U.S. patent application number 12/450954 was filed with the patent office on 2010-09-23 for multi component non-woven.
Invention is credited to Hanne Everland, Jens Hassingboe, Jacob Vange, Rong Weimin.
Application Number | 20100239560 12/450954 |
Document ID | / |
Family ID | 38686869 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100239560 |
Kind Code |
A1 |
Hassingboe; Jens ; et
al. |
September 23, 2010 |
MULTI COMPONENT NON-WOVEN
Abstract
The formation of a non-woven, free from organic solvent, formed
through parallel formation of fibers on a collection device is
disclosed. As the individual fibers are dry prior to contact with
other fibers, the different contents of the various fiber types do
not interact. However, when wetted, the fibers will start to be
dissolved, or swell, and the different contents will be released
and then interact. For the example of thrombin and fibrinogen, the
interaction will initiate the formation of a fibrin coagulum by the
cleavage of fibrinogen through the action of thrombin to form
fibrin monomers that spontaneously polymerize to form a three
dimensional network of fibrin.
Inventors: |
Hassingboe; Jens; (Graested,
DK) ; Vange; Jacob; (Helsingoer, DK) ;
Everland; Hanne; (Bagsvaerd, DK) ; Weimin; Rong;
(Bagsvaerd, DK) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W., SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
38686869 |
Appl. No.: |
12/450954 |
Filed: |
April 17, 2008 |
PCT Filed: |
April 17, 2008 |
PCT NO: |
PCT/DK2008/050090 |
371 Date: |
May 17, 2010 |
Current U.S.
Class: |
424/94.64 ;
514/1.1; 514/21.2 |
Current CPC
Class: |
A61L 15/32 20130101;
A61F 13/00987 20130101 |
Class at
Publication: |
424/94.64 ;
514/1.1; 514/21.2 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61K 38/00 20060101 A61K038/00; A61K 38/12 20060101
A61K038/12 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 20, 2007 |
DK |
PA 2007 00585 |
Claims
1. Multi component non-woven of fibers of a natural, water
dissolvable protein structure, comprising at least two fiber types
of different compositions.
2. Multi component non-woven according to claim 1, wherein the
different compositions constitute different contents of active
substance.
3. Multi component non-woven according to claim 1, wherein the
active substance is distributed homogeniously throughout the
fiber.
4. Multi component non-woven according to claim 1, wherein natural
protein structure is biodegradable.
5. Multi component non-woven according to claim 1, wherein the
natural protein structure is selected from the group consisting of
keratin, collagen, and gelatin.
6. Multi component non-woven according to claim 1, wherein
particles are suspended in at least one of the fiber
compositions.
7. Multi component non-woven according to claim 1, wherein the
particles have a mean diameter wider than the mean diameter of the
fibers.
8. Multi component non-woven according to claim 1, wherein the
particles are ECM particles.
9. Multi component non-woven according to claim 1, wherein at least
one of the fiber compositions comprises thrombin and at least one
other fiber composition comprises fibrinogen.
10. Multi component non-woven according to claim 1, wherein the
non-woven is cross-linked.
11. Multi component non-woven according to claim 1, wherein at
least one of the fiber compositions further comprises a
polycarboxylic acid.
12. Multi component non-woven according to claim 1, wherein at
least one of the fiber compositions has a larger diameter than at
least one other.
Description
BACKGROUND
[0001] In many instances it is desired to provide a product with
two different, but mixed components. One example is for control of
heamostasis. Here it is desired to provide a product with both
thrombin and fibrinogen to be released in in situ. However, care
has to be taken to ensure the active components do not interact
prior to placement in situ.
[0002] For this type of products any residual organic solvents in
the product can be harmful to the wound site, and should be
avoided.
[0003] WO99/56798 discloses a product of polysaccharides as
polyanionic polysaccharides, alginic acid, chitin, chitosan, and
dextran; synthetic materials as polyglycolide, polylactide,
polycaprolactone, and fibrin. The product is produced by admixing
of the components under high shear conditions to evenly disperse
the materials in an organic solvent.
SUMMARY
[0004] The present invention discloses the formation of a
non-woven, free from organic solvent, formed through parallel
formation of fibers on a collection device.
DETAILED DISCLOSURE
[0005] One aspect of the present invention relates to a multi
component non-woven of fibers of a natural protein structure,
comprising at least two fiber types of different compositions.
[0006] One aspect of the invention relates to a multi component
non-woven as described wherein at least one of the fiber types
comprises thrombin and at least one other fiber type comprises
fibrinogen.
