U.S. patent application number 11/633665 was filed with the patent office on 2007-07-05 for non-woven fabric for biomedical application based on poly(ester-amide)s.
This patent application is currently assigned to Cornell Research Foundation, Inc.. Invention is credited to Chih-Chang Chu, Patti Jo Lewis.
Application Number | 20070155273 11/633665 |
Document ID | / |
Family ID | 38225075 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070155273 |
Kind Code |
A1 |
Chu; Chih-Chang ; et
al. |
July 5, 2007 |
Non-woven fabric for biomedical application based on
poly(ester-amide)s
Abstract
Electrospun biodegradable poly(ester-amide) fabric is especially
suitable as a scaffold for tissue engineering and to incorporate
drug for burn or wound healing treatment to accelerate healing, or
to prevent tissue adhesion after surgery.
Inventors: |
Chu; Chih-Chang; (Ithaca,
NY) ; Lewis; Patti Jo; (Highland Park, NJ) |
Correspondence
Address: |
BACON & THOMAS, PLLC
625 SLATERS LANE, FOURTH FLOOR
ALEXANDRIA
VA
22314
US
|
Assignee: |
Cornell Research Foundation,
Inc.
Ithaca
NY
|
Family ID: |
38225075 |
Appl. No.: |
11/633665 |
Filed: |
December 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60750834 |
Dec 16, 2005 |
|
|
|
Current U.S.
Class: |
442/342 |
Current CPC
Class: |
A61L 2300/412 20130101;
A61F 2/0063 20130101; A61L 27/18 20130101; A61L 15/26 20130101;
A61L 27/3813 20130101; A61L 2300/602 20130101; A61L 15/26 20130101;
A61F 2/06 20130101; A61L 2400/12 20130101; Y10T 442/616 20150401;
A61L 15/44 20130101; D04H 3/16 20130101; A61L 27/18 20130101; C08L
77/12 20130101; C08L 77/12 20130101 |
Class at
Publication: |
442/342 |
International
Class: |
D04H 13/00 20060101
D04H013/00 |
Claims
1. A non-woven fabric consisting essentially of biodegradable
electrospun poly(ester amide) for use for biomedical application,
which is sterilizable and has an average fiber diameter ranging
from 0.1 to 10.0 micrometer, a median pore size ranging from 0.1 to
100 micrometer, a surface area ranging from 10 to 300 m.sup.2/g, an
average thickness ranging from 0.01 to 0.500 mm, a flexural
rigidity ranging from 10 to 80 mgcm, an average air permeability
ranging from 10 to 100 ft.sup.3/min/ft.sup.2, an average water
vapor transmission rate ranging from 200 to 500 g/m.sup.2/24 hr, a
wettability contact angle ranging from 40 to 80 degrees, tensile
stress property ranging from 0.01 to 0.10 kgf/mm.sup.2, tensile
strain property ranging from 100 to 800%, Young's modulus ranging
from 0.20 to 20.0 MPa and tensile toughness ranging from 0.50 to
3.0 MPa.
2. The non-woven fabric of claim 1 where the poly(ester-amide) is
able to be solution electrospun.
3. The non-woven fabric of claim 2 where the poly(ester-amide) has
a reduced viscosity ranging from 1.0 to 2.0 dL/g and is selected
from the group consisting of one or more subunits A, and one or
more subunits B, and combinations thereof, where the one or more
subunits A have the structure ##STR00007## where R.sup.1 is
(C.sub.2-C.sub.20) alkylene, and where R.sup.3 is hydrogen,
(C.sub.1-C.sub.20) alkyl, (C.sub.2-C.sub.6) alkenyl,
(C.sub.2-C.sub.6) alkynyl or (C.sub.6-C.sub.10) aryl
(C.sub.1-C.sub.6) alkyl; and where the one or more subunits B have
the structure ##STR00008## where R.sup.2 is hydrogen or
(C.sub.6-C.sub.10) aryl (C.sub.1-C.sub.6) alkyl and where R.sup.5
is (C.sub.2-C.sub.20) alkylene.
