Note: Descriptions are shown in the official language in which they were submitted.
CA 02207988 1997-06-16
CELLULOSE MEMBRANE AND METHOD
FOR MANUFACTURE THEREOF
FIELD OF THE INVENTION
The present invention relates to a method for the preparation of a
microbial cellulose membrane.
BACKGROUND OF THE INVENTION
For the treatment of burns, abrasions and surgical incisions, a variety
of products are currently available. The commonest type being the medicated
gauze-type dressing. This type of dressing requires frequent changes to
observe
the healing process and to apply medication to the wound. This is often
accompanied by discomfort to the patient since some adherence to the wound or
wound exudate is common. More importantly, gauze-type dressings do not protect
the wound from bacterial infection and as well lack moisture balance for
proper
healing of the wound.
More recently, various polymeric materials have been investigated for
wound dressing application. These can be subdivided into two broad classes of
materials, the first consisting of synthetic polymeric materials such as
polyurethanes and the other consisting of naturally derived polymeric
materials
such as collagen. Wound dressings have been made using combinations of
materials from these two classes of materials. Some of the currently available
products are described in the following papers: "Principles of Burn Dressings"
in Biomaterials, volume 6, p.369-377, 1985 by Quinn, Courtney, Evans and
Gaylor;
and "Modern Dressings: What to Use" in Australian Family Physician, volume 23,
p.824-839, 1994.
The production of cellulose pellicles by the bacteria Acetobacter
xylinum and their conversion into liquid loaded pads for use as wound dressing
was
disclosed in US Patent 4,788,146. Liquids loaded include various medications
that
are deemed useful in various functions in the process of wound healing.
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CA 02207988 1997-06-16
US Patent 4,912,049 describes a process for the production of
cellulose pellices from the bacteria Acetobacter xylinum and their processing
into
thin sheets for biomedical applications. This patent forms the basis of a
commercial
wound dressing product BiofiIIT"". This product has been shown to be effective
in
the treatment of a variety of wounds. However, a drawback to this material is
that
it is prone to being easily torn, especially when applied to areas of mobility
on the
body due to lack of sufficient elasticity of the material.
The production of cellulose by the bacteria Acetobacterxylinum has
been studied by numerous investigators. Their interests have been mainly on
the
cellulose generation mechanism of the bacteria. In a static reactor, cellulose
pellicles are formed at the air/liquid medium interface. The cellulose fibers
formed
under such condition have nominally infinite length and are intertwined
together to
form a pellicle. However, under agitation the cellulose fibers are broken up
into
microfibrils. The dimensions of the microfibrils are a function of the
reaction
conditions. Production of cellulose fibrils is described in US Patents
5,079,162 and
5,144,021 and a paper entitled "Properties and Uses of Bacterial Cellulose
Produced in Fermenters" presented by Johnson, Stephens and Westland in the
American Chemical Society Meeting in April, 1990. An advantage of bacterial
produced cellulose is that a high degree of control over product purity can be
obtained in comparison to other non-bacterial methods of cellulose production.
In
addition, control over the size and porosity of the cellulose is important for
medical
end uses due to the need to incorporate other medicinal agents into the
dressing.
Cellulose fibrils formed by Acetobacterxylinum (A. xylinum) are much
smaller than cellulose fibers from the standard pulping of wood as shown in
Table
1.
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CA 02207988 1997-06-16
Table 1
Cellulose Fiber Dimensions
Source Length Width
A. xylinum cellulose ~ 30 pm 0.1-0.2 pm
Birch 0.8-1.6 mm 14-40 pm
Pine 2.6-4.4 mm 30-75 pm
These lower molecular weight cellulose materials are much more
desirable than other sources of heavier molecular weight cellulose since the
former
are in principle more readily worked with due to their lower molecular weight
than
plant source cellulose. Cellulose fibers derived from plant sources can be
dissolved
in several solvent systems to form highly viscous solutions. These solutions
form
the basis of the regenerated cellulose fibre industry. Practical cellulose
dissolution
processes are described in the "Kirk-Othmer Encyclopedia of Chemical
Technology", Fourth Edition 1993, volume 5, p.476-563. These include the
viscose
process in which cellulose xanthate derivatives are formed and the cuen
process
which depends on the formation of copper complexes with cellulose. Due to the
nature of these solvent systems, cellulose dissolved in these solvent systems
tend
to degrade over time. A non-degrading solvent system for cellulose comprising
dimethylacetamide and lithium chloride was described in an article entitled
"Solution Studies of Cellulose in Lithium Chloride and Dimethylacetamide" in
Macromolecules, volume 18, p.2394-2401, 1985 by McCormick, Callais and
Hutchinson, Jr. and US Patent 4,857,201. Although these solvent systems are
suitable for cellulose of plant origin, they heretofore have not been applied
to
microbially produced cellulose.
