquid tissue dressing material, is free of organic
solvents such as alcohols, esters, alkanes, halogenated solvents,
amines, amides. It may, however, frequently contain an organic
acid; organic acids are not considered organic solvents in the
context of the present invention.
[0047] A preferred kit according to the invention comprises both a
solid or gel-like and a liquid tissue dressing material. In a
preferred method according to the invention, first a liquid and
subsequently a solid or gel-like tissue dressing material is
applied to the patient's tissue. Preferably in this method, the
solid or gel-like tissue dressing material is applied before the
liquid tissue dressing material has solidified. The inventors have
found that the liquid tissue dressing material can facilitate
attachment of the solid or gel-like tissue dressing material to the
target tissue. This is particularly true for water-soluble solid or
gel-like tissue dressing materials and as compared to an
alternative method in which the water-soluble solid or gel-like
tissue dressing material is wetted with water before attachment.
This is because the latter method has been found to frequently lead
to an undesirable deformation of the solid or gel-like tissue
dressing material, which deformation can be avoided by the
application of the liquid tissue dressing material for attachment
of the solid or gel-like tissue dressing material. Preferably, in
this kit and method, the liquid tissue dressing material is one of
the preferred liquid tissue dressing materials described herein.
Similarly, in this kit and method, the solid or gel-like tissue
dressing material is one of the preferred solid or gel-like tissue
dressing materials described herein. Preferably, the liquid tissue
dressing material and/or the solid or gel-like tissue dressing
material and the detachment solvent are provided in separate
containers.
[0048] The tissue dressing material, solid, gel-like or liquid, in
some embodiments of the invention is a mixture or a compound
material comprising several constituents, in the case of a liquid
tissue dressing material preferably several constituents other than
water. Preferably, the tissue dressing material comprises at least
one constituent other than water and chitosan. In one embodiment,
the tissue dressing material comprises glycerol. This
advantageously can accelerate the transformation of the tissue
dressing material from a water-soluble to a form that is only
soluble in an acidic medium.
[0049] In a preferred embodiment, the tissue dressing material
comprises at least one pharmaceutically active and/or bioactive
constituent other than chitosan. Suitable bioactive constituents
may e.g. be proteins, peptides or derivatives thereof, nucleic
acids or derivatives thereof, low molecular weight compounds active
as drugs, such as antibiotics or anti-inflammatory drugs, or
agonists or antagonists of the innate immune system, or stimulating
or differentiating growth factors for stimulating or
differentiating growth of at least one sub-type of cells, or resins
with affinity to certain components to be extracted from a wound
surface, or dissolved or dispersed compounds or polymers with
decorative functions such as light absorbing, fluorescent or
phosphorescent or light reflecting particles. Alternatively, or in
addition, the tissue dressing material may comprise biological
cells.
[0050] In one preferred embodiment of the invention, the tissue
dressing material (solid, gel-like and/or liquid) comprises a
pH-sensitive dye for visually indicating the pH at the site of the
tissue where the tissue dressing material is applied. The pH can be
used as a proxy for indicating the condition of the tissue covered
by the wound dressing material. For example, it is known that the
pH in a wound can indicate the wound's present phase within the
wound healing process.
[0051] In a preferred embodiment of the invention, the tissue
dressing material has a pH of below 6.3, preferably below 6,
particularly preferably around 5 to 5.5. The preferred pH is above
4.0, more preferably above 4.5. It is an achievable advantage of
this embodiment of the invention, that the pH is close to that of
the surface of healthy skin, thereby avoiding irritation or damage
of the tissue to which the tissue dressing material is attached.
This embodiment of the invention preferably applies to external
applications of the tissue dressing material.
[0052] In a preferred embodiment of the invention, the tissue
dressing material has a pH below 8.5, preferably below 8,
particularly preferably around 7 to 7.5. The preferred pH is above
6.0, more preferably above 6.5. It is an achievable advantage of
this embodiment of the invention, that the pH is close to that of
healthy tissue, thereby avoiding irritation or damage of the tissue
to which the tissue dressing material is attached. This embodiment
of the invention preferably applies to internal applications of the
tissue dressing material.