[0007] In one embodiment of the invention, one of the fibre types
comprises an analgesic. For a wound care device for treatment of
pain, this is particularly advantageous. The analgesic incorporated
into the fibers of the present invention is released over time
locally to the wound. Preferably, the release of the analgesic is
so low that no systemic effect is seen. Thus, the concentration of
analgesic in the device of the invention may be so low that little
or no effective systemic plasma concentration can be found. This
will reduce or even eliminate the possible systemic side effects of
the analgesic, and at the same time provide the patient with
maximum safety, as oral doses or topical doses on intact skin can
be taken at the same time. Thus, the device renders it possible to
ingest additional medication, if needed, orally or topically of the
same type as in the wound care device, without the risk of
overdosing. Furthermore, side effects are lowered and compliance
will be better as well as the HQoL.
[0008] For different analgesics, the plasma concentration for
systemic effect in the lowest range is reported to be as follows
given as examples: Acetylsalicylic acid: 270 .mu.g/ml; Ketoprofen:
3 .mu.g/ml; Ibuprofen: 10 .mu.g/ml; Piroxicam: 1 .mu.g/ml. Thus, a
wound care device for treatment of pain in a wound releasing
analgesics locally to a wound site may be designed in such a way
that the plasma concentration is under the lowest range for
systemic effect in the body.
[0009] This is also true for other anti-inflammatory pain reliving
compositions being suitable for incorporation into the multi
component non-woven (the device) of the invention.
[0010] Prostaglandins, leukotrienes, and thromboxanes are key
inflammatory mediators produced from arachidonic acid. Inhibition
of the synthesis of these mediators is the target of the most
highly prevalent class of anti-inflammatory drugs, the NSAIDs.
Inflammatory mediators will stimulate pain nociceptors and as a
result pain is produced.
[0011] Inflammatory pain is believed to be important for the
actually feeling of chronic or persistent wound pain. It is
believed that tissue injury as e.g. seen in chronic wounds triggers
the release of multiple inflammatory mediators that themselves,
alter nociceptor function. The level of inflammation is therefore
elevated and may be lowered by addition of anti-inflammatory drugs
locally to the wound that would lead to pain relief.
[0012] Preferably the pain relieving composition comprises an
anti-inflammatory painkilling agent that blocks the production of
inflammatory mediators produced from arachidonic acid.
[0013] NSAIDs (non-steroid anti-inflammatory drugs) generally have
analgesics and antipyretic properties along with their
anti-inflammatory capabilities. Anti-inflammatory pain killing
agents interact with enzyme targets such as
cyclooxygenase-inhibiting NSAIDs. The enzymes PGHS (prostaglandin H
synthease), commonly know as COX (cyclooxygenase), is responsible
for processing arachidonic acid into inflammatory mediators. COX
comes from two isoforms COX 1 and COX 2. COX 1 is produced in a
more or less constant level at all times and is involved in forming
the prostaglandins that perform several important functions,
including protection of the gastric mucosa and support of renal
function. Consequently, inhibitors of COX 1 may interfere with the
gastric mucosa and renal function. COX 2, which is inducible, is
expressed after tissue injury and promotes inflammation. Thus,
selective inhibition of COX-2, with sparing of COX 1 activity,
should be expected to block inflammation without gastric and renal
side effects upon oral administration. However, use of COX 1
locally in an open wound setting will not produce any systemic side
effects. Classical NSAIDs acts on both COX 1 and COX 2 whereas
newer drugs work selectively on COX 2.
[0014] Thus, in one embodiment of the invention the analgesic may
be capable of inhibiting mediators responsible for processing
arachidonic acid into inflammatory mediators.
[0015] In preferred embodiment of the invention the analgesic may
be capable of inhibiting COX 1 and COX 2.
[0016] In one embodiment of the invention the analgesic may be
capable of specifically inhibiting COX 2. The analgesic may
comprise one or more compounds chosen from the group of
anti-inflammatory compositions such as Phenylpropionic acids,
Phenelacetic acids, Indoleacetic acids, Pyrroleacetic acids,
N-Phenylacetic acids, Salicylates, Enolic acids, Phenols, Non-acids
or Coxibs.
[0017] Examples of such compounds for the analgesic may be:
Propionic acid derivatives such as Naproxen, Ibuprofen, Ketoprofen,
Fenoprofen, Flurbiprofen Dexibuprofen or Tiaprofenic acid, Acetic
acid derivatives such as Diclofenac, Alclofenac, Fenclofenac,
Etodolac, Aceclofenac, Sulindac or Indomethacin, Pyrroleacetic
acids such as Ketorolac or Tolmetin, N-Phenylacetic acids such as
Mefenamic acid, Salicylates such as Acetyl salicylic acid
(Aspirin), Salicylic acid or Diffunisal, Pyrazolon derivatives such
as Phenylbutazone, Oxicam derivatives such as Piroxicam,
Tenooxicam, Meloxicam or Lornoxicam, Enolic acid derivatives
Aminopyrene or antipyrene, Phenols such as Acetaminophen or
Phenacetin, Non-acid derivatives Nabumeton, Coxib derivatives such
as Celecoxib or Rofecoxib.
[0018] Compounds inhibiting COX 2 specifically may be Coxib
derivatives such as Celecoxib or Rofecoxib.
