Note: Descriptions are shown in the official language in which they were submitted.
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FILAMENTARY MEANS FOR INTRODUCING AGENTS INTO CELLS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/119,082, filed February 8, 1999.
STATEMENT REGARDING FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
Not Applicable.
BACKGROUND OF THE INVENTION
This invention relates to means for the delivery of agents into a living body.
More
specifically it relates to filaments comprising porous bioabsorbable polymers,
which
facilitate the implantation of living cells and other agents, such as drugs,
into specific
tissues, including skin and bone, for the purposes of site-specific drug or
cell release, gene
therapy, and the facilitation of the regeneration of tissue, including the
regeneration of
bone and hair tissue.
Current means for the delivery of agents such as drugs, growth factors,
genetically
modified cells, and the like into a living body include various pharmaceutical
dosage
forms, such as ingestable tablets, patches designed to deliver agents
transdermally, and
surgically implantable devices designed to deliver agents to an implant site.
Early
implantable devices were not bioabsorbable, and had to be surgically removed
after they
had been used for their intended purpose. More recently, implants of
bioabsorbable
polymers have been developed. Such implants are absorbed by the host in which
they are
implanted after, or in the course of serving their intended purpose. One such
device,
disclosed by Dunn et al. in U.S. Pat. No. 5,599,552 ("Dunn et al. '552"), is
an implant of a
porous core of a bioabsorbable polymer, surrounded by a non-porous surface
skin of the
bioabsorbable polymer. That particular device is designed for use in
delivering a
biologically active agent to a living host when implanted therein. The device
disclosed by
Dunn et al. '552 is also designed so that it can act as a matrix to promote
tissue
regeneration at an implant site. (Id.)
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Two other types of implantable devices of bioabsorbable polymers are disclosed
in
U.S. Pat. No. 5,847,012 ("Shalaby et al. '012"). One such device consists of a
bioabsorbable microporous polymeric foam with open-cell pores. The other such
device
consist of an implant with a modified surface, consisting of a surface layer
of
bioabsorbable microporous polymeric foam with open-cell pores. (Id.) The
implants of
Shalaby et al. '012 are designed to accept the agent to be delivered, such as
a medicament
or growth factor, and to deliver the agent to a living patient after
implantation therein.
Textile technologies have also been adapted for use in making biodegradable
woven fabrics as tissue engineering scaffolds. See Introduction of Peter X. Ma
and
Ruiyun Zhang in J. Biomed. Materials Res. 46(1):60-72 (July 1999). The
diameter of the
biodegradable fibers used to produce such woven scaffolds is about 15 pm. Ma
and
Zhang demonstrated that fibers with a considerably smaller diameter, ranging
from 50 to
500 nm could be created from biodegradable aliphatic polyesters. The woven
scaffolds of
Ma and Zhang, and those described therein were designed for use as scaffolds,
and not as
means for delivery of agents to tissue.
The advantage of all the bioabsorbable devices described above was that they
could be implanted into a living host and left in place to do what they were
designed to do,
without the necessity of removal therefrom. The devices would be absorbed by
the host
over time. Of the bioabsorbable devices disclosed in the references described
above, only
the device of Dunn et al. '552 is designed to act as both scaffolding and
delivery agent.
That device has limited flexibility, because of the way it is designed. What
is needed is a
bioabsorbable fiber or composite thereof, which is capable of being processed
into a
scaffolding for tissue formation, and which is capable of delivering agents to
a living host
when implanted therein. The present invention meets that need.
The present invention also meets a need for an inexpensive and relatively
painless
means for regenerating hair. Plastic surgery is one of the few means available
to correct
male pattern baldness, today. In that particular surgical procedure, the
amount of
permanently hair-bearing donor tissue available can significantly affect the
feasibility and
outcome of the procedure. 1~2 vitro growth techniques have been developed to
increase the
amount of hair follicle cells available for use in such procedures. See, e.g.,
Seigi Arase, et
al. Tokushima J. exp. Med 36: 87-95 (1989); and Edoardo Raposio, et al.
Plastic and
Reconstructive Surgery pp. 221-226 (July 1998). What is needed is a relatively
painless
and inexpensive means for the regeneration of hair, one which does not involve
plastic
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surgery or other painful and expensive implantation techniques, preferably, a
technique
which produces hair which looks realistic and similar to other hair on the
same host. The
present invention utilizes a modified form of the bioabsorbable polymeric
means
developed for use in implantable devices, as described above, to deliver hair
follicle cells
transdermally and to promote the regeneration of hair therein.
As is shown in the next section, below, the present invention provides a new
means
for the introduction of agents into a living host, a means which offers
several advantages
over known means in use today, such as those described briefly above.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a filamentary means for the introduction of
agents
into a living host, comprising a filament comprising a solid core and a porous
sheath
which coats at least a portion of the solid core. When the filamentary means
is to be
permanently implanted into a living host, both the solid core and the porous
sheath are
bioabsorbable. When the filamentary means is to be temporarily implanted into
the skin
of a living host to deliver agents, such as cells, therein, the porous sheath
is preferably
bioabsorbable but the core need only be biocompatable, not bioabsorabable.
The solid core is preferably wire when the filamentary means is designed to be
used to deliver an agent, such as hair follicle cells, into the skin of a
living host. The solid
core is preferably glass or ceramic wh;,n the filamentary means is to be used
to deliver an
agent, such as cells or pharmaceutical agents, into bone through implantation
of the
filamentary means into the body of the host.