4. The non-woven fabric of claim 3 when seeded with NHEK cells show
cell attachment and proliferation in a Calcein-AM assay.
5. The non-woven fabric of claim 4 where the polyester amide has
the structure (I) where R.sup.1 is (CH.sub.2).sub.8, R.sup.3 is
##STR00009## and R.sup.4 is C.sub.4-C.sub.8 alkylene.
6. The non-woven fabric of claim 1 with drug or other agent that
accelerates healing, matrixed therein.
7. The non-woven fabric of claim 1 where the pore size is tailored
for the utility by variation of electrospinning conditions.
8. A method for making the non-woven fabric of claim 1 comprising
solution electrospinning of poly(ester-amide) and varying thickness
of the non-woven fabric and/or solution concentration and/or
collection distance and/or voltage and/or fiber diameter to vary
pore size.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/750,834, the whole of which is
incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention is directed to poly(ester-amide)
structures fabricated for biomedical application.
BACKGROUND OF THE INVENTION
[0003] Biodegradable poly(ester-amide)s are known for use for
administration of drugs admixed with or chemically linked thereto
applied as a drug eluting film or coating and for use in the
manufacture of medical devices. See WO 02/18477A2; U.S. Pat. No.
6,503,538; and Katsarava, R., et al., Journal of Polymer Science,
Part A, Polymer Chemistry 37, 391-407 (1999). They have not
heretofore been fabricated into a form suitable for burn treatment,
wound coverage, artificial skin, or scaffolds for tissue
engineering.
SUMMARY OF THE INVENTION
[0004] It has now been discovered that the field for biomedical
application of biodegradable poly(ester-amide)s can be enlarged
from the uses previously proposed, by fabricating the biodegradable
poly (ester-amides) into non-woven fabric of electrospun fibers.
For example, it has been discovered that the poly(ester amide)s as
claimed in U.S. Pat. No. 6,503,538 can be fabricated into useful
non-woven fabrics by electrospinning.
[0005] In one embodiment herein, denoted the first embodiment, the
invention is directed to a non-woven fabric consisting essentially
of biodegradable electrospun poly(ester amide) for use for
biomedical application, which is sterilizable and has an average
fiber diameter ranging from 0.1 to 10 micrometer, e.g., 1.0 to 4.0
micrometer, a median pore size ranging from 0.1 to 100 micrometer,
e.g., 2 to 100 micrometers, a surface area ranging from 100 to 300
m.sup.2/g, e.g., 150 to 300 m.sup.2/g, an average thickness ranging
from 0.01 to 0.500 mm, e.g., 0.05 to 0.200 mm, a flexural rigidity
ranging from 10 to 80 mgcm, an average air permeability ranging
from 10 to 100 ft.sup.3/min/ft.sup.2, an average water vapor
transmission rate ranging from 200 to 500 g/m.sup.2/24 hr, a
wettability contact angle ranging from 40 to 80 degrees, e.g., 50
to 80 degrees, tensile stress property ranging from 0.01 to 0.10
kgf/mm.sup.2, tensile strain property ranging from 100 to 800%,
Young's modulus ranging from 0.20 to 20.0 MPa and tensile toughness
ranging from 0.50 to 3.0 MPa.
[0006] As used herein the term "biodegradable" means capable of
being broken down into innocuous products by various enzymes such
as trypsins, lipases and lysosomes in the normal functioning of the
human body and living organisms (e.g., bacteria) and/or water
environment.
[0007] As used herein the term "biomedical application" means
application to clinical medicine.
[0008] The poly(ester-amide) of the non-woven fabric is preferably
one that can be solution electrospun into fibers.