[0019] In one embodiment of the invention the analgesic is
Ibuprofen.
[0020] In another embodiment of the invention the analgesic is
Ketoprofen.
[0021] Thus, a particular embodiment of the invention relates to a
non-woven wound dressing with fibers comprising an analgesic. A
particular useful construction comprises a first wound-contacting
layer of fibers with an analgesic, and a second layer of fibers
without the analgesic. The first layer may have a lower
cross-binding and/or smaller diameter (and thereby a higher
release) than the fibers of the second layer.
[0022] The solution provided herein is a solid product, a
non-woven, which is easier to handle and administer as opposed to
double-syringe applicators known in the art. As the individual
fibers are dry prior to contact with other fibers, the different
contents of the various fiber types do not interact. However, when
wetted, the fibers will start to be dissolved, or swell, and the
different contents will be released and then interact. For the
example of thrombin and fibrinogen, the interaction will initiate
the formation of a fibrin coagulum by the cleavage of fibrinogen
through the action of thrombin to form fibrin monomers that
spontaneously polymerize to form a three dimensional network of
fibrin.
[0023] The contents of the fibers will be released as the fibers
get dissolved. Hereby is a controlled release obtained.
[0024] Materials applicable to present invention are natural
protein structures, alone or in combinations, particular preferred
are those originating from the ECM. Examples of such materials are
collagen, keratin, fibrin, elastin, laminin, vimentin, vitronectin,
reticulin, fibrinogen and derivatives of these and the like found
in a native or denaturated form. In one embodiment the natural
protein is not fibrin.
[0025] As illustrated in the examples, the invention is
particularly well suited for gelatin, why it is particularly
preferred that the natural protein structure is gelatin. Gelatin is
an example of a poor fiber forming material, which by the described
process in this patent can be made into a fibrous non-woven
material. The gelatin fiber is still wet and sticky when it leaves
the nozzle. The fiber formation is therefore enhanced if the
collection of a fiber is not in a small area, but spread over the
collection device. This can be obtained if the fiber ejected from
the nozzle hits the collecting device at an angle as described in
example 4 where the fibers are sprayed on the inside of an almost
vertical rotating cylinder that is close to parallel to the nozzle,
or if the collecting device is perpendicular to the nozzle, it has
to move at a sufficient speed to spread the fibers. Too slow a
speed will result in the fibers sticking together while still wet
and forming a more film-like structure.
[0026] A particular advantage to Gelatin is the diversity of
products possible, with different degrees of x-binding. A non-woven
with very little cross-binding will be almost dissolvable, whereas
a heavily cross-bound non-woven will stay in the body for a month
or two, making sustained release for a long time at a particular
site possible. This is particularly useful in the present invention
by adding various degrees of polycarboxylic acid to the spinning
solution for each fiber type. Hereby a burst release (low
cross-binding, small amounts of polycarboxylic acid) can be
obtained along with a sustained release (higher cross-binding,
higher amounts of polycarboxylic acid).
[0027] In one aspect of the invention the fibers are biodegradable
fibers. This means a fiber that disappears; is hydrolysed, is
broken down, is biodegraded/bioresorbable/bioabsorbable, is
dissolved or in other ways vanish from the wound site when in
contact with wound exudates, blood or other body fluids. This is a
huge clinical advantage as there is nothing to remove from the
wound. It is typically preferred that the scaffold is broken down
during 1 day to 10 weeks--depending on the application. For open
wound applications, it is preferred that the scaffold is broken
down during 1-10 days, such as 2-7 days.
[0028] One way of manufacturing the present non-woven is by
producing fibers of a natural protein structure comprising the
steps of: [0029] (a) ejecting an aqueous solution of the natural
protein structure through a nozzle, wherein the aqueous solution
comprising <25% low molecular weight alcohol; while [0030] (b)
emitting pressurized air from air jet bores to attenuate or stretch
the natural protein structure fiber; while [0031] (c) collecting
the fibers on a collecting device.
[0032] This method allows very thin fibers to be extruded by a
method that can be run in commercial scale. The method according to
the invention is equally applicable to protein structures that are
poor fiber makers as well as naturally fiber forming proteins.
Gelatin has low cohesive strength and has been hard to manufacture
fibers of. The present technique has proven applicable to make
gelatin fibers even from water without the use of organic
co-solvents.
[0033] The multi component non-woven is produced by setting up two
systems and, at the same time, ejecting the fibers to the
collection device. Hereby is obtained that the fibers are formed,
optionally gelled, and dried prior to being in contact with each
other.
[0034] Non-woven fibrous structures are produced by extruding a
material through a nozzle, which due to its structure allows air
from nozzles adjacent to the extruding nozzle to enhance the fiber
formation by drawing and swirling the material.
[0035] The pressurized air is emitted from air jet bores. This
attenuates or stretches the natural protein structure fiber by
letting pressurized air be ejected from the air jet directed
downwardly and substantially tangential to the nozzle (WO94/04282).