The porous sheath is preferably in the form of reticulated foam that is well
adhered
to the core but is capable of separating from the core after a period of
several days in vivo.
When the agent to be delivered with the filamentary means is a drug, the
porous sheath is
preferably in the form of a hydrogel and the porosity is on a molecular size
scale.
The filamentary means of the present invention provides means for delivery of
cells or other agents from outside the body of a living host into the skin of
the host, such
as a mammal, with minimal trauma to the host. When the filamentary means is
comprised
of a bioabsorbable core with a bioabsorbable porous sheath which coats at
least a portion
of the core, the filamentary means can be implanted into specific tissue
within a living
host and used to deliver agents to the specific tissue when implanted therein.
The
implantable embodiment of the filamentary means can serve as a surface for
osteoblast
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attachment and as a scaffold for bone regeneration upon implantation into the
bone tissue
of a living host. The above-cited features of the filamentary means of the
present
invention enable it to be used for a variety of purposes including, but not
limited to, hair
follicle regeneration, gene therapy and encapsulated cell delivery, bone
regeneration, and
traps-dermal drug delivery.
Another embodiment of the present invention is a method of making a
filamentary
means for introducing an agent into a living host, comprising providing a
solid filament, a
bioabsorbable polymer, and a pore-forming agent, mixing the bioabsorbable
polymer with
the pore-forming agent, coating the resulting mixture onto at least a portion
of the
filament, and substantially removing or decomposing the pore-forming agent.
Other embodiments of the present invention include devices designed to enable
one to use the filamentary means of the invention to deliver various agents
into a living
host, methods for making such devices, and methods for using the devices of
the present
invention to deliver agents into a living host. The devices and methods of the
present
invention enable one to regenerate hair follicles, to introduce genetically
altered cells or
encapsulated cells to a living host transdermally, to regenerate bones, and to
deliver drugs
transdermally. The devices and methods of the present invention are described
briefly
below.
One embodiment of the present invention is a hair follicle cell implant device
designed to implant hair follicle cells into the skin of a living host.
Another embodiment
of the hair follicle cell implant device of the present invention is a method
of making the
implant device. Yet another embodiment is a method of using the implant device
to
deliver hair follicle cells to implant such cells into the skin of a living
host, preferably into
the scalp of a human being suffering from male pattern baldness. The hair
follicle cell
implant device of the present invention comprises a plurality of filaments,
each of which
has a first end and a second end and comprises a solid core and a
bioabsorbable porous
sheath which coats the solid core, and a semi-rigid backing with the second
end of each
filament embedded therein such that the first end of each filament protrudes
therefrom.
The first end of each filament protrudes from the semi-rigid backing a
sufficient length to
penetrate the skin of a living host when the device is in contact therewith.
The filaments
are preferably spaced the same distance apart as hairs on the normal surface
of skin of the
living host.
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The hair follicle cell implant device of the present invention is preferably
made by
the steps comprising: providing a plurality of filaments, each of which has a
first end and a
second end and comprises a solid core and a bioabsorbable porous sheath which
coats the
solid core; and fixing the second end of each of the plurality of filaments in
a semi-rigid
backing such that the filaments are spaced the same distance apart as hairs on
the skin of a
normal living host, and such that the second end of each of the plurality of
filaments
protrudes from the semi-rigid backing at a depth sufficient to penetrate the
skin of the
living host when placed into contact therewith.
The hair follicle cell implant device is used to stimulate hair growth
according to a
method comprising: seeding the boabsorbable porous sheath at the first end of
each
filament with hair follicle cells, introducing the cells into the skin of a
living host by
puncturing the skin with the first end of each filament, and removing the
device from the
skin after sufficient time has passed to allow the porous coating within the
skin to separate
from the solid core of each filament, leaving the porous coating and hair
follicle cells in
the skin.
One advantage of the hair follicle cell implant device and methods of making
and
using the same is that they provide means for delivering cultured cells
harvested from hair
follicles into the skin of a host, such as the bald scalp of a human male,
such that said cells
are able to generate new hair follicles. The new hair follicles, once
generated, will
continue to grow and be maintained in the dermis of the host. Another
advantage of these
embodiments of the present invention is that they provide a means for the
simultaneous
implantation of hair follicle cells into multiple sites in the skin such that
the spacing
between each individual implant is approximately the same as the spacing
between the
hair follicles in the normal scalp. Regenerated hair grown from follicle cells
implanted in
such a pattern have a natural, cosmetically appealing look. Thus, the present
invention
provides an efficient device and method of restoring a normal density of
normally
functioning hair follicles in the hairless scalp as an effective, natural, and
permanent
remedy for baldness.
In another aspect, the present invention is a method of using the filamentary
means
of the present invention to deliver genetically modified cells into normal
healthy skin,
such that the cells deliver a therapeutically efficacious systemic level of
the desired gene
product. In this embodiment, a filamentary means comprising a filament
comprising a
solid core having a first end and a second end and a porous sheath which coats
at least the
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first end of the solid core, wherein the porous sheath is bioabsorbable, is
used to deliver
genetically modified cells into the skin of a living host, according to the
steps comprising:
providing the filament, seeding the porous sheath at the first end of the
filament with the
genetically transformed cells, introducing the cells into the skin of the
living host by
puncturing the skin with the first end of the filament, and removing the
filament from the
skin after sufficient time has passed to allow the porous coating to separate
from the
filament at the first end of the solid core of the filament, leaving the
porous coating and
genetically transformed cells in the skin.