Currently, a variety of substances and procedures exist for the
treatment of wounds. Research over the years has led to a consensus on the
ideal
characteristics of wound dressings. It is agreed that a wound covering should
be
adherent, elastic, pliable, impermeable to bacteria, easy to handle, non-
toxic, allow
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CA 02207988 1997-06-16
for proper water vapour permeability for moisture balance and possess
mechanical
properties so it can be used on areas of high movement. The wound dressings
available today all attempt to meet the criteria of an ideal wound dressing
but such
a dressing is yet to be developed. It would be very advantageous to provide a
method for producing membranes for wound dressings from bacterially-produced
cellulose. Cellulose membranes and films can be made by casting these
solutions
onto a flat surface.
OBJECTIVES OF THE INVENTION
An objective of the present invention is to provide a bacterially
produced cellulose membrane, and method for production, that may be used as a
wound dressing that is effective in providing a favourable environment for the
healing of the wound. Another objective is to provide a cellulose membrane
that has
good mechanical properties including flexibility, similar to the mechanical
properties
of human skin, and can be used effectively in areas of high mobility. Still a
further
objective is to provide a wound dressing that can be used on large area
wounds.
Another further objective is to provide a process for the preparation of the
aforementioned cellulose membrane. These and other objectives will readily
become apparent to those skilled in the art in view of the teachings
hereinafter set
forth.
SUMMARY OF THE INVENTION
The materials of this invention comprise a film of microbially produced
cellulose, particularly cellulose produced from the culture of
Acetobacterxylinum
in a stirred tank. The film is made by dissolving the cellulose in a solvent
system
comprising dimethylacetamide and lithium chloride, casting the resulting
solution
onto a flat surface and regenerating the film in a gelation bath. Humectant is
incorporated into the film by solvent exchange. The film is then
depyrogenated,
sterilized and packaged for long term storage.
4
CA 02207988 1997-06-16
In one aspect of the invention there is provided a method for
producing a cellulose membrane from microbially produced cellulose. The method
comprises the steps of providing a microbially produced cellulose; dissolving
the
cellulose into a solvent system comprising a lithium salt and
dimethylacetamide;
casting the solution onto a surface and drying to produce a membrane;
coagulating
the membrane in a gelation bath; and incorporating a humectant into the
membrane
by solvent exchange.
In this aspect of the invention the lithium salt is preferably lithium
chloride. The microbially produced cellulose is preferably produced by the
bacteria
Acetobacter xylinum.
In another aspect of the invention there is provided a cellulose
membrane useful as a wound dressing. The membrane is produced by a process
comprising the steps of providing a microbially produced cellulose; dissolving
the
cellulose into a solvent system comprising a lithium salt and
dimethylacetamide;
casting the solution onto a surface and drying to produce a membrane;
coagulating
the membrane in a gelation bath; and incorporating a humectant into the
membrane
by solvent exchange.
In another aspect of the invention there is provided a wound dressing
comprising a microbially produced cellulose membrane and a humectant
incorporated into the membrane. In this aspect of the invention the
microbially
produced cellulose may be produced by the bacteria Acetobacter xylinum.