The air also dries the fibers. Preferably, the pressurized air is
blown from a source as close to the orifice as possible, creating a
substantially tangentially, downwardly oriented pressurized air
flow.
[0036] The process and the apparatus is disclosed in detail in
WO94/04282.
[0037] When the aqueous solution is ejected from the nozzle a thin
fiber is formed. Given the high surface area to volume ratio of
these fibers, solvent evaporation occurs relatively quickly even
when operating with aqueous solutions at ambient temperature and
atmospheric pressure. It is appropriate to adjust temperature of
both ejected polymer and air such that the formed fibers are dry
enough to maintain the formed structure, but not dried too fast.
When fibers are not too rapidly dried the gelatin molecules will
have time to orient on a molecular level. This is related to the
inherent gel-sol properties of gelatin. When subsequent treating
the fibers with heat the fibers will cross-link more effectively if
the gelatin has been allowed to gel.
[0038] The present method avoids the need for biologically toxic
solvent systems. Thus, the present process allows real-time
fabrication of hybrid protein-cell constructs, and constructs of
biologically active constituents: discrete ECM regions, enzymes,
analgesics or the like.
[0039] In one aspect of the invention particles are suspended in
the aqueous solution prior to ejection. As the diameter of the
nozzle is wider than the diameter of the formed fibers, the
particles can have any diameter, up to the diameter of the nozzle,
or the particles can be smaller than the diameter of the fibers.
Wet, soft, and pliable particles of even larger diameter than the
nozzle may be ejected. Thus, in one aspect of the invention the
particles suspended in the aqueous solution have a mean diameter
wider than the mean diameter of the fibers.
[0040] It is preferred that the particles are ExtraCellular Matrix
(ECM) particles. ECM is the non-cellular portion of animal or human
tissues. The ECM is hence the complex material that surrounds
cells. In broad terms there are three major components in ECMs:
fibrous elements (particularly collagen, elastin, or reticulin),
link proteins (e.g. fibronectin, laminin) and space-filling
molecules (usually glycosaminoglycans' (GAG's)). ECMs are known to
attract cells and to promote cellular proliferation by serving as a
reservoir of growth factors and cytokines as well as providing the
cells with a scaffold.
[0041] The ECM material can be obtained from any mammal. It could
be derived from, but not limited to, intestinal tissue, bladders,
liver, spleen, stomach, lymph nodes or skin. ECM may be derived
from human cadaver skin, porcine urinary bladder submucosa (UBS),
porcine urinary bladder matrix (UBM), porcine small intestinal
submucosa (SIS).
[0042] Particles included in the present method can also be, or
contain, biological signal molecules e.g. chemo attractants,
cytokines and growths factors, polysaccharides, peptides and
derivatives of these and the likes. Examples of such materials
could be but are not limited to GAG's (chondroitin sulfate,
dermatan sulfate, heparan sulfate, hyaluronan, heparin etc.),
thrombin, fibrinogen, fibrin, fibronectin, vitronectin,
vimentin.
[0043] The particles could either consist of one material,
cross-linked if necessary, or found in combinations, mixed or
cross-linked together.
[0044] It is also preferred that the method further comprises the
step of cross-linking the non-woven natural protein structure.
Various methods of cross-linking exist like glutaraldehyde or
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC),
but in this context it is particularly preferred that the
cross-linking is done by heat treatment or ultraviolet irradiation
or both. Ultraviolet irradiation can be done as a post treatment or
as an in-line continuous treatment. Hereby is avoided to use
chemicals that are not compatible with introduction into the body
as tissue replacements or with introduction onto the body as
dressings.
[0045] When using the method for producing gelatin non-woven, the
best effect of heat treatment is obtained when the produced gelatin
fibers are not dried too rapidly such that sufficient time for
gelation, which is an orientation taking place on the molecular
level, is ensured. This mean, in essence, that the flow and
processing temperatures are adjusted to allow for sufficient slow
drying to allow gelation. Non-wovens of gelatin produced and
treated this way can be beta sterilized with 25 kGy and still be
sufficiently cross-linked. This also applies to other structures
made of gelatin for instance by freeze-drying. If the drying of
fibers is too rapid cross-linking by subsequent heat treatment will
still occur but to a lesser degree. Similarly, cross-linking by
heat treatment of freeze dried structures will occur to a lesser
degree, if the gelatin solution is not allowed to gel before
freezing. One aspect of the invention relates to a process of
cross-linking a natural protein structure comprising the step
allowing the natural protein to gel prior to drying follow by the
step freeze-drying.