An advantage of this embodiment of the present invention is that it provides a
means for the delivery of encapsulated or otherwise immunoprotected cells into
normal
healthy skin such that the cells delivered therewith take over the function of
cells in other
organs that have lost their required function due to disease such as diabetes.
Another
advantage of this embodiment of the invention is that it also provides a means
for the
delivery of genetically modified cells into diseased or ulcerated skin to
treat the disease or
provide growth factors to heal the ulcers.
An advantage of another embodiment of the present invention is that it
provides
that a rigid scaffold with a highly porous surface, which can be implanted
into a living
host, and maintained for a long enough period of time to facilitate new bone
formation.
The porous surface enables this embodiment of the invention to be used as a
means for the
delivery of osteoblasts and/or other osteoinductive substances into bone
defects, gaps, or
fusion devices.
Another embodiment of the present invention is a device for delivery of a drug
through the skin, comprising: a plurality of filaments each of which has a
fist end and a
second end and comprises a solid core and a bioabsorbable polymer sheath in
which the
drug is soluble and permeable; a semi-rigid backing having a first side and a
second side,
wherein the second side of the semi-rigid backing defines a reservoir, wherein
each of the
plurality of filaments is fixed in the semi-rigid backing such that the first
end of each
filament protrudes from the first side of the semi-rigid backing and the
second end of each
filament extends to the second side of the semi-rigid backing such that it is
in contact with
the reservoir. An advantage to the device of delivery of a drug through the
skin of the
present invention is that it provides a means for the continuous delivery of
drugs through
the skin that normally do not penetrate skin from a reservoir placed on the
surface of the
skin.
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Other advantages of the filamentary means for the delivery of agents into a
living
body of the present invention will become apparent upon disclosure of the
invention as
described below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. la is a schematic view of the filament in longitudinal section, showing
the
solid core (1) and the porous sheath (2), without details of the porous
structure.
FIG. lb is a schematic view of the filament in transverse section, showing the
solid
core (1) and porous sheath (2).
FIG. 2 is a schematic sectional view of a hair implant device with an array of
the
core-sheath filaments (3) shown imbedded in a semi-rigid backing (4) which
maintains the
filaments in a stable, rigid parallel configuration.
FIG. 3 is a schematic sectional view of the hair implant device with the array
of
filaments (3) imbedded in the semi-rigid backing (4), immersed in a vessel (5)
containing
a tissue culture broth of free floating cells (6) derived from the hair
follicles of a living
host.
FIG. 4a depicts a sectional view of a single filament with cultured cells (6)
contained within the porous sheath (2) surrounding the solid core (1) of a
filament, which
has been implanted in the skin through the full thickness of the dermis (7).
The filament
has been present in the skin for several days during which time the epidermis
(8) has
begun to grow down the outside of the filament.
FIG. 4b depicts a sectional view of the implant site after the filament core
(1) has
been removed by pulling out the semi-rigid backing (4) to which it is attached
out of the
dermis (7) and epidermis (8). Note the resulting separation of the porous,
cultured cell
laden sheath (2) from the core. As shown in this figure, the implanted sheath
has been
present for a long enough time that new matrix cells (9) are beginning to
elongate a new
hair shaft (10).
FIG. Sa depicts a sectional view of a single filament with cultured cells (6)
contained within the porous sheath (2) surrounding the solid core (1) of the
filament,
which has been implanted in the skin through the full thickness of the dermis
(7). In this
case the filament is shown in the state in which it would be if it had been
present in the
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skin only long enough for the porous coating to soften and detach from the
solid core, but
not long enough for the epidermis (8) to grown down the outside of the
filament.
FIG. Sb depicts the implant site after the filament core has been removed by
pulling out the semi-rigid backing to which it was attached as shown in FIG.
Sa. In this
case, pulling out the semi-rigid backing and core has resulted in separation
of the cell
laden porous sheath (2) from the solid core. Sufficient time has elapsed that
the epidermis
(8) has grown over the implant site, the porous bioabsorbable coating has
resorbed, and
the implanted cultured cells (6) have survived and are functioning properly.
FIG. 6 is a schematic representation of filaments comprised of a solid core
(1) and
a porous coating (2) that are bonded together. The process that is utilized to
create the
bonds between the filaments, for example by heating and cooling, preferably is
the same
process that is used to create porosity in the coating
FIG. 7 is a scanning electron micrograph (SEM) of the device described in
Example l, at a scale of 1 mm.
FIG. 8 is an SEM of the device described in Example 1, viewing the wires on
end
showing the exposed tips of the wires and the surrounding coatings of porous,
bioabsorbable polymer, at a scale of 100 pm.