[0009] In a preferred case of the first embodiment, the poly(ester
amide) of the non-woven fabric has a reduced viscosity ranging from
1.0 to 2.0 dL/g, e.g., 1.2 to 2.0 dL/g and is selected from the
group consisting of one or more subunits A, and one or more
subunits B, and combinations thereof, where the one or more
subunits A have the structure
##STR00001##
where R.sup.1 is (C.sub.2-C.sub.20) alkylene, and where R.sup.3 is
hydrogen, (C.sub.1-C.sub.20) alkyl, (C.sub.2-C.sub.6) alkenyl,
(C.sub.2-C.sub.6) alkynyl or (C.sub.6-C.sub.10) aryl
(C.sub.1-C.sub.6) alkyl; and where the one or more subunits B have
the structure
##STR00002##
where R.sup.2 is hydrogen or (C.sub.6-C.sub.10) aryl
(C.sub.1-C.sub.6) alkyl and where R.sup.5 is (C.sub.2-C.sub.20)
alkylene.
[0010] For purposes of scaffold for tissue engineering, the
poly(ester-amide) of the non-woven fabric, in one case has the
structure (I) where R.sup.1 is (CH.sub.2).sub.8, R.sup.3 is
##STR00003##
and R.sup.4 is C.sub.4-C.sub.8 alkylene.
[0011] The poly(ester-amide) within the preferred case described
above, determined to be most preferred of the poly(ester-amide)s
tested, for a scaffold for tissue engineering, has the
structure
##STR00004##
[0012] Suitability for a scaffold for tissue engineering is shown
by cell attachment and proliferation in a Calcein-AM assay of cells
seeded on the non-woven fabric. The seeding and assay for cell
attachment and proliferation are described below. Calcein-AM is
available from Molecular Probes (Eugene, Oreg.). The cells used as
model cells to show attachment and proliferation were
keratinocytes, particularly Normal Human Epidermal Keratinocyte
cell line (NHEK cell line), Catalog No. CC-2501 from Cambrex Bio
Science Walkersville, Inc. (Walkersville, Md.) or Biolife
Solutions, Inc. (Oswego, N.Y.). These cells were chosen for testing
because of easy availability and ease of manipulation. This testing
is relevant to use for a scaffold for tissue engineering because
tissue engineering involves seeding of cells into a scaffold for
proliferation into tissues.
[0013] Other utilities for the non-woven fabrics herein are burn
treatment including adjunct therapy for burn treatment, wound
coverage, partial thickness wound repair, healing acceleration,
artificial skin, barrier to prevent tissue adhesions after surgery
and administration of drugs or other agent physically or chemically
associated therewith, for these purposes.
[0014] The non-woven fabrics herein are advantageous over films of
the same poly(ester-amide) in providing a three dimensional porous
network structure that a two dimensional film does not have.
[0015] So far as scaffolds for tissue engineering are concerned,
the non-woven fabrics herein are advantageous over films of the
same poly(amide-ester) because they have a larger surface area for
the cells to attach to and proliferate.
[0016] So far as utility for administration of drug or other agent
is concerned, the drug or other agent, e.g., drug or other agent is
matrixed into the non-woven fabric.
[0017] We turn now to determination of the various properties of
the non-woven fabrics herein.
[0018] Sterilizability is determined as follows: Samples of
nonwoven fabrics are cut to the size of the bottoms of wells of a
96-well microplate (1/4 inch). The samples are attached around the
bottom edges with 15% poly(ester-amide) in chloroform solution. A
microplate with samples therein is placed into a Medi-Plus ethylene
oxide bag, the bag is sealed and air plasma sterilization is
carried out using a Harrick Plasma Cleaner model PDC-32G (Harrick
Scientific, N.Y.) on high setting for five minutes.
[0019] Fiber diameter and pore size are determined as follows based
on scanning electronmicroscopy (SEM) pictures using Scion Image for
Windows (www.scioncorp.com/pages/scionimagewindows.htm): Pore size
of nonwoven and average fiber diameter are measured from the SEM
images using the Scion program. Electrospun fibrous
poly(ester-amide) mats are sputter coated with gold for thirty
seconds using a BAL-TEC sputter coater (Manchester, N.H.) (Bal-Tech
SCD050), and the sputter coated mats are observed using a Hitachi
S4500 (Mountain View, Calif.) scanning electron microscope and an
accelerating voltage of 10 kV.