[0046] In one embodiment cross-linking is obtained by
heat-treatment. The heat-treatment is even better if a
polycarboxylic acid is added to the spinning solution. Such heat
treatment could be at 150.degree. C. for 3 h. In one embodiment, it
is heat-treated in vacuum. The benefit is that cross-linking is not
taking place in the solution, as will be the case with for example
glutaraldehyde. Thus a fiber, a non-woven or for instance a
freeze-dried structure can be processed with the cross-linking
additive present without activating it. Activation for
cross-linking can be done as a simple post treatment neither
involving hazardous wet chemistry nor needs for energy consumption
due to drying of the product nor induction of deformations of the
already obtained structure by for example swelling. Examples of
usable polycarboxylic acid are poly acrylic acids, citric acid, and
carboxy-methyl-cellulose (CMC) but not excluding others.
[0047] In one embodiment the cross-linked fiber non-woven is a
non-woven that if submerged in 20.degree. C. tap water for 2
minutes, it can be pulled up in 1 piece.
[0048] Suitable crosslinkers are polycarboxylic acids, either the
free acid or salts thereof. These could be (but are not limited
to): Synthetic: poly(acrylic acid), poly(methacrylic acid),
poly(methyl-vinyl ether-co-maleic anhydride) (various grades of
Gantrez AN), copolymers of acrylic acid and vinylic monomers
(vinylpyrrolidone, alkyl vinyl ethers alkylacrylates,
alkylmethacrylates, styrene, maleic anhydride, maleic acid, fumaric
acid, hydroxyalkylacrylates, hydroxyalkylmethacrylates), copolymers
of methacrylic acid and vinylic monomers (vinylpyrrolidone, alkyl
vinyl ethers, alkylacrylates, alkylmethacrylates, styrene, maleic
anhydride, maleic acid, fumaric acid, hydroxyalkylacrylates,
hydroxyalkylmethacrylates) and copolymers and blends of the above;
Natural (acidic polysaccharides most preferred): Pectin,
carboxymethylcellulose, sodium alginate, gum arabic, Hyaluronic
acid, dermatan sulfate, heparin sulfate, heparan sulfate,
chondroitin sulfate and blends of the above.
[0049] Crosslinking can be chemical crossbinding, where chemical
groups react and form covalent bounds. However, the same effect of
stabilization will be obtained when complexes are formed resulting
in in-solubility. Such complex formation is also considered
crosslinking herein.
[0050] The thrombin used in preferred embodiments of this aspect of
the invention can be of animal or human origin. For example,
thrombin obtained from one mammalian species (e.g., bovine, pig,
sheep) can be incorporated into compositions of the invention used
to treat another mammalian species, for example, humans. More
preferably, the thrombin used in the composition is from the same
species for which the composition is intended to be used. The term
"thrombin" as used herein includes natural thrombin molecules
derived from animal or human plasma, and synthetic forms such as
those produced by recombinant DNA technology including functionally
active analogs that effectively maintain clotting activity in an
animal or human. Thrombin is present in the hemostatic composition
of the invention in a concentration of 1 to 100 U/cm.sup.2, and
preferably between 10 to 50 U/cm.sup.2. A unit of thrombin, as used
herein, is defined as the amount of thrombin required to clot a
standardized 15 1 ml fibrinogen (=250 mg/ml) solution in 15
seconds.
[0051] In other preferred embodiments of this aspect of the
invention, the hemostatic composition includes fibrinogen. The
fibrinogen can be of animal or human origin, and is preferably from
the same species for which the composition is intended to be used.
By the term "fibrinogen," as used herein, is meant to include
natural fibrinogen molecules derived from animal or human plasma,
and synthetic forms such as those produced by recombinant DNA
technology including functionally active analogs that effectively
maintain clotting activity in an animal or human. The fibrinogen
used in the compositions of the invention can be highly purified,
can contain small amounts of clotting Factor XIII, or can be
enriched with clotting Factor XIII. Also preferred is enrichment
with Factor VII. Typically, the amount of clottable fibrinogen is
present in the hemostatic composition of the invention in a
concentration between about 0.05 and 20 mg/cm.sup.2, preferably
between about 1 and 20 mg/cm.sup.2, and more preferably between
about 5 and 15 mg/cm.sup.2.
[0052] What happens is that the natural protein structure comes out
through a somewhat wide nozzle. The width of the nozzles also
allows the particle to come through without clogging the nozzle.
The thinness of the fibers is obtained through the combination of
the air-flow emitted and the consequent stretching the fiber as
well as the spinning process forming the non-woven. Thus, the
diameter of the fibers is a consequence of the production process:
the air-pressure, the ejection speed, the viscosity of the
solution. One aspect of the invention relates to a non-woven with
an average fiber diameter of 0.5 and 300 .mu.m, such as 10 to 30
.mu.m. In one aspect none of the fibers have a diameter of less
than 0.5 .mu.m and/or more than 300 .mu.m. In another aspect none
of the fibers have a diameter of less then 10 .mu.m and/or more
than 30 .mu.m.