[0053] The preferred tissue dressing material has a water uptake
capacity of less than 1500% by weight, more preferably less than
100%, more preferably less than 80%. Thereby it is advantageously
achievable that a degree of humidity that is favourable for wound
healing can be maintained under tissue dressing material as applied
to a wound site. Preferably, the tissue dressing material in a
solid or gel-like form has a water-uptake capacity of more than
25%, more preferably more than 50%. Advantageously this embodiment
of the invention is suitable for absorbing exudative fluids and
toxants. In a particularly preferred embodiment of the invention,
the water-uptake capacity of the tissue dressing material is
between 65 and 75%.
[0054] In a preferred embodiment of the invention, the tissue
dressing material is transparent, in particular in the solid,
gel-like or solidified form. In a kit that comprises both a solid
or gel-like and a liquid tissue dressing material, preferably both
the solid or gel-like tissue dressing material and the liquid
tissue dressing material after solidification are transparent.
Advantageously, this can make it easier for a physician to inspect
the tissue treated with the tissue dressing material, in particular
if it is a wound tissue. In some embodiments, the material is a
transparent solid film. In others it is a mixture such as a
dispersion, a suspension or a solution that forms a transparent
film when applied to the tissue. Also, in the case that the tissue
dressing material comprises a pH-sensitive dye the colour of the
dye can be judged due to the transparency of the tissue dressing
material.
[0055] In a preferred embodiment of the invention, the tissue
dressing material is part of a tissue dressing that comprises a
first layer, which layer is formed of the tissue dressing material,
and at least another layer formed of another material, this other
layer acting as a support. In particular, the support
advantageously can help preventing premature detachment of the
tissue dressing material from the tissue. The support preferably is
located at the side of the layer of the tissue dressing material
opposite to the side that is in contact with the tissue.
Preferably, the support is adjacent to the tissue dressing
material. The support according to the invention is particularly
advantageous if the respective type of chitosan, preferably
deacetylated native chitosan, is provided in the tissue dressing
material in the form of the chitosan base, as the chitosan base in
general adheres less well to tissue than a chitosan salt containing
tissue dressing material. The support may for example be a woven
fabric, foam or a perforated film. The support may for example be
of natural materials such as cotton or a natural or synthetic
polymer. Suitable polymers include biodegradable polymers, such as
polyesters, polyorthoesters, polycarbonates, polyanhydrides,
polyurethanes, polyphosphazenes, polyphosphoesters,
polysaccharides, polypeptides, as well as derivatives, copolymers,
and blends based on these polymers. Suitable polymers also include
biodissolvable polymers, such as polyvinyl alcohol, polyvinyl
acetate, poly-N-vinyl pyrrolidone, polyethylene glycol,
polypropylene glycol, polysaccharides, polypeptides, as well as
derivatives, copolymers, and blends based on these polymers.
Furthermore, the support may consist of a
non-biodegradable/non-biodissolvable polymer, such as silicones,
polyurethanes, polyethylene terephthalate, polytetrafluorethylene,
polysulfones, polyethersulfones, polyether ether ketones,
polycarbonates, polymethacrylates, polysaccharides, polypeptides,
as well as derivatives, copolymers, and blends based on these
polymers.
[0056] A preferred tissue dressing according to the invention
comprises a first layer, which layer is formed of the tissue
dressing material, and another layer formed of another material,
this other layer acting as an at least partial moisture barrier. In
other words the other layer can prevent or at least delay the
evaporation of water in the tissue dressing material during
treatment of the tissue with the tissue dressing material according
to the invention. This can be of particular advantage when the
tissue dressing is applied to dry wounds. The other layer
preferably is located at the side of the layer of the tissue
dressing material opposite to the side that is in contact with the
tissue. Preferably, the other layer is adjacent to the tissue
dressing material. The invention also encompasses tissue dressings
that have both a support layer and another layer that acts as an at
least partial moisture barrier. Of course both functions, that of a
support and that of an at least partial moisture barrier, can also
be fulfilled by a single other layer. The other layer may for
example be of silicone or another polymer or polymer composition
from the groups of polymers listed above. Typically the other layer
is between 10 and 1000 .mu.m thick, preferably between 50 and 500
.mu.m. In some embodiments of the invention, the other layer is
perforated. The holes of the perforation typically are between 10
and 1500 .mu.m in diameter, preferably between 50 and 1000 .mu.m.