FIG. 9 is an SEM of the end of a single wire of the device described in
Example 1,
showing the morphology of the porous coating, at a scale of 20 Vim.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides filamentary means for delivery of various
agents
into a living host, a means comprising a filament comprising a solid core and
a
bioabsorbable porous sheath. When the solid core is made of bioabsorbable
material, it is
preferably material selected from the group consisting of glass, ceramic, and
polymeric
material. When the solid core is made of a biocompatable material, it is
preferably
material selected from the group consisting of metals or alloys containing the
elements of
iron, nickel, aluminum, chromium, cobalt, titanium, vanadium, molybdenum,
gold, and
platinum. The core of the filamentary means is preferably made of
bioabsorbable material
when the filamentary means is to be used as or as part of an implant to be
permanently
implanted into the body of a living host. The core of the filamentary means is
preferably
made of biocompatable material when the filamentary means is to be used in the
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transdermal delivery of an agent. The bioabsorbable nature of the material of
the core and
sheath of the preferred permanent implant devices of the present invention
enable the
living host into which they are implanted to absorb the implant over time.
The bioabsorbable porous sheath is preferably comprised of a bioabsorbable
polymer, more preferably a bioabsorbable polymer selected from the group
consisting of
poly(lactic acid), poly(glycolic acid), poly(trimethylene carbonate),
poly(amino acids,
tyrosine-derived poly(carbonate)s, poly(carbonate)s, poly(caprolactone),
poly(para
dioxanone), poly(ester)s, polyester-amides, poly(anhydride)s, poly(ortho
esters,
collagen, gelatin, serum albumin, proteins, carbohydrates, polyethylene
glycol)s,
polypropylene glycol)s, poly(acrylate esters, poly(methacrylate esters,
polyvinyl
alcohol), and copolymers, blends and mixtures of said polymers.
A particularly preferred bioabsorbable polymer for use as a bioabsorbable
porous
sheath coating on the solid core is poly(lactic acid) or any of the various
known
copolymers of lactic and glycolic acids such as a copolymer of L-lactide with
dl-lactide
known as poly(L/DL-lactide). Such bioabsorbable polymers have a long history
of safe
clinical use in the form of synthetic absorbable suture materials and have
been utilized
successfully in a number tissue engineering research experiments. Moreover,
these
polymers are thermoplastic and soluble in a variety of organic solvents
enabling their use
in coating wires by known extrusion and solution based processes.
An advantageous feature of the filamentary means of the present invention is
the
porosity of the bioabsorbable porous sheath. Here again the application of
proven
technology can be beneficial in achieving the desired pore size and void
volume of the
porous sheath. A preferred method for creating porosity in the bioabsorbable
polymer
coating involves the use of "blowing agents". These are chemical additives
that
decompose at known temperatures with the liberation of gases that cause
foaming in the
molten polymer and porosity in the resultant cooled material. A number of
useful blowing
agents are commercially available under the trade name of CelogenTM (Uniroyal
Chemical
Co.). One example of a traditional blowing agent is azodicarbonamide. Another
blowing
agent that may be especially useful in the present invention due to its
compatibility with
bioabsorbable polymers is urea dicarboxylic acid anhydride, described in U.S.
Patent
4,104,195, the teachings of which are incorporated herein. The use of blowing
agents can
produce both open cell and closed cell foams. In the present invention open
cells are
desired and closed cells are to be avoided. Thus the conditions used in the
manufacture of
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the porous coating are preferably optimized to achieve an open cell structure
known as
"reticulated" foam.
The filamentary means of the present invention can be designed to deliver a
variety
of different agents, depending upon the porosity and composition of the porous
sheath.
The agent delivered with the filamentary means of the present invention is
preferably
selected from the group consisting of: cells, growth factors, drugs,
recombinant molecules,
cell recognition factors, cell binding site molecules, cell attachment
molecules, cell
adhesion molecules, proteins, glycoproteins, carbohydrates, naturally
occurring polymers,
synthetic polymers, semi-synthetic polymers, and recombinant polymers.
The porous sheath of the filamentary means is designed to deliver the agent
into a
living host when the agent is coated on the outer surface of the sheath, or
mixed,
dissolved, or imbedded within the porous sheath. The porous sheath preferably
defines
pores which are substantially interconnected and large enough to admit the
agent. The
pores of the porous sheath are preferably open pores produced using blowing
agents, as
described below. The pores are preferably large enough to admit molecules
ranging in
molecular weight from about 100 to about 3,000,000 Daltons, more preferably
ranging
from about 500 to about 100,000 Daltons. Alternatively, the porous sheath
preferably
defines pores which range in size from about 0.1 micrometers to about 500
micrometers,
more preferably which range in size from about 10 to 200 micrometers.
The extent to which the porous sheath coats the core of the filamentary means
varies according to the application in which the filamentary means is to be
used. For
example, when only one end of a filament is to be combined with an agent
before being
used to implant the agent into a living host by puncture the skin of the host,
only that end
of the filament need include the porous sheath. In contrast, when an entire
filament is to
be used to deliver an agent to a host, such as happens when the entire
filament is
implanted into the host, the entire length of the core of the filament is
preferably coated
with the porous sheath.
A discussion of more specific preferred features of the filamentary means of
the
present invention, as used in additional embodiments of the present invention
follows. It
is contemplated that additional advantages and features of the present
invention will
become evident to one of ordinary skill in the art of the present invention
upon review of
the present disclosure.
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Hair follicle regeneration.
One of the embodiments of the present invention provides materials and methods
for a tissue engineering approach to hair follicle regeneration. Tissue
engineering is
generally defined as the technology of restoring defective or missing tissues
or organs by
the implantation of living cells that have been cultured and multiplied
outside the body. A
more detailed description of the philosophy and techniques of tissue
engineering has been
published by C.W. Patrick Jr., A. G. Mikos and LV. McIntire, eds., Frontiers
in Tissue
En ing eerin~, Elsevier Science, Inc., New York, 1998, the teachings of which
are
incorporated herein.