[0053] One aspect of the invention relates a multi component
non-woven as described wherein at least one of the fiber
compositions has a larger diameter than at least one other. As
illustrated in Example 10, the smaller diameter, the softer the
material. When applied e.g. as a wound dressing, softness is key to
avoid pressure marks to the surrounding skin and to avoid pain in
the open wound. Additionally, smaller diameter provides for a
higher surface area in the wound dressing. This, in turn, provides
a higher release of substance and a higher absorption.
[0054] In one aspect of the invention particles are suspended in
the aqueous solution prior to ejection. As the diameter of the
nozzle is wider than the diameter of the formed fibers, the
particles can have any diameter, up to the diameter of the nozzle,
or the particles can be smaller than the diameter of the fibers.
Wet, soft, and pliable particles of even larger diameter than the
nozzle may be ejected. Thus, in one aspect of the invention the
particles suspended in the aqueous solution have a mean diameter
wider than the mean diameter of the fibers.
[0055] As illustrated in FIG. 3, the thin fiber will have bulbs of
particles, where the particles are coated with the natural protein
structures. It is preferred that the particles are compatible with
the natural protein structures, such that coating is strong. That
is, the strength of the fiber will be lowered if the particles are
not compatible with the natural protein structures. When the term
`particle` is used, it includes materials in the form of flakes,
fibers, particles, powder or the like.
EXAMPLES
Example 1
[0056] An aqueous solution of type B porcine gelatin with 260 bloom
of pharmaceutical grade from Gelita in the ratio of 20 g gelatin to
30 g water and 3 g propanol was prepared. The gelatin was allowed
to dissolve in the liquids by heating to 50.degree. C. for several
hours. The dissolved gelatin solution was transferred to a can,
which fit a small lab-size bulk melter built especially for this
purpose. The size of the can used with the bulk melter is
approximately from 0.5 liter to 1 liter. The bulk melter heats only
the upper surface of the material in the can, which then becomes a
viscous liquid and therefore can be pumped to a dispensing unit,
mounted hereto. The dispensing unit is a CF-200 Controlled
Fiberization Gun provided from Nordson Corp, equipped with a nozzle
with 0.012 inch orifice and 6 air holes. The temperature of the
bulk melter can be controlled in its different parts. The
temperature of the gun can be controlled and the temperature and
rate of the air is controlled.
[0057] The temperature of the gelatin was kept at approx.
50.degree. C. and the air was not heated. The obtained non-woven
was rigid and the resulting fibers had a diameter from 100 to 200
.mu.m.
[0058] The air-flow is controlled by a valve. A maximum of
approximately 201 atmospheric air per min is used.
Example 2
[0059] In a setup similar to the one described in example 1a nozzle
with an orifice of 0.030 inch (6 air holes) was used. The obtained
non-woven was similar in structure to the one described in example
1.
Example 3
[0060] In a setup similar to the one described in example 1 the
temperature of the gelatin was kept at approximately 92.degree. C.,
and the air was heated to approximately 92.degree. C. The obtained
non-woven was less rigid than in example 1 and fibers were
approximately 100 .mu.m wide. A similar result was obtained using a
nozzle with an orifice of 0.030 inch.
Example 4
[0061] An aqueous solution of porcine gelatin with bloom 300 from
Gelita was prepared similar to example 1. The solution contained
30% gelatin and 5% propanol. A nozzle with an orifice of 0.018 inch
was used with the equipment mentioned in example 1. A fibrous
non-woven structure could be obtained when the collecting device
was held in a parallel position to the fiber extruding direction.
It was found that an easy way to process a non-woven sheet was when
a rotating cylinder was used as a collecting device. In this case
the non-woven sheet was collected on the inner vertical surface of
the rotating cylinder. While the cylinder was rotating it was
furthermore moved in the vertical direction alternating from an
upward movement to a downward movement. When a constant rate of the
movements of the collecting device was maintained and the rate of
fiber output was kept constant it was possible to create a
non-woven sheet, which has a uniform appearance.
[0062] The powerfulness of this process is seen by the fact that in
approximately 5 minutes a non-woven gelatin with an area of app.
1350 cm.sup.2 and an approximate thickness of app. 2 mm is
made.
Example 5
[0063] In another experiment similar to example 4a 30% gelatin
solution was made in pure water (70%) without alcohol. The fibers
of the resulting non-woven had diameters from 3 to 7 .mu.m.
Example 6
[0064] In another experiment similar to example 5a 35% gelatin
solution was made. The fibers of the resulting non-woven had
diameters from app. 4 to app. 17 .mu.m with an average of app. 9
.mu.m. The non-woven was cross-linked by a heat-treatment. The
heat-treatment was done over night in a vacuum oven, which upon
evacuation of air was heated to 120.degree. C. The cross-linked
fibers swell upon hydration but do not dissolve, which on the other
hand was seen with untreated gelatin non-woven.