In an alternative embodiment of the invention, instead of the
moisture barrier a layer is provided that can take up fluid, e.g.
wound exudate. A suitable material may for example be
polysaccharide-based hydrogels or hydrocolloids including cellulose
derivatives, or polyurethane foams. This can be of particular
advantage when the tissue dressing is applied to wet wounds.
[0057] In a preferred embodiment of the invention, the tissue
dressing material, preferably the entire tissue dressing, is
provided in a container that can prevent transformation of the
tissue dressing material from its liquid or water-soluble state to
its water-insoluble state as long as it is in the container and the
kit's shelve life has not yet expired. Preferably, the container is
vapour proof, more preferably it is essentially airtight.
[0058] Moreover, in some embodiments of the invention, the tissue
dressing material on its side which is intended to be applied in
contact with the patient's tissue is covered with a strippable
cover sheet. The cover sheet is vapour proof, more preferably
air-impermeable. This can contribute to preventing premature
transformation of the tissue dressing material from its liquid or
water-soluble state to its water-insoluble state before it is
applied to the patient's tissue.
[0059] The tissue dressing material according to the invention in
particular can exhibit one or a combination of the following
advantageous properties: transparency; neutrality of odor; adhesion
to the tissue to which it is applied; permeability for gas, in
particular oxygen; locally confined antibiotic properties;
hemostatic properties; regulation of humidity; and dissolvability
at moderate pH. It may be non-cytotoxic, non-cytostatic and
non-inflammatory. The material may inhibit or, alternatively,
promote the growth cells of the tissue treated. It may also act as
a barrier to protect from bacterial infection from inside and
outside the tissue treated and as a mechanical protection. In
particular it may protect and cover superficial wounds, lesions,
abrasions that are at risk of infection, and burns. The tissue
dressing material and the tissue dressing according to the
invention can provide protection in cases where conventional wound
dressings prove ineffective or are at least less effective, such as
in the treatment of ulcerous tissue, wounds caused by viruses which
tend to become ulcerous, mucosal tissue, the genital area, and body
cavities. The material can be suitable for sterilization; it may be
coloured with a dye;
[0060] The wound dressing material, in particular the liquid wound
dressing material according to the invention may be free of aseptic
agents, antioxidants and surfactants, thereby reducing the risk of
toxic or allergic reactions. It may be pH-neutral to the skin,
exhibiting a pH between 4 and 6, preferably around 5.5. It may be
suitable for inhalation or for internal application by injecting or
swallowing the material.
[0061] In particular, the liquid tissue dressing material according
to the invention can exhibit one or a combination of the following
advantageous properties: it may be water-based, free of organic
solvents; have film-forming properties; be removable with an
aqueous solution. It can be applied to areas of the human or animal
body that are difficult to reach or where it is difficult to apply
a solid material such as a film.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] The invention is illustrated in greater detail with the aid
of the following figures:
[0063] FIG. 1 shows an .sup.1H NMR spectrum of native chitosan as
purchased;
[0064] FIG. 2 shows an .sup.1H NMR spectrum of native chitosan
essentially deacetylated after further hydrolysis steps;
[0065] FIG. 3 illustrates the cell viability of keratinocytes on
chitosan materials of various degrees of acetylation, relative to
tissue culture polystyrene controls (PS=100%);
[0066] FIG. 4 illustrates the controlled dissolution of tissue
dressing material according to the invention by applying a gauze
soaked with acetate buffered solution;
[0067] FIG. 5 shows a tissue dressing comprising a tissue dressing
material according to the invention before (5a), during (5b), and