[0020] Surface area is determined as follows: Surface area is
analyzed using a Brunauer, Emmett and Teller (BET) surface area
analyzer from Porous Materials, Inc. (Ithaca, N.Y.). A poly(ester
amide) sample is cut and weighed and is then placed in a BET tube.
The test is run at -195.76.degree. C. with adsorbate nitrogen gas
entering the system at 20 microns/minute under vacuum. One mat is
used for providing all the samples for obtaining average surface
area data.
[0021] Thickness is determined according to ASTM D1777-96 as
follows: Measurements are carried out using a Sherman W. Frazier
Compressometer using a circular, 9.525 mm diameter presser foot.
Ten measurements are made at a pressure of 0.023 MPa (3.4 pounds
per square inch) to obtained an average value.
[0022] Flexural rigidity is measured according to ASTM D1388 as
follows: Standard commercially available spun bonded polypropylene
(40 GMS, i.e., 40 gms per square meter) nonwoven samples are cut
into 6.times.1 inch strips. Poly(ester amide) constructs are cut
into 3.times.1 inch strips. The strips are mounted on a horizontal
platform with one end sloping at a 45 degree angle. Each strip is
slowly pushed off the platform in such a way that it overhangs. The
fabric bends down and from the length and weight per square
centimeter, the flexural rigidity G (stiffness) is calculated
according to the following equation.
G=Wxc.sup.3
where W=mg/cm.sup.2 (the weight per unit area) and c is the bending
length--the length of overhang in cm/2. The flexural rigidity of
the spun bonded polypropylene is used as a control. The units are
denoted with the terminology mgcm. Five measurements are made to
obtain an average value.
[0023] Air permeability is measured according to ASTM D737-96 as
follows: Circular samples with a minimum diameter of 3.5 inches are
mounted on a Frazier precision instrument (Silver Spring, Md.). The
rate of airflow through the fabric is measured under a differential
pressure range of 0-1.0 inches of water. The data is expressed in
ft.sup.3 air/min/ft.sup.3. The air permeability of spun bonded 40
GSM polypropylene nonwoven material is used as a control. Five
measurements are made to obtain an average value.
[0024] Water vapor transmission rate is determined according to
ASTM D6701-01 as follows: Samples are cut into 2.5 inch diameter
circles and weighed to obtain sample density (gms/m.sup.2)10 mL of
distilled water is placed into the bottom of model 305 water vapor
permeability cups. Then the samples are mounted onto the cups. Each
assembled system including cup, fabric and water is weighed at 0,
0.5, 1, 3, 5, 12, 24 and 48 hours. The test is performed at
21.degree. C. and 65% relative humidity. Three measurements are
made to obtain an average value for water vapor transmission rate
(water vapor permeability). The water vapor transmission rate is
calculated according to the equation (G/t)A where G is the weight
change in grams, t is the time during which G occurred, in hours,
and A is the test area (cup's mouth area) in m.sup.2. The results
are expressed in grams moisture/m.sup.2 fabric/24 hours. The water
vapor transmission rate of spunbonded polypropylene 40 GSM
nonwoven, is used as a control.
[0025] Wettability contact angle is determined as follows: Film of
each poly(ester amide) is cast by pouring a layer of approximate
thickness of 0.500 mm of 7% weight/weight, poly(ester-amide) in
chloroform solution, onto a Teflon.RTM. plate. When a uniform
thickness layer is obtained, the plate is covered by a watch glass
to decrease evaporation rate. Each film layer is dried at room
temperature for twenty-four hours. After the twenty-four hour
drying time, each film is pulled from its Teflon.RTM. plate and cut
into three 0.5 inch by 0.5 inch samples. The samples are mounted
onto the contact angle analyzer (Hingham, Mass.) stage with double
sided tape. Each stage is inserted into the analyzer and a small
amount of distilled water or methylene iodide (CH.sub.2I.sup.2) is
dropped onto the sample and brought into the viewing area. The
height and 1/2 the width of the droplet are measured and the
contact angle is calculated according to the following
equation:
cos .theta. = x 2 - y 2 x 2 + y 2 where ##EQU00001##
x=1/2 drop width, y=drop height, .theta.=contact angle.
