The present invention relates to water-insoluble biocompatible
compositions formed from one or more chemically modified polyanionic polysaccharides,
and more specifically to compositions of these chemically modified polyanionic polysaccharides
and hydrophobic bioabsorbable polymers.
Polyanionic polysaccharides are polysaccharides, also called
glycans, containing more than one negatively charged group (e.g., carboxyl groups
at pH values above 4.0); they consist of long chains having hundreds or thousands
of basic repeat units. These molecules may differ in the nature of their recurring
repeat units, in the length of their chains, and in the degree of branching. There
are two major types of polyanionic polysaccharides: homopolysaccharides, which contain
only a single type of monomeric unit, and heteropolysaccharides, which contain two
or more different types of monomeric units.
Polysaccharides naturally occur in a variety of tissues
in the body and in some cases associate with proteins in complex macromolecular
structures. Examples include proteoglycans, found in the jellylike ground substance,
or extracellular matrix, filling the space between the cells of most tissues. Proteoglycans
are also present in cartilage, tendons, skin and in the synovial fluid. Likewise,
glycosaminoglycans are water-soluble polysaccharides found in the ground substance
of connective tissue, and are highly charged linear polyanions having the general
formula (AB)n, where A is a uronic acid residue and B is a hexosamine.
Hyaluronic acid (HA) and its salt sodium hyaluronate is
an example of a naturally occurring glucosaminoglycan, or mucopolysaccharide that
is a common extracellular matrix component. HA is ubiquitous within the human body
and exists in a wide range of forms in a variety of tissues including synovial fluid,
vitreous humor, blood vessel walls, pericardial fluid, and umbilical cord.
Hyaluronic acid in chemically modified ("derivatized")
forms, is useful as a surgical aid, to prevent adhesions or accretions of body tissues
during the post-operation period (e.g., U.S. Patent No. 5,017,229). The derivatized
HA in the form of a gel or membrane is placed over and between damaged tissue surfaces
in order to prevent adhesion formation between apposing surfaces. To be effective,
the gel or film must remain in place and prevent tissue contact for a long enough
time so that when the gel finally disperses and the tissues do come into contact,
they will no longer have a tendency to adhere.
Chemically modified HA can also be useful for controlled
release drug delivery. Balazs et al., 1986, U.S. Patent No. 4,582,865, states that
"cross-linked gels of HA can slow down the release of a low molecular weight substance
dispersed therein but not covalently attached to the gel macromolecular matrix".
Sparer et al., 1983, Chapter 6, pages 107-119, in Roseman et al., Controlled
Release Delivery Systems, Marcel Dekker, Inc., New York, describes sustained
release of chloramphenicol covalently attached to hyaluronic acid via ester linkage,
either directly or in an ester complex including an alanine bridge as an intermediate
Danishefsky et al., 1971, Carbohydrate Res., Vol.
16, pages 199-205, describes modifying a mucopolysaccharide by converting the carboxyl
groups of the mucopolysaccharide into substituted amides by reacting the mucopolysaccharide
with an amino acid ester in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride ("EDC") in aqueous solution. They reacted glycine methyl ester with
a variety of polysaccharides, including HA. The resulting products are water-soluble;
that is, they rapidly disperse in water or in an aqueous environment such as is
encountered between body tissues.
Proposals for rendering HA compositions less water-soluble
include cross-linking the HA. R.V. Sparer et al., 1983, Chapter 6, pages 107-119,
in T.J. Roseman et al., Controlled Release Delivery Systems, Marcel
Dekker, Inc., New York, describe modifying HA by attaching cysteine residues to
the HA via amide bonds and then cross-linking the cysteine-modified HA by forming
disulfide bonds between the attached cysteine residues. The cysteine-modified HA
was itself water-soluble and became water-insoluble only upon cross-linking by oxidation
to the disulfide form.
De Belder et al., PCT Publication No. WO 86/00912, describe
a slowly-degradable gel, for preventing tissue adhesions following surgery, prepared
by cross-linking a carboxyl-containing polysaccharide with a bi- or polyfunctional
epoxide. Other reactive bi- or polyfunctional reagents that have been proposed for
preparing cross-linked gels of HA having reduced water-solubility include: 1,2,3,4-diepoxybutane
in alkaline medium at 50°C (Laurent et al., 1964, Acta Chem. Scand.,
vol. 18, page 274); divinyl sulfone in alkaline medium (Balazs et al., U.S. Patent
No. 4,582,865, (1986); and a variety of other reagents including formaldehyde, dimethylolurea,
dimethylolethylene urea, ethylene oxide, a polyaziridine, and a polyisocyanate (Balasz
et al., U.K. Patent Application No. 84 20 560 (1984). Mälson et al., 1986,
PCT Publication No. WO 86/00079, describe preparing cross-linked gels of HA for
use as a vitreous humor substitute by reacting HA with a bi- or polyfunctional cross-linking
reagent such as a di- or polyfunctional expoxide. Mälson et al., 1986, EP 0
193 510, describe preparing a shaped article by vacuum-drying or compressing a cross-linked
Reference may also be made to the following: Herrmann,
K., et al., Journal of Materials Science: Materials in Medicine, 5, (1994),
728-731, which relates to heparin-modified polylactide as biodegradable hemocompatible
biomaterial; WO-A-94/01468, which relates to a biomaterial comprising an interpenetrating
polymer network, wherein one of the polymer components is an acidic polysaccharide
or a derivative thereof; WO-A-94/21299, which relates to a biocompatible composition
for tissue augmentation; and WO-A-96/02286, which relates to compositions and methods
for a bioartificial extracellular matrix.