after (5c) the application of the detachment solvent;
[0068] FIG. 6 schematically illustrates a wound to which a liquid
tissue dressing material according to the inventions has been
applied;
[0069] FIG. 7 schematically illustrates a wound to which a solid
tissue dressing material according to the inventions has been
applied;
[0070] FIG. 8 schematically illustrates a wound to which a
non-perforated wound dressing according to the invention has been
applied; and
[0071] FIG. 9 schematically illustrates a wound to which a
perforated wound dressing according to the invention has been
applied.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
1. .sup.1H NMR Spectroscopy
[0072] The chitosan used as a starting material in the examples
below was obtained in the form of fine flakes from Cognis
(Germany). The degree of acetylation (DA) was determined by .sup.1H
NMR spectroscopy. FIG. 1 shows an .sup.1H NMR spectrum obtained
from this commercially available chitosan. FIG. 2 shows a
corresponding .sup.1H NMR spectrum obtained from chitosan
deacetylated after further hydrolysis steps applied to the
commercial product as described further below. In both cases,
chitosan was analyzed in a mixture of 0.25% DCl in D.sub.2O at a
chitosan concentration of approximately 0.5% (w/v). The spectra
were recorded using a Bruker AC200 spectrometer. NMR chemical
shifts (.delta., in ppm) were referenced to the signal of HDO
(.delta.=4.8 ppm). The DA, calculated by comparing the integrated
area under the peaks associated with H2-H6 of the D-glucosamine
subunit with that of the methyl group, was determined as 14.5% for
the native chitosan as purchased, and 1.5% for the deacetylated
native chitosan.
2. Synthesis of Low-DA Chitosan
[0073] For further hydrolysis, 50 g (grams) of the chitosan flakes
as obtained from the supplier Cognis were placed in a glass
container, and 500 g of a 45% aqueous sodium hydroxide solution
were added. The glass container was well shaken to mix the
components, and placed in an oven for 2 hours at 100.degree. C. It
was then removed from the oven, and 500 mL (milliliters) of
distilled water were added. The mixture was filtered through a
glass frit. Then, the chitosan was washed with distilled water
until the pH of the filtrate reached 6.5, and dried at 100.degree.
C. for 4 h (hours). This hydrolysis treatment was then repeated,
resulting in 42 g of deacetylated native chitosan having a DA of
1.5% as determined by .sup.1H NMR spectroscopy.
3. Cell Viability on Low-DA Chitosan
[0074] Human HaCaT keratinocytes were cultured in serum-free medium
(Gibco) supplemented with 0.2 ng/mL (nanograms per milliliter) rEGF
and 25 .mu.g/mL (micrograms per milliliter) bovine pituitary
extract. The calcium concentration was adjusted to 0.02 mM and the
pH to 7.2-7.4. Cells were seeded at a density of 1.times.10.sup.6
cells per 20 mL medium and incubated at 37.degree. C. in air
containing 10% CO.sub.2. Cells were passaged once per week, and
passages 20-25 were used for analysis.
[0075] Chitosan films having DAs of 1.5, 4.0, and 14.5%,
respectively, were placed in 24-well cell culture plates, and human
HaCaT keratinocytes were seeded at a density of 5.times.10.sup.4
cells per cm.sup.2 and cultured for 2 days. Cell viability was
determined using the MTS assay (Promega). After 4 h of MTS
incubation with the cells, the light absorbance at 490 nm was
measured by an ELISA plate reader and subtracted from that of the
controls (without cells) to yield the corrected absorbance. Five
samples of each DA were studied. FIG. 3a shows the relative light
absorbencies .alpha. at 490 nm (PS=100%) for the three samples and
a control using polystyrene (PS).
4. Preparation of a Solution of the Tissue Dressing Material
(Material A)
[0076] 7.5 g of the thus obtained native chitosan having a DA of
1.5% were dissolved in 500 mL of a 0.5% aqueous acetic acid by
gently shaking for 24 h. A portion of the solution was filtered
first through a glass fiber filter (pore size approximately 1
.mu.m), and then through a 0.22 .mu.m filter for sterilization,
resulting in a solution of a tissue dressing material essentially
consisting entirely of deacetylated native chitosan. Below, the
material is referred to as tissue dressing material A.