[0065] To evaluate the cell morphology and 3D growth of fibroblasts
on gelatin fibers, biopsies were punched out and seeded with
primary human fibroblasts (passage 3) on the surface with a density
of 2.5.times.10.sup.4 cells/cm.sup.2 in a small volume of growth
medium (10% FCS in DMEM) containing antibiotics (penicillin,
streptomycin and Amphotericin B). The scaffolds were incubated at
37.degree. C. at 5% CO.sub.2 before additional growth medium was
added. Evaluation of the cells attachment, morphology, growth and
population of the scaffold were preformed on day 1, 3 and 7 by
staining the cells with neutral red followed by evaluation using an
Leica DMIRE2 inverted microscope fitted with a Evolution MP cooled
colour camera (Media Cybernetics). Digital images were taken using
Image Pro Plus 5.1 software (Media Cybernetics).
[0066] The fibroblasts were adhering to the fibers as
spindle-shaped cells growing on single fibers except in regions
where several fibers were crossing each other. These cells were
growing across the fibers. There was a continuous increase in cells
number from the start of the study at day 1 to day 7.
Example 7
[0067] In another experiment with a setup similar to the one
described in example 6a 24% gelatin solution was used. In the
gelatin solution particles of porcine urinary bladder matrix (UBM)
was mixed in. The dry matter of the UBM particles was 30% of the
dry matter of gelatin. The average particle size of the particles
was approximately 150 .mu.m. A nozzle with an orifice of 0.030 inch
was used. The fibers were cross-linked by a heat-treatment similar
to the one described in example 6. The resulting non-woven had
fibers with diameter from app. 3 .mu.m to app. 15 .mu.m with an
average of app. 7 .mu.m.
[0068] In order to evaluate the cell morphology and 3D growth of
fibroblasts on gelatin fibers +/-UBM particles, biopsies were
punched out of each type of the scaffolds and seeded with primary
human fibroblasts (passage 3) on the surface with a density of
2.5.times.10.sup.4 cells/cm.sup.2 in a small volume of growth
medium (10% FCS in DMEM) containing antibiotics (penicillin,
streptomycin and Amphotericin B). The scaffolds were incubated at
37.degree. C. at 5% CO.sub.2 before additional growth medium was
added. Evaluation of the cells attachment, morphology, growth and
population of the scaffold were preformed on day 1, 3 and 7 by
staining the cells with neutral red followed by evaluation using an
Leica DMIRE2 inverted microscope fitted with a Evolution MP cooled
colour camera (Media Cybernetics). Digital images were taken using
Image Pro Plus 5.1 software (Media Cybernetics).
[0069] The cell growth showed on both types of gelatin fibers
(+/-UBM particles) and on all days tested adherent cells growing as
spindle-shaped cells. The cells were growing around the fibers and
in areas where several fibers were crossing each other the cells
were stretching across the fibers. At the first days of the study
no difference was seen between having UBM particles in the scaffold
or not but at day 7 it was apparent that the cells were more
dispersed in the scaffold containing UBM particles compared to the
pure scaffold and also contracted this scaffolds more. There were a
continuously increase in cells number from day 1 and to day 7.
[0070] One large SPF pig (crossbred of Durac, Yorkshire and Danish
landrace at Lab Scantox, Denmark) had circular full-thickness
wounds approximately 20 mm in diameter. The non-woven with UBM (20
mm disc), tested in duplicates, was carefully applied on top of the
wound-bed. To obtain optimal contact to the wound-bed, each
material was held in place by a 20 mm pre-wetted foam plug and
covered by foam dressings. On day 2 the top-foam dressing was
removed and the foam plug was very carefully removed, so as not to
disturb the healing and to ensure that the sample materials remain
in full contact with the wound bed. The wounds were covered by a
hydrocolloid dressing (Comfeel Plus) and changed on day 3, 6, 8,
10, 12 and 15. Following euthanasia, each wound was cut free as a
block separated from skeletal muscle tissue and fixed in 10%
neutral buffered formalin. The fixed samples were paraffin embedded
and sectioned in 5 .mu.m slices stained with haematoxylin and eosin
(HE) for general structure of tissue, Masson's trichroma for newly
formed collagen and von Willebrand factor for angiogenesis. The
evaluation was preformed by a trained pathologist at Lab
Scantox.
[0071] Massive amounts of granulation tissue developed was observed
consisting mainly of large numbers of thin-walled blood vessels and
fibrocytes/fibroblasts (fibrovascular connective tissue). Moderate
amounts of newly formed collagen and slight angiogenesis were
present in the wounds. A minimal presence of foreign material
likely to be test item was recorded and minimal numbers of clear
vacuoles were observed in the profound granulation tissue.
[0072] In the superficial parts of the wounds a moderate to marked
inflammation was found. In the deeper parts of the wounds a marked
inflammation was present. Marked numbers of giant cells were seen
and minimal to slight haemorrhage was recorded.
[0073] The re-epithelialisation was slight and the thickness of the
epithelium was marked in some cases with rete-ridge formation.