[0026] Tensile properties are determined as follows: Tensile
stress, tensile strain, Young's modulus and tensile toughness of
poly(ester amide) and spunbonded 40 GSM polypropylene are measured.
The samples are cut into 1.times.6 cm rectangular shapes and are
mounted with vertical orientation in an Instron testing machine,
model 1166. Tests are performed using a gauge length of 50 mm and a
cross-head speed of 50 mm/minute. Average fabric thickness is used
for calculating the tensile properties. The strength of a
spunbonded polypropylene 50 GSM nonwoven material is used as a
control. Five specimens are tested to obtain the average tensile
properties.
[0027] Biodegradability is determined by in vitro
.alpha.-chymotrypsin catalyzed hydrolysis as described in
Katsarava, R., et al., Journal of Polymer Science: Part A. Polymer
Chemistry 37, 391-407 (1999).
[0028] Reduced viscosities are determined as follows:
[0029] Each poly(ester amide) (PEA) polymer is dissolved with
m-cresol to a 0.25 g/dL concentration. After the PEA polymers are
dissolved, the solution is poured into a model C572 Glass Cannon
capillary viscometer. The capillary viscometer is placed into a VWR
Scientific Model 1120 Constant Temperature Circulator and the
temperature held constant at 25 degrees Celsius. Suction is applied
to the solution until it is past the top mark. Once the solution
flows past the mark, timed collection is started. The timed
collection is ended when the solution passes the second mark. The
procedure is repeated 5 times for the pure solvent (m-cresol) and
each polymer solution. The reduced viscosity is calculated using
the following equations.
.eta.=.eta..sub.s/.eta..sub.p Equation 1
.eta..sub.s=solution time (seconds) .eta..sub.p=m-Cresol time
(seconds)
.eta..sub.sp=.eta.-1 Equation 2
.eta..sub.reduced=.eta..sub.sp/concentration (0.25 g/dL) Equation
3
REFERENCE
[0030] Jan F. Rabek, "Experimental methods in polymer chemistry",
Wiley-Interscience, NY, 1980, Chapter 9 "Viscosimetric methods",
pp. 123-136.
[0031] In another embodiment herein, denoted the second embodiment,
the non-woven fabric of the first embodiment is made by a method
comprising solution electrospinning of poly(ester-amide) and
varying thickness of the non-woven fabric and/or solution
concentration and/or collection distance and/or voltage and/or
fiber diameter to vary pore size.
DETAILED DESCRIPTION
[0032] The polymerizations to provide the poly(ester-amide)s can be
carried out by an interfacial technique or by active
polymerization.
[0033] The poly(ester-amide)s described above, can be prepared by
active polymerization as described in Katsarava, R., et al.,
Journal of Polymer Science: Part A: Polymer Chemistry 37, 391-407
(1999); U.S. Pat. No. 6,503,538; and in WO 02/18477A2.
[0034] We turn now to the interfacial technique. This is described
at pages 270-271 of Seymour/Carraher's Polymer Chemistry, Fifth
Edition (2000) which is incorporated herein by reference.
Description of the technique there includes the following "Many of
the reactions can be carried out under essentially nonequilibrium
conditions. The technique is heterophasic, with two fast-acting
reactants dissolved in a pair of immiscible liquids, one of which
is usually water. The aqueous phase typically contains the Lewis
base--a diol, diamine or dithiol--along with any added base or
other additive. The organic phase consists of a Lewis acid, such as
an acid chloride, dissolved in suitable organic solvent, such as
toluene, octane or pentane. Reaction occurs near the
interface."