In a preferred embodiment of the present invention, cells harvested from a few
hair
follicles are multiplied in vitro by tissue culture techniques and re-
implanted into bald
skin, thereby generating hundreds of hairs from each hair that is harvested to
seed the
tissue cultures. A remarkable feature of the present invention is the means by
which the
appropriate cells are introduced into the skin and the unique ability of the
implants to
facilitate regeneration of the cellular architecture of normal hair follicles.
The hair follicle cell implant device of the present invention is comprised of
three
preferred parts: (1) a fine stainless steel or other suitably biocompatible
wire or stiff fiber
that is capable of easily penetrating the skin; (2) a porous coating on said
wire comprised
of a bioabsorbable polymer that is suitable as a support for cell attachment
and growth;
and (3) a means for the semi-rigid backing of multiple coated wires in an
array to facilitate
the implantation of said multitude of wires into the skin and their subsequent
removal at an
appropriate time post-implantation. The stainless steel wire is the core, and
the porous
coating the porous sheath of this particular embodiment of a filament of the
present
invention. The wire and filament produced therefrom have a first end and a
second end.
The porous bioabsorbable coating on the wire can be selected from a variety of
known polymers and the process of applying the coating and forming the porous
structure
can be selected from a variety of known processes. Similarly, the degree of
porosity, the
thickness of the coating, and the diameter of the wire can be varied as
desired to obtain the
optimum performance of the implant and the most efficient neogenesis of hair
follicles. In
general, the wire diameter will be similar to the diameter of the hair that is
to be
regenerated for each individual patient. The thickness of the coating will be
equivalent to
the sum of the various cellular layers comprising the hair follicle that
surround a normal
hair shaft. The preferred coating will not cover the entire length of the
wire. Instead, the
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coating will begin at a position distal to the first end of the wire,
depending on the desired
depth at which the cells are to be delivered from that end of the wire.
The first end of the filament is preferably sharp to facilitate entry into the
skin of
the living host. The sharp shape of the first end can be produced by cutting
the first end of
the wire off at an angle after the wire is coated with the porous
bioabsorbable polymer.
However, it is more preferably produced by cutting the first end of the wire
at an angle
prior to coating the first end with the porous bioabsorbable polymer. The
coating
preferably does not cover the most distal end of the first end of the wire, in
order to allow
first end of the wire to enter the skin of a living host using the lowest
possible injection
force.
A preferred wire for use as the core of the filamentary means of hair follicle
cell
implant device of the present invention is 316L stainless steel. This
particular metal alloy
has been widely used clinically in the form of surgical skin staples. A
typical diameter of
wire for use in this invention that is similar to the diameter of human hair
is about 3 to 5
thousandths of an inch. Although such a fine wire might seem too flexible for
use in
penetration of the skin, the depth of penetration needed is shallow enough to
permit the
implant to have a relatively low aspect ratio (length divided by diameter)
such that
bending of the wire is unlikely to occur. In addition, penetration of the skin
by such a fine
wire sharpened by cutting the end off at an angle will be relatively
atraumatic. As an aid
to penetration and patient comfort, the skin can first be anesthetized and
softened by the
application of an analgesic lotion and covered with an occlusive dressing for
about an hour
prior to implantation of the wires.
A novel method of creating the porous sheath on the wire core to create the
filaments used to make the device of the present invention is to select a wire
core that can
be heated electrically, such as nickel-chromium alloy wire. The wire core is
then coated
with a mixture of polymer and blowing agent below the decomposition
temperature of the
blowing agent. The porous coating is then formed by connecting the wire to an
electrical
current to obtain the precise rate of heating, duration of heating time, and
ultimate
temperature to produce the desired effect. Conducting this operation under the
flow of an
inert atmosphere of nitrogen or argon, or submerged in oil, is beneficial in
protecting the
polymer from oxidation and providing rapid cooling and solidification of the
highly
porous structure created at the instant of blowing agent decomposition.
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Upon obtaining a plurality of fibers, each comprising a desired wire coated
with a
bioabsorbable porous sheath, the fibers are imbedded in a semi-rigid backing
as follows.
The first end of each fiber is placed in a mold. The mold is comprised of a
block of any
suitable material, such as TeflonTM, with defining holes in a surface of the
block that are
just sufficiently large in diameter to accommodate the coated wires, and of a
depth that
corresponds to at least the desired depth that the first end of each fiber is
to penetrate the
skin when the hair follicle implant device is used to implant hair follicle
cells into the skin
of a living host. The spacing between the holes preferably corresponds to the
spacing
between hair follicles in the normal scalp. When the first end of each fiber
is placed in the
mold, the second end of each fiber protrudes from the surface of the mold.
Once the
plurality of fibers has been placed into the mold, a layer of silicone resin
or other suitable
liquid pre-polymer is then coated over the protruding second end of each
filament and
cured. The resulting implant device is preferably removed from the mold by
placing a
layer of adhesive tape with a backing that is puncture resistant over the
cured resin, and
then removing the tape therefrom. Removal of the tape pulls out the wires from
the mold
to yield the finished implant device (see Figure 2). The implant device is
preferably
packaged in an appropriate container for protection of the delicate wires
until use, and
sterilized by ethylene oxide or other suitable means prior to use.