BRIEF DESCRIPTION OF THE DRAWINGS
The product wound dressing and method of producing the wound
dressing in accordance with the present invention will now be described, by
way of
example only, reference being had to the accompanying drawings, in which:
Figure 1 shows a stress-strain curve for a sample UWO-000 produced
in accordance with the present invention;
Figure 2 is a plot of stress at a constant strain level of 30% for the
5
CA 02207988 2001-O1-22
samples prepared with various periods of drying time;
Figure 3 shows stress-strain curves for a sample (UWO-000) of a
microbially produced cellulose membrane produced in accordance with the
present
invention in both the direction of draw (UWO-000-00) and perpendicular to the
direction of draw (UWO-000-90);
Figure 4 shows the stress-strain curves for the films soaked in
different bath mediums;
Figure 5 compares the mechanical properties of a film prepared from
a plant derived cellulose, with zero drying time, and a bacteria derived
cellulose
according to the present invention;
Figure 6 shows the fracture strain, measured using an Instron tensile
tester, for the sample UWO-000-00 compared with that of human skin and
BiofiIIT'",
a cellulose wound dressing derived from the cellulose pellicle produced by A.
xylinum;
Figure 7 shows the fracture strain data of Figure 6 for the sample
UWO-000-00 on an expanded scale;
Figure 8 compares the fracture strain of human skin, rat skin, sample
UWO-000-00 and commercial BiofiIIT"' ;
Figure 9 compares the rates of water permeation of the cellulose films
(UWO-000, UWO-240) prepared according to the present invention compared with
the BiofiIIT"~ dressing of Figure 6, the membranes were subjected to pressure
in a
permeation cell and water permeation rates were determined;
Figure 10 shows the effect of sterilization on the stress-strain
behaviour of the films produced according to the present invention;
Figure 11 shows the effect of depyrogenation on the stress-strain
behaviour of the films produced according to the present invention; and
Figure 12 shows the results of the biocompatibility studies with the
films of the present invention compared to controls.
6
CA 02207988 1997-06-16
DETAILED DESCRIPTION OF THE INVENTION
Cellulose Dissolution and Preparation
In preparing the wound dressings of the present invention, cellulose
produced by the bacteria Acetobacter xylinum (CellulonT"", NutraSweet Kelco
Company, 2025 East Harbor Drive, San Diego, California, 92113-2123) was
dissolved in a solvent system comprising dimethylacetamide and a lithium salt,
preferably lithium chloride. The lithium chloride concentration in the solvent
is in the
range from about 5% to about 12%, and preferably about 9%. Concentration of
cellulose in the resulting solution was in the range from about 2% to about
8%. The
solution was filtered with 25 pm millipore filter. This ensured the removal of
any
undissolved cellulose before the solution was cast.
The solution is spread onto a solid smooth surface using a casting
knife with a built-in gap width. Depending on the gap width of the casting
knife
used, solution films of a wide range of thickness can be made. After a drying
period
of between approximately 0-360 minutes, the solution film is then transferred
into
a gelation bath comprising water. Cosolvents and additives can also be added
to
the bath. Examples of these are alcohols, ketones and ethers that are miscible
with
water. The cosolvents are believed to assist in the formation of an open
capillary
system in the membrane to provide a porous structure. The coagulated film is
then
transferred into distilled water, rinsed thoroughly and then soaked in a bath
comprising a humectant such as glycerol, polyvinyl alcohol, polyethylene
glycol etc.
followed by air drying. The role of the humectant is to displace water from
the
capillary structure and to replace it with a hygroscopic material having
hydroxy
groups.
To produce films of large surface area, casting may be done on a
continuous casting machine which those skilled in the art will understand.
This
invention is further illustrated by the following non-limiting examples.
7
CA 02207988 1997-06-16
Example 1
A 4% solution of cellulose produced by A. xylinum was prepared by
the following procedure. Wet cellulose (CellulonT"") (40 g, cellulose content
17.85%) was soaked in distilled water (100 ml). After 24 hours, the cellulose
was
filtered and transferred into methanol (70 ml), stirred and filtered. This
procedure
was repeated three more times. After this, the cellulose was transferred into
dimethylacetamide (70 ml), stirred and filtered. This procedure was repeated
four
more times. The cellulose was then dried in a vacuum oven until cellulose
concentration was about 30-45%. The actual concentration of cellulose was
determined gravimetrically by drying a sample in a vacuum oven at
100°C. The
resulting cellulose was used to prepare the 4% cellulose solution in a solvent
system of 9% lithium chloride in dimethylacetamide.