In one aspect, the present invention provides a water-insoluble
biocompatible composition characterised in that it comprises a combination of:
- (a) a water-insoluble polyanionic polysaccharide derivative in the form of a
gel which does not contain covalent cross-links between the polyanionic polysaccharide
molecules, the gel being prepared by combining hyaluronic acid, a polyanionic polysaccharide
and a carbodiimide activating agent;
- (b) a hydrophobic bioabsorbable polymer selected from polyglycolide, polylactide
(D, L, DL), polydioxanones, polyestercarbonates, polyhydroxyalkonates, polylactones
and copolymers thereof.
In preferred embodiments, the polyanionic polysaccharide
is selected from carboxymethylcellulose (CMC), carboxymethylamylose (CMA), chondroitin-6-sulfate,
dermatin sulphate, heparin, heparin sulfate, heparan sulfate, or dermatin-6-sulfate.
More preferably, the polyanionic polysaccharide is CMC or CMA. Also in preferred
embodiments, the biocompatible composition comprises two or more polyanionic polysaccharide
derivatives, e.g. HA and CMC or HA and heparin. Preferred hydrophobic bioabsorbable
polymers include polyglycolide or polylactide, or a copolymer or polyglycolide-caprolactone
or polyglycolide and polylactide, polylactide-polycaprolactone. The compositions
of the invention can be provided in the form of an adhesion prevention composition,
e.g, in a membrane, foam, film, or composition suitable for extrusion. The composition
containing a water-insoluble polyanionic polysaccharide derivative can also be produced
in the form of fibers, or knitted or weaved fabric.
The compositions of the invention which contain a water-insoluble
polyanionic polysaccharide derivative can also be provided as a composite matrix
to support cell and tissue growth and proliferation. For example, any desired cell
type may be cultured in vitro in the presence of one of the water-insoluble
compositions of the present invention to form a water-insoluble matrix that is coated,
impregnated or infiltrated with the cells. Preferably, the cells are derived from
a mammal, and most preferably from a human. In one example, fibroblast infiltrated
matrices may be placed at the site of a skin lesion (e.g., wound or ulcer) to promote
healing of the lesion. Other cell types that can be cultured on the matrices of
this invention include but are not limited to, osteocytes, chondrocytes, keratinocytes
and tenocytes. Matrices impregnated with these cells can be used to aid in the healing
of bone, cartilage, skin and tendons and ligaments, respectively. Matrices can also
be generated which contain a mixture of cell types, e.g., to mimic the cellular
makeup of a desired tissue. The matrices of this invention can also be seeded with
non-differentiated mesenchymal cells that can differentiate into a variety of tissue
specific types upon implantation, or seeded with fetal or neonatal cells of the
desired type. One advantage associated with the use of the water-insoluble compositions
as cellular matrices in vivo is that the matrix is completely biocompatible
and is reabsorbed by the body. Alternatively, matrices impregnated with various
cell types are useful for in vitro diagnostic applications. For example,
matrices infiltrated with fibroblasts can be used to test the efficacy and/or toxicity
of various pharmaceutical or cosmetic compounds.
The compositions of the invention may further include a
drug for use as a drug delivery system. The particular drug used is a matter of
choice depending on the intended use of the composition. Preferred drugs include,
but are not limited to, proteins (e.g., growth factors, enzymes), steroids, non-steroidal
anti-inflammatory drugs, cytotoxic agents (e.g., anti-tumor drugs), antibiotics,
oligonucleotides (e.g., antisense), and biopolymers. When provided for cell and
tissue growth and proliferation, the compositions of the invention may further include
growth factors, and cell attachment proteins or peptides.
In a second aspect, the invention provides a method of
making such a biocompatible composition by combining one or more polyanionic polysaccharides
with a hydrophobic bioabsorbable polymer under conditions sufficient to form the
biocompatible composition. Preferably, the polyanionic polysaccharide is in the
form of a film or foam.