[0074] In conclusion, no significantly difference in the
histopathological wound healings parameters assessed were detected
between the non-wowen and the untreated control wounds. However a
tendency towards more giant cells were seen in the treated wounds
compared to control wounds, probably reflecting a foreign reaction
to the non-woven, a common and naturally reaction to materials left
in wounds.
Example 8
Multi-Nozzle Set Up for Combined Scaffolds
[0075] Multi component gelatine scaffold may be produced by
combining several lab-size bulk melters in a set up. In each
lab-size bulk melter there is a gelatine solution/gel containing
different components.
[0076] One example is using two containers and two nozzle systems.
In one container there is a 30% gelatine solution in water
containing 1000 IU thrombin/ml. In the other container there is a
30% gelatine solution in water containing 6 mg fibrinogen/ml. The
solutions may also contain an alcohol for regulating the viscosity
and fast evaporation of the solvent. When forming the fibres it is
important that the fibres are dry when touching each other on the
support layer otherwise the polymerisation reaction of fibrinogen
may initiate.
[0077] If the scaffold/non woven should be used as a haemostasis
sheet depending on the situation the scaffolds may be cross-linked
as described in example 6 but using lower temperature but longer
time or if a fast reaction is needed it may remain
un-cross-linked.
Example 8
[0078] Another example is the possibility to combine fibres with
different diameters by using nozzles with different orifice. The
scaffold/non-woven is cross-linked and used as scaffold for tissue
regeneration.
Example 9
[0079] An aqueous solution of porcine gelatine with bloom 300 from
Gelita was prepared similar to example 1. The concentration of
gelatine is 22%. A nozzle with an orifice of 0.012 inch was used in
the equipment. A fibrous non-woven structure could be obtained when
the collecting device was held in a vertical position to the fiber
extruding direction (see the drawing). In this case the non-woven
sheet was collected on a piece of textile which was wrapped on the
outside surface of a rotating plastic barrel. The rotation
direction of the barrel was same as the fiber's extruding
direction. While the barrel collected the fibers, it was rotating
and moving from side to side. The advantages of this collecting
process is that the collected non-woven fiber sheet is
homogeneous.
Example 10
[0080] In another experiment with a set up similar to the one
described in example 9, the pump speed, which was used to pump 22%
gelatine solution in water to the dispensing unit, run at half
speed compared to the pump speed at example 1. A nozzle with an
orifice of 0.012 inch was applied in this experiment. The diameter
of the obtained fibers at a low pumping speed was approximately 5
.mu.m. The softness of the non-woven sheet increased due to the
fiber diameter was decreased.
[0081] The conclusion was that the diameter of fibers decreased
with decreasing the speed of pumping gelatine solution to the
dispensing unit. The diameter of fibers at a low gelatine pumping
speed was uniform. The softness of non-woven sheet increased with
decreasing the diameter of the fibers.
Example 11
[0082] In another experiment with a system with dual set up similar
to the one described in example 1 were established for dispensing
gelatine fibers and gelatine fibers with thrombin.
[0083] One of the set ups had a 22% gelatine solution in water, and
the other was a 22% gelatine solution in water containing 1000
IU/ml thrombin. The gelatine solution and the thrombin solution
were pumped to the dispensing units individually. Each of
dispensing unit had a nozzle with an orifice of 0.012 inch. The
distance between two was 10 cm. The experiment was carried out in a
clean room with a low humidity.
[0084] A non-woven sheet collector similar to the one in example 9
was used to collect the dispensed fibers.
[0085] A home use vacuum cleaner was connected to the sidewall of
the barrel to suck the ambient dry air through the multi holes
(I.D. 5 mm) on the surface of the barrel into the inside of the
barrel. The ambient air dried the dispensed fibers rapidly on the
textile to prevent a cross contamination between the gelatine
fibers and the thrombin fibers.
[0086] The benefit of implementation of a vacuum system in the
collection device is to speed up the process of drying the
dispensed fibers for preventing a cross contamination between the
multi components fibers, and cut down the risk of spreading the
thrombin in the ambient air during the production processes.
FIGURES
[0087] FIG. 1: Labscale production of gelatin non-woven using the
inside of a rotating cylinder as the collecting device.
[0088] FIG. 2: Gelatin non-woven made from 30% aqueous gelatin.
Light microscopy 10.times.magnification. Scale bar is 50 .mu.m.
[0089] FIG. 3: Gelatin non-woven with UBM particles. Scale bar is
100 .mu.m.
[0090] FIG. 4: Picture showing examples of nozzles used to make
gelatin non-woven. The orifice is found on the top of the raised
center part. There are either 6 or 12 air holes in a circle around
the orifice.
[0091] FIG. 5: Is a schematic drawing of a production setup.
[0092] FIG. 6: The collected non-woven sheets from example 10
(left) and example 9 (right).
[0093] FIG. 7: Is a detailed drawing of a production setup.
[0094] FIG. 8: Cross section of multi components.
* * * * *