Cellulose solution films were prepared using a casting knife with a gap
width of 100pm. The films were coagulated in a water bath after a range of
drying
time of 0-240 minutes. Afilm, with a drying time of 0 minutes, coagulated in a
50%
DMAc and 50% water bath was also prepared. The films were then solvent
exchanged in a 10% glycerol solution and then were laid flat to air dry. Also
a
dense film was prepared by allowing the solvent to be completely removed by
vacuum without the use of a water bath. Plant derived cellulose films were
prepared
in a similar manner for purposes of comparison. Inspection of these films are
summarized in the following Table 2. All samples made in accordance with the
present invention are labelled UWO-XXX-XX, the second three digits represents
the drying time in minutes for that particular sample, and the last two digits
designate the direction of draw of the cellulose solution during film
preparation and
may be either 00 or -90 corresponding to either being parallel to, or
perpendicular
to the direction of draw respectively.
8
CA 02207988 1997-06-16
Table 2
Sample Drying Time Thickness Appearance
UWO-000 0 ~ 10 pm clear
UWO-060 60 minutes ~ 10 pm clear
UWO-240 240 minutes ~ 10 pm clear
Mechanical Properties Of Prepared Films
Strips 5 mm x 25 mm were cut from the cellulose films for testing,
using a surgical blade and a template. The strips were cut from the film in
both the
draw direction and perpendicular to the draw direction. The thickness was
measured using a lever apparatus which measures the displacement due to the
thickness of the specimen. The displacement is converted to a measurement in
mm.
The measurement was accurate to ~0.001 mm. The mechanical properties of
cellulose solution films were determined using an Instron tensile tester
(Model
1125) with a load versus crosshead distance travelled setup. The gauge length
of
the specimen was recorded before the start of each test. The gauge length is
the
length of the specimen between each grip. At this point the specimen was
straightened and the load was zero. Preconditioning was performed on each
strip
before testing to ensure repeatability of uniaxial tests. The specimens were
loaded
from 0-25 -0 g, for 5 cycles, at the same crosshead speed used for the
mechanical
tests. Load versus elongation was monitored for verification.
Five stress-strain tests were pertormed on each of different types of
specimens, at extension rates of 5 mm/min with a 500 g load cell. The tensile
tests
were performed on dry samples. Stress-strain curves were obtained by straining
the
specimen from a load of 0 g to the load at which failure occurred. The
engineering
stress was obtained from the recorded load and defined as:
Stress (MPa) _ (Load{g)* 9.81 (g/m2))/(width(mm)*thickness(mm)* 1000)
where load in g is recorded from the load cell, width and thickness is the
initial
dimensions of the specimen.
9
CA 02207988 2001-O1-22
Engineering strain is defined as:
Strain = I - Ig./ Ig
where I is the length and Ig is the recorded gauge length at zero load. The
stress-strain curves were plotted as strain versus stress (MPa). The curves
for
specimen cut in the x and y direction were both determined. The ultimate
tensile
strength (stress at failure), the elastic modulus (slope of the initial curve)
and
percent elongation (maximum strain) were calculated from these curves.
A typical stress strain curve of the cellulose films with zero drying time
is shown in Figure 1. These cellulose films exhibited small strain increments
in the
elastic lower stress regions followed by increased strain increments in the
higher
plastic stress regions. The results of the stress-strain tests for the
cellulose films
with various drying times and commercial cellulose BiofiIIT"', a trademark of
BiofiIIT""
Productos Biotecnologicos, Curritiba, Parana, Brazil, are presented in Table
3.