[0035] The poly(ester-amide)s made for testing herein are as
follows:
##STR00005## ##STR00006##
[0036] GJ1, GJ2, GJ4 and GJ5 were synthesized with the lysine unit
being in the benzyl ester form. For GJ1 and GJ2, the benzyl ester
of the lysine unit was more than 90% converted by hydrogenolysis to
the free acid form for electrospin processing. For GJ4, 40% of the
benzyl ester in the lysine unit was converted by hydrogenolysis to
the free acid form for electrospin processing. GJ5 was left
entirely with the lysine unit in the benzyl ester form for
electrospin processing.
[0037] GJ1, GJ2, GJ4 and GJ5 were made by active
polymerization.
[0038] (8P4) and (8P6 ACP) were made by active polymerization.
[0039] (8P4 ICP) was made by the interfacial technique.
[0040] Reduced viscosities for the above are set forth in Table 1
below.
TABLE-US-00001 TABLE 1 Structure .eta..sub.red (dL/g) GJ1 1.698 GJ2
1.571 GJ4 1.616 GJ5 1.415 8P4 1.433 8P6 ICP 1.768 8P6 ACP 1.554
[0041] The molecular weight can be determined from the reduced
viscosities.
[0042] We turn now to electrospinning of the poly(ester-amide)s
into fibers and formation of non-woven fabric.
[0043] Solution or melt electrospinning can be used.
[0044] Described below is the lab set up for the non-woven fabric
production herein by solution electrospinning. However, any
solution electrospinning system including conventional ones can be
used.
[0045] For admixture of drug or other agent in the non-woven
fabric, for example, drug or other agent that accelerates wound
healing, the drug or other agent can be incorporated into polymer
solution prior to solution electrospinning.
[0046] In the experiments carried out, polymer solutions were
placed in a horizontally oriented 5 cc glass syringe fitted with a
24 gauge blunt end needle. The collection plate was a wire mesh
taped to three layers of wax paper on the collecting face. The wire
mesh was connected to a grounding wire and was positioned 10-15 cm
from the needle. The voltages applied to the needle ranged from 9
to 20 kV. Flow rates tried ranged from 0.01 mL/min to 0.10 mL/min.
Preferred conditions determined were 0.02 mL/min flow rate, 15 cm
distance between needle end and collection plate and 11 kV voltage
applied to the needle.
[0047] Droplets are formed at the needles end. The charge on the
needle provides an electric charge in the droplets emitting
therefrom to overcome the surface tension of a droplet to produce a
jet of polymer giving rise to unstable flow toward the collecting
plate manifested by a series of electrically induced bending
instabilities/whipping motions and evaporation of solvent and
production of elongated polymer fibers and deposit thereof on the
wax paper of the collection plate as a non-woven fabric of the
polymer.
[0048] The solvent selected for dissolving a poly(ester-amide) for
the solution electrospinning should provide dissolution within 24
hours at room temperature and solution viscosity and evaporation
rate suitable to produce fiber by solution electrospinning. A
solution viscosity of 1-20 poise, a surface tension for the
solution of 33-35 dyne/sm and a solvent evaporation rate of at
least 1.0 g/m.sup.2/h are aimed for.
[0049] Since comparative data was being determined, a solvent was
sought that would dissolve all seven specific poly(ester-amide)s in
24 hours at room temperature. This criterion was found to be met by
both dimethylformamide (DMF) and chloroform. Chloroform was
selected for use in experiments because it produced higher
viscosity poly(ester-amide) solutions compared to an equal
concentration of the same poly(ester-amide) in DMF and a higher
evaporation rate so that fibers would more likely solidify and dry
before reaching the collection plate.
[0050] The most uniform poly(ester-amide) fibers were observed at
12.5, 15% and 17% concentration of poly(ester-amide) in chloroform
and 15% and 17% were chosen for further test work.
[0051] GJ1 was not able to be solution electrospun. It wouldn't
form fibers and the solution would just create spray droplets.
However, it may be able to be melt electrospun.
[0052] GJ4 provided the best solution electrospinning results--a
single thin filament pulled out of the droplet during
electrospinning.