5. Preparation of a First Example of a Solid Film-Type Tissue
Dressing Material (Material B)
[0077] Two portions of 144 mL each of the non-filtered solution of
deacetylated native chitosan prepared above were poured into two
square-shaped moulds, 24.times.24 cm.sup.2 (square centimeters) in
size, and left in a dust-free environment for drying at room
temperature. The resulting film was removed from the first mould,
and sterilized using a 10 kGy (kilogray) electron beam. An
approximately 80 .mu.m thick transparent film of tissue dressing
material essentially consisting entirely of deacetylated native
chitosan acetate salt was obtained. Below, the material is referred
to as tissue dressing material B.
6. Preparation of a Second Example of a Solid Film-Type Tissue
Dressing Material (Material C)
[0078] The dried film from the second mould was placed for 2 hours
in a bath containing a solution of 1.5% ammonia in methanol/water
90/10 (v/v). The film was then removed from the bath and dried by
storage at room temperature. The film was sterilized using a 10 kGy
electron beam. An approximately 80 .mu.m thick transparent film of
tissue dressing material essentially consisting entirely of
deacetylated native chitosan base was obtained. Below, the material
is referred to as tissue dressing material C.
7. Preparation of a Third Example of a Solid Film-Type Tissue
Dressing Material (Material D1) 144 mL of the filtered solution of
deacetylated chitosan prepared as above was poured into a
square-shaped mould, 24.times.24 cm.sup.2 in size, and left in a
dust-free environment for drying at room temperature. After 3 days
of storage, the resulting film was removed from the mould,
transferred in a plastic bag that was then tightly sealed, and
sterilized using a 25 kGy (kilogray) electron beam. An
approximately 80 .mu.m thick transparent film of tissue dressing
material essentially consisting entirely of deacetylated chitosan
acetate salt was obtained. Below, the material is referred to as
tissue dressing material D1.
8. Preparation of a Fourth Example of a Solid Film-Type Tissue
Dressing Material (Material D2)
[0079] In a slightly modified procedure, 4% (w/w) glycerol was
added to the filtered solution of deacetylated chitosan before
pouring it into the square-shaped mould. Subsequent treatment as
described above for tissue dressing material D1 resulted in a
transparent film of tissue dressing material essentially consisting
entirely of a mixture of deacetylated chitosan acetate salt and
glycerol. Below, the material is referred to as tissue dressing
material D2.
9. Preparation of a Fifth Example of a Solid Film-Type Tissue
Dressing Material (Material D3)
[0080] In a further modified procedure, the glycerol containing
solution of deacetylated chitosan was poured into a square-shaped
mould which was covered with a two-layered film consisting of
polyurethane/polyethylene (Platilon U073 PE, Epurex,
Bomlitz/Germany), with the polyurethane side up and the
polyethylene side fixed to the bottom of the mould. Subsequent
treatment as described above for tissue dressing material D1
resulted in a transparent film of tissue dressing material
essentially consisting entirely of a mixture of deacetylated
chitosan acetate salt and glycerol which was attached to the
polyurethane/polyethylene support film. Below, the material is
referred to as tissue dressing material D3. Upon use, the
polyethylene layer is removed. The remaining polyurethane layer is
gas-permeable.
10. Preparation of a Sixth Example of a Solid Film-Type Chitosan
(Chitosan D4)
[0081] In a slightly modified procedure to the preparation of
chitosan film D2, 1% (w/w) glycerol was added to the filtered
solution before pouring it into the square-shaped mould. Subsequent
treatment as described above for chitosan D1 resulted in a
transparent film essentially consisting entirely of a mixture of
deacetylated chitosan acetate salt and glycerol. Below, the
material is referred to chitosan D4.
11. Preparation of Two Examples of a Solid Film-Type Tissue
Dressing Material with Higher DA (Materials E1 and F1)
[0082] Two further examples of tissue dressing materials were
produced by the procedure leading to material D1 with the only
modification that in one case the hydrolysis step was shortened,
leading to a DA of 4% (material E1), and in the other case the
hydrolysis step was entirely omitted, leading to a DA of 16%
(material F1).
12. Water Uptake of Tissue Dressing Material C
[0083] Tissue dressing material C, produced as described in the
above example, was weighted, and then placed in distilled water for
15 min. The weight of the wet film was compared to the weight of
the dry film, and the water uptake was determined to be 72% by
weight.