Table 3
Sample Ultimate TensileElastic ModulusFracture Strain
Strength (Mpa) (MPa) (%)
UWO-000-00 53.2 3.5 276.8 28.6 98.4 5.4
UWO-000-90 37.2 2.9 206.3 23.5 143.2 6.2
UWO-060-00 54.8 8.2 295.4 31.2 99.1 3.5
UWO-240-00 38.3 5.3 297.7 29.5 38.8 4.1
UWO-1200-00 39.7 4.3 293.8 31.5 37.3 2.2
BIOFILLT~~ 159.4 6.9 1678 5.1 15.8 2.3
The mechanical properties of the specimens varied with the drying
time. From Table 3, the measured properties show that an increase in the
drying
time caused a decreased fracture strain and an increased elastic modulus. The
zero
drying time had the highest strain to fracture and the lowest elastic modulus.
This
resulted in a sample with elasticity and extensibility.
CA 02207988 1997-06-16
As the drying time increased the level of stress at a particular level of
strain increased. This result is illustrated in Figure 2 where stress is
plotted at a
constant strain level of 30% for the samples prepared with various periods of
drying
time. The level of stress steadily increased as the drying time during the
preparation was increased. It can be concluded from the observed results that
the
mechanical properties of the cellulose films can be controlled by the
preparation
conditions. As the drying time increases, the stiffness of the films increased
and the
extensibility decreased.
The fracture strain of the films ranged from 40 % to over 100%. The
results can be interpreted in terms of polymer organization in the cast
cellulose film.
Initially following casting, the polymer chains are loosely aligned. Long
drying times
allow the polymer chains to become more organized, leading to an increase in
stiffness and a decrease in fracture strain. The strong hydrogen bonding of
cellulose leads to this organization. Some of the prepared samples showed
adequate strength and extensibility to be used as a wound dressing.
Referring now to Figure 3, the stress-strain curve for sample
UWO-000-00 produced according to the present invention in both the direction
of
draw (UWO-000-00) and perpendicular to the direction of draw (UWO-000-90). As
may be seen from Figure 3 the cellulose films exhibited an anisotropic
behaviour.
The films are hence stronger in the direction of draw but more elastic
perpendicular
to the direction of draw. In other words, the draw direction exhibited a
higher
strength and the direction perpendicular to the draw direction exhibited a
higher
amount of extensibility.
Linear polymers such as cellulose tend to line up in the direction of
draw. Mechanical strength in this direction is proportional to the inherent
strength
of the polymer chain. Perpendicular to the draw direction, neighbouring
polymer
molecules are held together mainly by intermolecular hydrogen bonding. Hence,
it
would be expected that mechanical properties of the film are anisotropic. The
observed results indicate that the polymer chain is stronger than the
intermolecular
11
CA 02207988 2001-O1-22
hydrogen bonds between neighbouring polymer chains. It is interesting to note
that
human skin also exhibits anisotropic behaviour.
The results of a typical stress-strain curve for the films soaked in the
different bath mediums is illustrated in Figure 4. It is clear that there is
no clear
difference in the samples. The DMAc provided no improvement in the mechanical
properties. The film that was allowed to dry completely to form a dense film
showed
clear differences in its mechanical properties compared to the film soaked in
the
water bath. As shown in Figure 4, the dense film has both lower stress~and
strain
values and hence was less suitable, in terms of mechanical properties, for the
purpose of a wound dressing.
The different bath mediums used in the preparation conditions had no
clear effect on the mechanical properties. Previous reports indicated that for
some
films, additional swelling in a solvent can improve its mechanical properties,
see W.
Zhao et al. Chemtech, March 23, (1996). In the present preparation, DMAc had
no
further swelling effect to alter the mechanical properties of the ~Im. The ~Im
that
was prepared without a bath medium and was allowed to dry to form a dense film
had a lower level of elasticity and strength. Water was used as the bath
medium of
choice for the wound dressing preparations.
The mechanical properties of a film prepared from a plant derived
cellulose, with zero drying time, is shown in Figure 5. The film was prepared
under
conditions identical to the preparation conditions of the bacterial derived
cellulose.
The comparison between the bacteria source cellulose film and the plant source
cellulose derived film revealed that they have very different mechanical
properties.
The plant derived cellulose films were stiffer and had lower ductility. It can
be
concluded that the bacteria derived cellulose produced films possess more
suitable
mechanical properties as a wound dressing than the plant derived cellulose
films.