In preferred embodiments of this aspect of the invention,
methods for combining the hydrophobic bioabsorbable polymer and polyanionic polysaccharide
include coating the polyanionic polysaccharide with the hydrophobic bioabsorbable
polymer, e.g., by spraying or brushing the polyanionic polysaccharide with a hydrophobic
bioabsorbable polymer solution; applying hydrophobic bioabsorbable polymer coating
to only one side of the polyanionic polysaccharide composition; admixing the hydrophobic
bioabsorbable polymer with a solution of the polyanionic polysaccharide composition;
dispersing fibers of hydrophobic bioabsorbable polymer into a solution of the polyanionic
polysaccharide composition; and compressing a film of the hydrophobic bioabsorbable
polymer onto the polyanionic polysaccharide composition, e.g., by heat compression
with elevated temperature to ensure the hydrophobic polymer flows onto the polyanionic
polysaccharide composition. Using a water-insoluble derivative of a polyanionic
polysaccharide, the method of the invention can also involve dipping the insoluble
composition into a hydrophobic bioabsorbable polymer solution to coat both sides
of the insoluble polyanionic polysaccharide composition simultaneously. After application
of the hydrophobic bioabsorbable polymer, the composition is dried to remove solvent,
leaving a polyanionic polysaccharide hydrophobic bioabsorbable polymer matrix.
The hydrophobic bioabsorbable polymer solution is made
by dissolving the polymer, polymers, or copolymers in a volatile solvent such as
methylene chloride at a concentration of 0.1 to 50% (w/w); preferably 0.5 to 20%
(w/w); more preferably 0.5 to 5% (w/w); and most preferably 1.0 to 3.0% (w/w).
In another aspect, the invention provides a method for
promoting cell growth and proliferation in vitro. In this aspect, the method
includes the steps of obtaining a sample of cells, admixing the cells with the water-insoluble
biocompatible matrix containing a water-insoluble derivative of a polyanionic polysaccharide
combined with a hydrophobic bioabsorbable polymer, and then culturing the admixture
under conditions sufficient to promote growth and infiltration of the cells into
the matrix. Cells which may be grown according to the method of the invention include
any cell type which can be cultured in vitro; preferably, the cells are mammalian;
and most preferably, they are derived from a human.
In still another aspect, the invention includes a method
for promoting cell growth and proliferation in vivo at the site of an injury, e.g.,
in a mammal, preferably a human. This method includes the steps of obtaining a sample
of cells capable of promoting healing of the injury, admixing the cells with a water-insoluble
biocompatible matrix containing a water-insoluble derivative of a polyanionic polysaccharide
combined with a hydrophobic bioabsorbable polymer, and placing the admixture at
the site of injury in the mammal to promote growth and proliferation of cells at
the site in order to facilitate the healing of the injury.
Embodiments of this aspect of the invention include obtaining
the cell sample directly from the desired tissue and admixing the sample with the
water-insoluble biocompatible matrix; obtaining the cell sample from the desired
tissue and culturing the cells in vitro prior to admixture with the water-insoluble
biocompatible matrix; and obtaining the cell sample from an established cell line
and admixing the cells with the water-insoluble biocompatible matrix. Preferably,
the admixture containing the cell sample and the water-insoluble biocompatible matrix
is cultured in vitro under conditions sufficient to promote proliferation
and infiltration of the cells into the matrix prior to placement at the site of
The cells admixed with the biocompatible matrix for this
aspect of the invention can be of any cell type which is capable of supporting cell
growth and proliferation at the site of injury. For example, the source of the cells
can be xenogeneic to the mammal, but preferably the cells are allogeneic, and most
preferably the cells are immunologically compatible with the mammal. Further, the
infiltrated matrix can contain cells of the same cell type as the cells found at
the site of injury (e.g., from the same tissue), or the matrix can contain cells
which are of a different cell type but which deposit extracellular matrix components
within the biocompatible matrix to serve as a scaffold for cell growth
In preferred embodiments of this aspect of the invention,
the cells are fibroblasts and the infiltrated matrix is placed at the site of a
skin lesion (e.g., a wound, burn, surgical incision, or a dermal ulcer), the cells
are osteocytes, and the infiltrated matrix is placed at the site of a bone injury;
the cells are chondrocytes and the infiltrated matrix is placed at the site of an
injury to cartilaginous tissue; the cells are keritinocytes and the infiltrated
matrix is placed at the site of a skin lesion; the cells are tenocytes and the infiltrated
matrix is placed at the site of an injury to a tendon; or the cells are non-differentiation
The biocompatible matrix used in the methods of the invention
can further contain one or more drugs, e.g., a growth factor to further enhance
growth of the cells and/or an antibiotic to reduce the risk of infection at the
site of placement.