[0053] In the experiments carried out, the poly(ester-amide)
non-woven fabrics obtained had average fiber diameter ranging from
2 to 4 micrometer, a median pore size of 50 micrometer, a surface
area of 220 m.sup.2/g, an average thickness of 0.1 mm, a flexural
rigidity ranging from 4 to 65 mgcm, an average air permeability
ranging from 25 to 90 ft.sup.3/min/ft.sup.2, an average water vapor
transmission rate ranging from 280 to 430 g/m.sup.2/hr, a
wettability contact angle ranging from 60 to 75 degrees, tensile
stress property ranging from 0.035 to 0.095 kgf/mm.sup.2, tensile
strain property ranging from 125 to 795%, Young's modulus ranging
from 0.9 to 14.5 MPa and tensile toughness ranging from 0.90 to
2.10 MPa.
[0054] Pore size can be varied by varying thickness of the
non-woven fabric. The greater the thickness, the smaller the pore
size. Increasing the solution concentration causes increase in
fiber diameter. Fiber diameter is related to collection distance.
For example, in experiments carried out on poly(ester amide) 8P4,
electrospun at 15% concentration in solution, use of a collection
distance of 15 cm created fiber diameter of 2 .mu.m whereas use of
a collection distance of 10 cm created a fiber diameter of 2.35
.mu.m. At 20 cm, the fiber diameter becomes larger because the
collection plate is beginning to be moved outside the electrical
charge field resulting in lesser force to draw the fiber to its
full extension. Thus the collection distance had an initial inverse
effect on fiber diameter as the collection distance increased.
Fiber diameter increases with voltage increase. In experiments
carried out on poly(ester amide) 8P4 at 15% concentration in
solution at voltage levels of 9, 11, 15 and 20 kV, as applied
voltage was increased, the fiber diameter first is increased and
then is decreased and number of bead defects increased with
increasing voltage. Variation in fiber diameter can be used to vary
porosity. For a given coverage, g/m.sup.2, increase in fiber
diameter can provide increase or decrease in pore size.
[0055] Testing for cell attachment and proliferation (tissue
engineering scaffold utility) was carried out as follows:
[0056] Firstly, all seeding was carried out as follows: Normal
human epidermal keratinocytes (NHEK cells) were plated in a
monolayer in 75 cm.sup.2 tissue culture flask and cultured till the
cells reached three passages. After the third passage, the cells
were removed by trypsin treatment, counted, and seeded onto the
constructs at a density of 10,000 cells/well. The constructs were
maintained in an incubator at 37.degree. C. with 5% CO.sub.2. The
medium was changed every three days.
[0057] Cell attachment as a result of seeding was determined as
follows:
[0058] To assay the cells attached, the medium was removed and
wells were rinsed with Hanks Balanced Salt Solution (HBSS) without
Mg.sup.2+, Ca.sup.2+, and phenol red. The constructs were exposed
to a Calcein-AM solution (1:250 HBSS without phenol red) for thirty
minutes. Cell numbers were indicated directly by the relative
fluorescence units (RFU) obtained from a Spectrafluor. Calcein-AM
fluorescent pictures were obtained by Zeiss optical fluorescent
microscope.
[0059] To determine cell proliferation, the cells were assayed at
day 1, 3 and 7. A Spectrafluor and Zeiss optical fluorescent
microscope collected the readings.
[0060] Of the poly(ester amide)s specifically described above, 8P4
was considered the model for nonwoven fabric production and the
following results were obtained on nonwoven fabric from 8P4.
[0061] Results are set forth below:
[0062] For nonwoven fabric from 8P4 from electrospinning from 15%
concentration at feed rate 0.02 mL/min, 11 kV, 10 cm collection
distance, absorbencies (RFU) denoting cell proliferation were
14,000, 18,000 and 31,000 at days 1, 3 and 7 as compared to
absorbencies of 22,000, 26,000 and 44,000 for a well with only NHEK
cells and no scaffold.