12
CA 02207988 2001-O1-22
The Comparison of Mechanical Properties of The Prepared Films with Human
Skin and Commercial Products
Human skin consists of collagen fibres randomly arranged in layers.
Quantitative analysis of human abdominal skin revealed that there is an
overall
preferential orientation of fibres across the abdomen. This is the direction
of
minimum extensibility. Thus skin tissue shows mechanical anisotropy. As shown
previously in Figure 3 films prepared by the present method exhibit
anisotropic
behaviour and therefore is consistent with the properties of human skin.
A general quantitative analysis of the mechanical properties of human
skin is difficult due to the differences in human skin at different ages. With
increasing age the skin becomes less extensible and the tensile strength
increases.
Studies reveal age related changes in the form of the collagen fibres of the
dermis,
which accounts for their decreased mobility in elderly skin. In children's
skin, the
fibres are loosely arranged with less connections between the fibres. This
accounts
for the increased extensibility. On average, an adult's abdominal skin has a
tensile
strength of 7 MPa and an elongation of 80 percent.
The elastic modulus of the proposed wound dressing determines the
ability of the sample to be used as a wound dressing on areas of high
mobility. It is
thus suitable for use on wounds in areas of high mobility. Figure 6 compares
the
fracture strain for the sample UWO-000-00 , human skin and BiofiIIT"", a
cellulose
wound dressing derived from the cellulose pellicle produced by A. xylinum. It
can
be seen that UWO-000-00 has very similar fracture strain to the human skin but
much higher than that of BiofiIIT"~. A closer comparison between UWO-000-00
and
human skin is shown in Figure 7. The higher strength and fracture strairi
exhibited
by UWO-000-00 than human skin is advantageous when films produced by the
present method are used as wound dressings.
Figure 8 shows a comparison of the fracture strain of the cellulose film
(UWO-000-00), BiofiIIT"~ (commercial product), rat skin and human skin (see
"The
Biomedical Handbook" Yannas. CRC Press Inc. p. 2025-2045, 1995 and R. Hut, J.
13
CA 02207988 2001-O1-22
Biomechanical Engineering, 111, p. 136, 1989). The fracture strain of the
UWO-000-00 cellulose film is very similar to that of the rat skin and human
skin and
greatly exceeded that of the commercial product BiofiIIT"~. Therefore the
dressing
produced by the present invention is thus much more suitable for use on wounds
in areas of high mobility due to its improved extensibility and elasticity.
The cellulose that makes up the present films produced according to
the present invention and the BiofiIIT"" product were both derived from the
bacteria
Acetobacter xylinum. The results show clear differences in the mechanical
properties exist between them. The difference in mechanical properties and
appearance is due to the cellulose film preparation process. Regeneration of
cellulose films from a cellulose polymer solution allows for better control of
the
resulting film properties.
Water Permeation Studies
An AmiconT"~ ultrafiltration cell, model 8050, was used to determine
water permeation rates. The cell was lined with the sample and subjected to
water
at various pressures. At the end of a two minute time period, the mass of the
water
was measured. This procedure was repeated several times until a constant mass
was reached. This was performed at several different pressures. The permeation
rates were calculated from the data and recorded in units of g/m2-hr, where g
represents the mass in grams, hr is the time in hours and m is size of the
film in
meters. These results were then used to estimate the water vapour permeance
rates.
Water permeation rates were determined for the UWO-000 sample
preparations. Referring to Figure 9, for comparison, the permeation rates for
a
commercial wound dressing, BiofiIIT"", was also measured. It can be observed
from
the results in Figure 9 that the films are permeable to water under pressure.
A ratio
between these two results and the water vapour permeance value from the
literature for BiofiIIT"' was used determine the water vapour permeance value
for
14
CA 02207988 2001-O1-22
the UWO sample. The samples have a lower rate compared to BiofiIIT"". The
results
of the calculated water vapour transmission rates shown in Table 4 indicate an
adequate level of permeation, see for example A. M. Gatti et al. J. Materials
Science
in Medicine, 5, 190 (1994) and M. Jonkman et al., Biomaterials 9, 263, (1988).