In order to use the above hair follicle cell implant device to regenerate hair
follicles, a suspension of follicle progenitor cells is obtained. These cells
can be harvested
from some of the patient's normal follicles. Alternatively, progenitor cells
can be obtained
from the follicles of living donors or recently deceased organ donors. The
finding that
follicle progenitor cells were not rejected by an unrelated human recipient
has been
published by A.J. Reynolds, C. Lawrence, P.R. Caerhalmi-Friedman, A.M.
Christiano, and
?5 C.A.B. Jahoda, Nature 402: 33-34, Nov. 4, 1999, the teachings of which are
incorporated
herein.
Cells multiplied in a growth medium are loaded into the array of coated wires
by
seeding the porous sheath of the first end of each filament with the cells,
preferably by
wicking the growth medium containing the cells into the porous coatings. The
cells
optionally can be further multiplied by continuing the tissue culture process
after seeding
the porous sheath of each filament with the cells, to ensure that cell
attachment and
spreading within the porous matrix has occurred. The first end of each
filament is then
implanted into the dermis by pressing the seeded implantation device onto the
skin,
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preferably after softening and anesthetizing the skin with the use of an
analgesic lotion
under an occlusive dressing such as TegadermTM (3M Company). After several
days the
bioabsorbable coating separates from the wire. The wires and semi-rigid
backing can then
be removed by pulling off the tape to which they are attached, thereby leaving
the porous
bioabsorbable matrix and attached follicle cells in the dermis. New hair
follicles are
generated in the implant sites as the transplanted cells multiply and sort
themselves into
the appropriate functional layers under the influence of the follicular stem
cells earned
through from the original donor follicles. Each new hair follicle will then
grow a hair
shaft in the space that was formerly occupied by the wire. The hair will have
the same
color and consistency as the donor hair that was used to create the cell
culture. The hair
follicle cell donor is preferably the living host, and the hair regenerated
thereby has the
same color and consistency as that of the host.
Gene thera~,y and encapsulated cell delivery.
In a related embodiment of the present invention, the cells that are obtained
from
hair follicles of a living host can be genetically modified to express gene
products that
benefit the living host when implanted therein. Because the hair follicle
cells are rapidly
multiplying and renewing themselves as hair grows, the genetically modified
cells
implanted as described above will serve as a permanent and continuous source
of needed
substances. An example of such a substance is factor IX which would provide a
cure for
hemophilia B. In this case, new hair growth may not be needed on the scalp and
instead
could be established on other parts of the body such as on the back or the
legs. The
follicles harvested to produce the genetically engineered cultured cells can
be taken from
the same skin in which the new hair will be created. Thus the cosmetic effect
of the
presence of this superfluous new hair growth will be insignificant.
In another embodiment, cells that do not produce hair such as dermal
fibroblasts
can be similarly implanted in the skin. These cells would be suitable for
achieving a
temporary therapeutic effect. For example, bed sores, also known as decubitus
ulcers, are
a significant cause of patient discomfort, infection risk, and health care
cost in nursing
homes. Another major medical problem is non-healing skin ulcers primarily
found on the
lower extremities of patients with poor circulation due to disease such as
diabetes. It is
well known that growth factors such as platelet derived growth factors are
capable of
facilitating rapid wound healing but cannot heretofore be conveniently
administered to
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ulcers that are exuding fluid. In this embodiment of the present invention,
fibroblasts or
other suitable cells are genetically modified to express the desired growth
factors and are
implanted into the ulcer, thereby stimulating tissue regeneration and healing
the ulcers.
Other disorders and diseases of the skin that can be treated with similar
embodiments include lamellar ichthyosis, a disfiguring skin disease
characterized by
abnormal epidermal differentiation and defective cutaneous barner function.
This skin
disorder is caused by the deficiency of an enzyme known as keratinocyte
transglutaminasel (Tgasel), the replacement of which is a potential future
approach to
therapeutic gene delivery in human skin. Thus dermal keratinocytes can be
genetically
engineered to express the needed enzyme and implanted into the skin by the
methods of
the present invention.
In another embodiment, cells that have a very slow rate of division but
provide an
essential function can be transplanted from one part of the body of a donor
into the skin of
a living host. Once transplanted, the cells will benefit from the high
vascularity of the
surrounding tissue. An important example is the transplantation of pancreatic
islet cells as
a treatment for diabetes. When the donor and host are not the same individual,
as is the
case when pancreatic islet cells are transplanted into a living host suffering
from diabetes,
the cells must typically be immunoprotected by encapsulation prior to
transplantation.
Encapsulation prevents the patient's antibodies from destroying the foreign
cells, while
allowing lower molecular weight substances including insulin and glucose to
diffuse in
and out of the capsules containing the donor cells.
A serious deficiency of methods of the prior art of introducing such
encapsulated
cells into the patient is the difficulty of both delivering a large number of
cells and
providing a high surface area implant to ensure good exchange with the blood
supply.
Thus, encapsulated cells of the prior art have been implanted in a manner that
resulted in
an inadequate survival rate and an insufficient output of insulin to cure
diabetes. The
present invention solves these problems by providing a more effective means
for the
delivery of encapsulated cells. Thus a multitude of small implants each
comprising only a
few layers of encapsulated cells delivered by the methods of the present
invention ensures
that the donor cells receive optimal nutrition from the vasculature of the
dermis and
provide an efficacious, glucose responsive release of insulin into the blood
stream.