[0063] The nonwoven fabric from 8P4 from electrospinning at feed
rate of 0.02 mL/min, 11 kV and 10 cm collection distance had
properties as follows: Average fiber diameter of 3 micrometers,
median pore size of 50 micrometers, a surface area of 220m.sup.2/g,
an average fabric thickness of 0.102 mm, a flexural rigidity of
19.5 mgcm, an air permeability of 31.8 ft.sup.3/min/ft.sup.2, an
average water vapor transmission rate of 427 g/m.sup.2/24 hr, a
wettability content angle of 66.4 degrees, tensile stress of 0.073
kgf/mm.sup.2, tensile strain of 144.5%, Young's modulus of 14.31
MPa, and tensile toughness of 0.902 MPa.
[0064] As indicated above, the nonwoven fabric herein can
incorporate drug or other agent.
[0065] Examples of these are nonwoven fabric incorporating agent
for accelerating wound healing or for burn treatment or adjunct
therapy for burn treatment,, e.g., gallium nitrate, for
administration of nitroxyl radical (e.g.,
2,2,6,6-tetramethylpiperidine-1-oxy radical), e.g., to reduce
intimal hyperplasia in vascular grafts or to reduce tissue adhesion
by retarding smooth muscle cell proliferation, or for
administration of rapamycin (sirolimus) to prevent tissue adhesion
after abdominal or other surgery, or for administration of
therapeutic protein (as suggested by incorporation into the fabric
of the model protein albumin).
[0066] A working example of incorporation of gallium nitrate into
non-woven fabric within the scope of the invention follows:
[0067] Approximately 1.5 g of the poly(ester amide) (8P4) was
dissolved in 4 g of chloroform (CHCl.sub.3 99.8% HPLC grade),
purchased from Aldrich Chemical Co., Inc. Gallium (III) nitrate
hydrate, purchased from Sigma-Aldrich Inc. was dissolved in 500 mg
99.8% anhydrous N,N-dimethylformamide (DMF) (Aldrich), in amounts
of 10 and 500 mg. The DMF-dissolved gallium nitrate was slowly
added (droplet by droplet) to chloroform dissolved PEA without any
visible precipitation, to provide 1.2, 1.0 and 0.2 grams per gram
of poly(ester amide).
[0068] The homogeneous mixed drug (gallium nitrate)/polymer
solution was electrospun at 15 kV under a steady flow rate of 0.025
mL/min using a spinneret with an orifice of diameter 0.2 mm as the
jet with the distances of approximately 15-cm from the collecting
plate. The electrospun fibers were collected in the form of thin
fabric on a metal sheet (10 cm.times.15 cm) wrapped with wax paper.
The fibrous fabric is peeled off the collecting wax paper.
[0069] In order to determine release profiles of gallium nitrate
from the fabric, a calibration curve was prepared as follows:
Solutions of known concentrations of gallium nitrate in chloroform
(not completely dissolved) were extracted with 10-mL deionized
water. The electrical resistance of the aqueous solutions from
extraction was measured. A calibration curve was constructed based
on conductivity (inverse of measured resistance) and known
concentration of gallium nitrate.
[0070] Drug release was established at 1, 2, 3, 4, 5, 8, 12, 18 and
28 days by extraction with water and measuring conductivity and
generating gallium nitrate release profiles for 1.2 grams of
gallium nitrate per gram (8P4), 1.0 gram of gallium nitrate per
gram (8P4) and 0.2 gm of gallium nitrate per gram (8P4). In each
case there was a burst of drug release within the first 5 days. The
drug release profile suggests that release time is independent of
the concentration of gallium nitrate incorporation in the
fiber.
[0071] The role of gallium nitrate in promoting wound healing is
demonstrated by Staiano-Coico, L., J. Surgical Res. 103, 134-140
(2002).
Variations
[0072] The foregoing description of the invention has been
presented describing certain operable and preferred embodiments. It
is not intended that the invention should be so limited since
variations and modifications thereof will be obvious to those
skilled in the art, all of which are within the spirit and scope of
the invention.
* * * * *