Table 4: Water Vapour Permeation
Sample Water Vapour Permeation
(glkPa*hr*m2)
UWO-000 g.2
BiofiIIT"" 31
Human Skin 4
Op-SiteT"~t 11
t trademark of Smith & Nephew
A wound dressing should limit excessive evaporative water loss and
desiccation of the wound to promote healing. However, a wound dressing must
allow the passage of some water vapour to pre~~ent excessive accumulation of
exudate, which might cause the separation of the dressing from the wound. The
results in Table 4 show that the water vapour permeance for the wound dressing
produced in accordance with the present invention was lower than the two
commercial products and is closer to human skin. The water vapour permeation
data for human skin and the commercial product Op-SiteT"~ is disclosed in M.F.
Jonkman et al., Biomaterials, Vol. 9, p.263-267, 1998. The water vapour
transmission data for the commercial product BiofiIIT"" is disclosed in A.M.
Gatti et
al., Journal of Material Science: Materials in Medicine, Vol. p.190-193, 1994.
Sterilization And Depyrogenation
The ability of the cellulose film to withstand both sterilization and
CA 02207988 1997-06-16
depyrogenation processes are necessary and important steps towards making a
biomedical device. Often biomedical polymers have lower thermal and chemical
stability than other materials such as metals and ceramics and therefore they
are
harder to sterilize using conventional techniques. For any material used as a
wound
dressing, it must be free from pyrogens, bacteria and any possible
contaminants
that will intertere with the healing process. It is therefore important to
study the
effect of sterilization and depyrogenation on the properties of the cellulose
films.
The samples were steam sterilized by autoclaving in excess glycerol.
The glycerol acts as a humidient to ensure that the structural and dimensional
integrity is maintained. A series of runs for 15 minutes and 1 hour at 121
°C and a
pressure of 15 psi were used to determine the effects. The stress-strain
relationships of the sterilized samples were determined and compared to the
untreated samples. The results shown in Figure 10 indicate that the films can
be
sterilized without any substantial change in their desirable mechanical
properties.
It can be concluded that the samples were able to withstand sterilization and
depyrogenation without any significant changes to the mechanical properties.
The samples were depyrogenated in a 1 M and 5M concentrated
NaOH solution for 1 hour at 25°C and '~5°C, respectively.
Following
depyrogenation, the stress-strain relationships of the treated and untreated
samples
were compared. The results shown in Figure 11 indicate that these films can be
depyrogenated with a strong alkaline sodium hydroxide solution at an elevated
temperature without any substantial change in their desirable mechanical
properties.
Biocompatibility Tests
Friend erythroleukemia cells were grown in the presence and absence
of the cellulose film samples. The cells were incubated in Iscoves medium at
37°C
for 3 days. Cell counts were done with a hemacytometer with three independent
measurements taken per sample. A total of 1000 cells were counted for each
16
CA 02207988 1997-06-16
sample. No cell lysis was observed. Results collected in Table 5 and Figure 12
indicate that the samples do not affect normal cell growth.
Table 5: Biocompatlbility Results
Sample Cell Titer
Control 1 5.17 0.67 x 105
Control 2 4.60 0.67 x 105
UWO-240 4.71 0.67 x 105
UWO-240 A 3.48 0.67 x 105
Further tests were pertormed to determine if fibroblasts could grow in
the presence of the cellulose films. Mouse fibroblasts, 3TC cells, were placed
in a
petri dish in the presence of the samples. Over a period of time observations
were
performed, without any counts to determine the relative abundance of the
fibroblasts. These observations confirmed that the mouse fibroblasts were able
to
grow on the samples. The biocompatibility of the UWO membranes was thus
demonstrated by the ability of both the Friend erythroleukemia cells and mouse
fibroblasts to grow in the presence of the samples.
The foregoing description of the preferred embodiment of the
invention has been presented to illustrate the principles of the invention and
not to
limit the invention to the particular embodiment illustrated. It is intended
that the
scope of the invention be defined by all of the embodiments encompassed within
the following claims and their equivalents. It will be understood that the
bacterially
produced cellulose membranes disclosed herein may be used in most applications
for which known cellulose membranes are used.
17