13. Water Uptake of Chitosan D4
[0084] Chitosan D4, produced as described in the above example, was
weighted, and then placed in distilled water for 60 min. The weight
of the wet film was compared to the weight of the dry film, and the
water uptake was determined to be 1217% by weight 7 days after film
preparation, and 475% by weight 14 days after film preparation.
14. Application of Tissue Dressing Materials A, B and C
[0085] In Table 1 below, the outcomes of treatments of patients
with tissue dressing materials A, B and C are detailed. Material A
was sprayed directly onto a wound and then left uncovered to allow
the solvent to readily evaporate into the air. Materials B and C
were applied as small cuts of the film-like material in direct
contact with the wound. In the case of material B, the skin was
pre-wetted before the application of the material. In all examples,
the material was left uncovered after application.
TABLE-US-00001 TABLE 1 Type of chitosan Application of tissue
dressing Patient Wound the dressing Outcome A female, 49 yrs cut of
3.5 cm application of skin incision (years) length and 5 mm appr. 2
mL of completely closed depth (finger), tissue dressing A after 24
h moderate (one treatment) bleeding B male, 42 yrs cut of 1.5 cm
application of skin incision length and 3 mm tissue dressing B
completely closed depth (hand), (size 2 .times. 0.5 cm.sup.2) after
4 h weak bleeding on pre-wetted skin (one treatment) C male, 57 yrs
praeputial application of Wound and ulcus inflammation tissue
dressing C completely healed (size 1.5 .times. 1.5 cm.sup.2) after
48 h (treatment repeated after 24 h)
15. Dissolution of Chitosan Tissue Dressing
[0086] Controlled dissolution of tissue dressing materials B and C
was tested in dissolution experiments using distilled water, 0.9%
aqueous sodium chloride solution, and 0.5% acetic acid/acetate
buffered solution, respectively. The pH of the solutions was
adjusted to the values indicated in Table 2 using appropriate
amounts of 1 N hydrochloric acid or sodium hydroxide solutions.
Materials B and C were cut into rectangular samples having dry
weights between 5 and 10 mg each. A gauze soaked with a 100-fold
per volume excess of the respective solution to the dry weight of
the film was applied to each sample film and the time for complete
film dissolution was recorded.
TABLE-US-00002 TABLE 2 Material B Material C Material C pH of the
Material B (0.9% aqueous (0.9% aqueous (0.5% acetic dissolution
(distilled sodium sodium acid/acetate mixture water) chloride)
chloride) buffer) 4.0 n.a. n.a. n.d. 0.5 h 4.5 n.a. n.a. n.d. 0.5 h
5.0 n.a. n.a. n.d. 2 h 5.5 0.1 h 0.5 h n.d. 4 h n.a. = not analyzed
n.d. = no dissolution observed after 24 h
[0087] The controlled dissolution experiment with tissue dressing
material C and a mixture of 0.5% acetic acid/sodium acetate (right
column in Table 2), is illustrated in FIG. 4. The material has been
stained by storage in 0.01% aqueous indigocarmine solution for 1
hour for better visualization. Complete dissolution was observed
after 30 minutes at pH 4.0 and 4.5, after 2 hours at pH 5.0, and
after 4 hours at pH 5.5, respectively.
16. In Situ Conversion of Water-Soluble Tissue Dressing Material
into Water-Insoluble Tissue Dressing Material
[0088] Samples of tissue dressing materials A, D1, D2, D3, and D4,
respectively, were left unsealed on air at room temperature and a
humidity of 20-40%. Under these conditions, tissue dressing A was
drying to a solid film within several hours. Complete dissolution
in distilled water was analyzed at days 3, 7, and 14. Results are
summarized in Table 3.
TABLE-US-00003 TABLE 3 Tissue Dressing Day 3 Day 7 Day 14 A soluble
insoluble insoluble D1 soluble soluble insoluble D2 soluble
insoluble insoluble D3 soluble insoluble insoluble D4 soluble
insoluble insoluble
[0089] Similarly, conversion of the water-soluble into the
water-insoluble form of the wound dressings A, D1, D2, D3, and D4,
respectively, was observed after application of the wound dressing
on human skin. In the case of D3, the wound dressing was applied to
the skin with its chitosan side. Conversion of the water-soluble
into the water-insoluble form of the wound dressings A, D1, D2, D3,
and D4, respectively, was also observed after alkaline treatment or
storage in an alkaline atmosphere.