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Bone regeneration.
In another embodiment of the present invention, fibers of the present
invention are
fused together in a three dimensional structure which provides a highly porous
matrix for
bone regeneration (see Figure 6). In addition to the porosity created by the
spaces
between the fibers which is beneficial for bone ingrowth, the high surface
area of the
porous coating on the fibers facilitates osteoblast attachment. This allows
the option of
seeding the material with osteoblasts to provide a tissue engineered implant.
In this case
the porous coating is preferably selected from polycarbonates such as
poly(trimethylene
carbonate) and tyrosine-desaminotyrosine derived polycarbonates due to their
excellent
compatibility with new bone and the absence of acidic degradation products
that may
contribute to bone resorption or inflammation late in the bioabsorption
process. The core
fiber is preferably a biocompatible, osteoconductive ceramic or glass such as
those known
as "bioglass". The fiber may also be selected from a number of slowly
dissolvable or
bioabsorbable glasses such as calcium metaphosphate glasses.
The process for making the bone regeneration matrix involves providing an
osteoconductive ceramic or glass fiber and coating said fiber with a mixture
of polymer
and blowing agent. The coated fibers are then cut into short lengths and
formed into a
nonwoven web by any of several known methods. The web is then formed into the
desired shape and heated at a temperature that both melts and fuses the
coating on the
fibers and decomposes the blowing agent to yield a reticulated foam structure.
The solid
inorganic fibers are unaffected by this process other than becoming glued
together to form
a rigid structure. The device can then be sterilized, seeded with cells, and
stored in a
frozen state until needed for implantation to regenerate bone. The surgeon can
then sculpt
the material just prior to implantation so that it fits into the bone defect.
If the material is
to be used to regenerate bone within a spinal fusion device, such as an
interbody fusion
"cage", the matrix can be pre-formed to fit exactly the dimensions of the
cage.
Trans-dermal drug delivery.
Another embodiment of the present invention is a trans-dermal drug delivery
device, and a method of using the device. Many drugs could benefit from trans-
dermal
delivery but lack the properties that are required for penetration of the
skin. The present
invention overcomes this problem by providing a physical path through the
stratum
corneum and into the dermis.
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The traps-dermal drug delivery device of the present invention comprises a
semi-
solid backing with a plurality of filaments fixed therein, wherein each
filament comprises
a wire core coated by a porous polymer sheath in which the drug is soluble and
permeable.
Each filament has a first end and a second end. The second end of each
filament is fixed
in the semi-solid backing, such that the first end of each filament protrudes
from one
surface of the semi-solid backing. The semi-solid backing further comprises a
drug
reservoir which is in contact with the second end of each filament.
When the traps-dermal drug delivery device of the present invention is applied
to
the skin of a living host, the first end of each filament penetrates the
outermost barrier
layer of the skin and allow the drug to diffuse slowly through the porous
sheath of each
filament into the blood stream of the living host.
The traps-dermal drug delivery device of the present invention is preferably
made
according to a method similar to the method described above for producing the
hair
follicle cell implantation device of the present invention, disclosed above.
In the present
case, the second ends of each of the plurality of filaments are set in holes
in a release liner
film corresponding to mold cavity holes that are not as deep as those used to
make the hair
follicle cell implantation device. The polymer resin that is used to cover the
protruding
wire ends is a pressure sensitive adhesive with drug blended in, and the
puncture resistant
backing has adequate moisture vapor transmission for long term coverage of the
skin. The
device is then sterilized and packaged. To use the device the patient simply
separates the
protective release liner from the backing, thereby exposing the coated wires
and
drug/adhesive surface, and applies this to the skin in the same manner as with
other trans-
dermal drug delivery patches.
Alternatively, when the drug to be delivered with the traps-dermal drug
delivery
device of the present invention has a very low solubility in the polymer used
to make the
porous sheath of the filaments, the drug can be mixed with the coating polymer
as a
suspension prior to formation of the porous sheath. In this case a slow
release of the drug
is provided directly to the dermis from the drug loaded coating and from there
into the
blood stream. Suitable coating polymers for use in the drug delivery
application include
bioabsorbable hydrogels that have porosity on a molecular scale.
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EXAMPLE 1: Porous coated wires for implantation of follicle progenitor cells.
An array of 21 wires, 0.0035 inches in diameter (nickel-chromium alloy,
California
Fine Wire Co., Grover Beach, CA 93433), was made by imbedding the wires in
epoxy
resin contained in the cut-off end of a tuberculin syringe. The wires were
first placed in a
S 2mm thick disc with 0.0063 inch diameter holes arranged in a pattern of one
surrounded
by 7 surrounded by 13. The wires were pushed into the disc until flush with
the surface.
The opposite surface had various lengths of wire protruding. This surface was
placed in
contact with the liquid epoxy resin mixture such that the protruding wires
became
imbedded. The surface of the disc in contact with the epoxy was first coated
with a thin
layer of petrolatum to prevent adhesion. Upon curing of the epoxy resin, the
disc was
pulled off to expose 21 wires extending exactly 2 mm from the surface of the
4.5 mm
diameter plug of epoxy resin.