17. Dissolution of Tissue Dressing Material with Detachment
Solvent
[0090] Tissue dressing materials D1, E1 and F1 were dissolved by
storage in a 2% acetic acid/acetate buffered solution. The pH of
the solutions was adjusted to the values indicated in Table 4 using
appropriate amounts of 10% sodium hydroxide solutions. Films D1, E1
and F1 made from chitosans with different degrees of acetylation
(DA) were left on air for 14 days for conversion into the
water-insoluble form, cut into rectangular samples of 1.times.1
cm.sup.2 size and stored in approximately 10 mL of the respective
solution, and the time for complete film dissolution was
recorded.
TABLE-US-00004 TABLE 4 DA = 16% DA = 4% DA = 1.5% pH Time for
complete dissolution (min) 4.0 5 10 1 4.5 15 15 2 5.0 30 15 15 5.5
60 60 30 6.0 60 overnight overnight
[0091] In another dissolution experiment, a wound dressing material
film C (3.times.1 cm.sup.2) was fixed on the inside of a commercial
perforated band-aid (5.times.2 cm) which was then fixed on a Petri
dish. An acetic acid/acetate buffered solution (pH 5.5) was added
dropwise through the perforations of the band-aid causing the wound
dressing material film to dissolve. The side of the tissue dressing
comprising the tissue dressing material film is shown in FIG. 5a
before and in FIG. 5c after application of the solution. The
application of the solution to the band-aid side of the wound
dressing is shown in FIG. 5b.
[0092] In FIG. 6, schematically a tissue 1 comprising a wound 2 is
shown. For better illustration, FIGS. 5 to 8 are not drawn to
scale. The liquid tissue dressing material according to the
invention has been applied to the tissue 2 and the constituent
water has been allowed to evaporate, leaving behind a film 3 that
dresses the tissue 2 including the wound 3. In general, the film 3
is about 10 to 20 .mu.m thick. Advantageously, the film 3 tightly
snuggles to the tissue surface 4, including the wound surface
5.
[0093] FIG. 7 schematically shows a tissue dressing material in the
form of a solid film 6 that is applied to a tissue 1, comprising a
wound 2. The solid film is about 80 .mu.m thick. Cavities 7, 8
between the tissue 1 and the tissue dressing material 6 may be
filled with water or exudative fluid.
[0094] In FIG. 8, a tissue dressing 9 comprising a tissue dressing
material 6 of FIG. 7 as a first layer and a silicon film 10 as a
second layer is applied to a tissue 1, comprising a wound 2. The
silicon film 10 is about 50 .mu.m tick. Again, cavities 7, 8
between the tissue 1 and the tissue dressing material 6 may be
filled with water or exudative fluid. Finally, FIG. 9 shows a
tissue dressing 11 applied to a tissue 1 comprising a wound 2, the
tissue dressing 11 differing from that 9 of FIG. 8 in that the
silicon film 10 is perforated to allow an exchange of air between
the tissue 1 and the surrounding though the wound dressing material
6. The perforations have a diameter if between 50 and 100
.mu.m.
18. Preparation of an Example of a Liquid-Type Tissue Dressing
Material
[0095] 1000 ml of tissue dressing material A was diluted by
addition of 1000 mL of sterile distilled water, resulting in a
liquid-type tissue dressing material consisting of 0.75% chitosan,
0.25% acetic acid and 99% water. Using a mechanical dispenser, 20
mL of the solution was each filled into glass bottles which were
then equipped with a pump head, resulting in a gas-free
chitosan-based wound spray. Similarly, the liquid-type dressing
material was placed into a spraying apparatus containing a
pressurized gas.
[0096] The features described in the above description, claims and
figures can be relevant to the invention in any combination. The
reference numerals in the claims have merely been introduced to
facilitate reading of the claims and are by no means meant to be
limiting.
* * * * *