A mixture of poly(dl-lactide-co-50%-glycolide) (ResomerTM RG504, Boehringer
Ingelheim, D-55216 Ingelheim am Rhein, Germany) and 5% by weight of
azodicarbonamide (Aldrich Chemical Co., Milwaukee, WI 53201) was melt blended
by
stirnng in a test tube immersed in an oil bath maintained at a temperature of
180 °C. A
small amount of this mixture was placed in the bottom of a SOmI beaker and re-
heated to
give a viscous liquid. The above epoxy resin plug was inverted and the wires
dipped into
the molten polymer mixture to a depth of about 0.3mm and quickly removed. This
produced a thin coating of polymer mixture on the tips of the wires and fine
filaments
pulled away from the melt and attached to the tips.
Crystals of sodium chloride (Morton Popcorn Salt) were placed in an electric
coffee bean grinder and milled into a fine powder. A'/z inch diameter hex bolt
was placed
on end and a nut threaded onto the bolt just far enough to engage the threads.
The cavity
formed by the nut and bolt was filled with powdered salt. A thermometer was
lowered
into the salt and clamped in a vertical position with clamps on a ring stand.
The surface of
the salt was smoothed out with a spatula. The bolt was heated with a propane
torch until
the temperature of the salt reached 240 °C. The polymer-coated wires
were then dipped
into the hot salt, pushed about half way in and then quickly pulled out. The
adherent salt
was removed by dipping the wires in water until the salt dissolved. The water
was
removed from the device by gently blotting with tissue paper. The device was
placed back
in a tuberculin syringe barrel for protection and stored in a desiccator.
Scanning electron
micrographs of device are shown in Figures 7-9.
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EXAMPLE 2 - Use of the device of Example 1 to produce new hair follicles.
The device of Example 1 is soaked in ethanol for 5 minutes and then rinsed
with
sterile water. The excess water is removed from the porous structure by
blotting with a
sterile, lint-fee surgical sponge. This process serves both to sterilize the
polymer and to
improve the ability of cells to be wicked rapidly and completely into the
porous polymer.
Human dermal papilla cells that have been multiplied in culture are collected
and
resuspended in an isotonic buffer solution at approximately ten million cells
per cubic
centimeter. The pre-wet implant is then dipped into the suspension of cells
and
immediately injected into human skin where the growth of hair is desired.
Prior to implantation, the skin is wiped with gauze soaked in 70% isopropanol
and
then wiped with gauze soaked in BetadineTM. Lidocaine cream is then applied to
the skin
and covered with a TegadermTM dressing for a minimum of one hour. This pre-
implantation procedure serves to kill bacteria and to soften and anesthetize
the skin. After
implanting the device, the exposed epoxy resin plug is covered with a dressing
to prevent
it from being disturbed or dislodged.
Approximately 48 hours after implantation, the dressing is removed. The wires
are
then removed from the skin by pulling on the epoxy resin plug. The cell-laden
porous
bioabsorbable polymer tips remain under the skin, having separated from the
wires as a
result of tissue attachment to the porous polymer and moisture induced
loosening of the
polymer attachment to the metal.
During the next period of several weeks the implanted cells multiply and
organize
into a new hair follicle as the bioabsorbable polymer degrades and is
bioabsorbed. The
growth of new hair indicates that the restoration process is complete.
EXAMPLE 3 - Porous scaffold for tissue-engineered bone.
Poly(70%-L-lactide-co-30%-d,l-lactide) obtained from Purac Biochem, b.v.
(Gorinchem, Holland) is melt blended at about 170 oC with 5% by weight
azodicarbonamide (Aldrich Chemical Co.) and pelletized. A proprietary
bioabsorbable
glass fiber produced by MO-SCI Corp. (T'witty Industrial Park, Rolla, MO
65401) is
obtained as a monofilament single strand that is 100 microns in diameter.
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A cladding extrusion die is made such that the 100 micron diameter fiber can
pass
through the die while polymer is extruded to cover and clad the moving fiber.
The
polymer/azodicarbonamide blend is extruded as a cladding on the glass to give
a cross-
sectional area ratio of 2:1 core to sheath.
The fiber is cut into 3 to 5 millimeter lengths and added to a beaker of water
with
stirring. The resultant slurry is then poured onto a Buchner funnel equipped
with a coarse
glass frit and the water allowed to drain. This produces a "wet-laid" non-
woven web of
fibers. The web is dried under vacuum and carefully transferred into a wire
basket. The
basket is lowered into a beaker of peanut oil heated to 240 oC for about one
second and
then immersed in a beaker of hexane. The hot oil causes the polymer to melt
and the
azodicarbonamide to decompose into gas, thereby converting the polymer into an
open-
cell foam. The cool hexane causes the polymer to solidify, thereby preserving
the porous
structure and adhering the glass fibers together at every point that they
touch. The hexane
also dissolves and removes the oil from the structure without affecting the
polymer.
Chondrocytes are seeded onto the structure from an aqueous suspension causing
the cells to wick into the porous polymer coating on the glass fibers. The
structure can
then be implanted in a bone defect immediately or allowed to mature in culture
prior to
implantation. In either case the cells will be retained in the microporous
structure of the
coating while the macroporous structure of the adhered fibers will allow ample
room for
tissue ingrowth and osteoinduction of new bone due to the seeded cells.
While the present invention has now been described with some detail and
specificity, those skilled in the art will appreciate the various
modifications, including
variations, additions, and omissions, that may be made in what has been
described.
Accordingly, it is intended that the scope of the present invention be limited
solely by the
broadest interpretation that lawfully can be accorded the appended claims.