By the phrase "immunologically compatible" as used herein,
is meant that the cells are obtained from a histocompatible donor in order to minimize
the probability of rejection by the immune system of the mammal being treated. Preferably,
the cells are from an individual who has the same or a compatible HLA phenotype.
Most preferably, the cells are obtained directly from the mammal to be treated.
A "polyanionic polysaccharide" (PAS) as the term is used
herein, is a polysaccharide, including non-modified as well as chemical derivatives
thereof, that contains more than one negatively charged group (e.g., carboxyl groups
at pH values above about 4.0) and includes salts thereof, such as sodium or potassium
salts, alkaline earth metal salts such as calcium or magnesium salts.
A "polyanionic polysaccharide derivative", as the term
is used herein, is one or more polyanionic polysaccharides (PAS) that are chemically
modified from the native form. Such modifications can include the addition of functional
groups (e.g., substituted amide groups, ester linkages and amine groups); reactions
that increase the water insolubility of the PAS by covalently cross-linking the
PAS molecules; and reactions that increase the water insolubility of the PAS by
non-covalent interactions as described herein.
By "non-modified polyanionic polysaccharide" is meant a
polyanionic polysaccharide with its native chemical structure intact.
The term "film", as used herein, means a substance formed
by compressing a foam to a thin membrane, by casting into a flat mold and air drying
to a thin membrane, or by compressing a gel or fibers, or by allowing or causing
a gel or fibers to dehydrate.
The term "foam", as used herein, means a substance with
a porous structure formed, for example, by lyophilization of the polyanionic polysaccharide
solutions suspensions, gels, or fibers of the invention.
The term "hydrophobic", as used herein, refers to compounds
or compositions which lack an affinity for water.
The term "bioabsorbable", as used herein, refers to the
ability of a tissue-compatible material to degrade in the body after implantation,
into nontoxic products which are eliminated from the body or metabolized (Barrows,
"Synthetic Bioabsorbable Polymers", p. 243 In High Performance Biomaterials -
A Comprehensive Guide to Medical and Pharmaceutical Applications, Michael Szycher,
ed., Technomic Publishing: Lancaster, PA, 1991).
The term "polymer" as used herein refers to a molecule
made by the repetitive bonding of at least two, and preferably more than two, repeating
monomeric smaller units (e.g., monosaccharide, amino acid, nucleotides, alkenes,
or organic acid units) . Accordingly, the term copolymer refers to a polymer formed
by combination of two or more copolymerized monomeric or polymeric species.
A "biocompatible" substance, as the term is used herein,
is one that has no medically unacceptable toxic or injurious effects on biological
A "water-soluble" film or foam, as the term is used herein,
is one which, formed by drying an aqueous solution of 1% weight/weight ("w/w") unmodified
polyanionic polysaccharide in water, and having dimensions 3 cm x 3 cm x 0.3 mm,
when placed in a beaker of 50 ml distilled water at 20°C, and allowed to stand
without stirring, loses its structural integrity as a film after 3 minutes and becomes
totally dispersed within 20 minutes. A "water-insoluble" film as used herein of
the invention, as that phrase and like terms are used herein, is formed using a
1% aqueous solution of a polyanionic polysaccharide, modified as previously described,
having the same dimensions and similarly allowed to stand without stirring in a
beaker of 50 ml distilled water at 20°C, is structurally intact after 20 minutes;
the film boundaries and edges are still present after 24 hours.
The foams, films and other forms of the invention can be
prepared in colored form, by including a dye or stain in the reaction mixture. Such
colored films and gels can be more easily seen when in place or during placement,
making them easier to handle during surgical procedures than colorless ones.
In general, the compositions of the invention have improved
biocompatible and physical properties over previous compounds. Therefore the compositions
of the invention are especially useful in methods of preventing adhesion formation
between injured tissues. One or more of the compositions of the invention can be
placed between or among injured tissues that tend to form adhesions (e.g., surgical
incisions and trauma) in an amount sufficient to prevent adhesions of the tissues
during the healing process. The compositions act as a temporary barrier between
the tissues and remain in place long enough so that once the composition has been
reabsorbed and the tissues come into contact, the tissues no longer have the tendency
Additional uses include designing nerve guides by forming
the foams, films or gels into tubes or matrices for guidance of axons following
nerve trauma, to foster growth cone elongation while reducing the risk of neuroma
formation. They are also useful as scaffolding for cell proliferation and migration,
e.g., skin regeneration, as well as tendon, ligament and cartilage regeneration.
These substances are also suitable as a vehicle for drug delivery, since the drug
may be introduced either before or after the biocompatible composition has been
formed, allowing a controlled release of the drug to be administered.
The water-insoluble polyanionic polysaccharide compositions
combined with the hydrophobic bioabsorbable polymers have the following additional
advantages over uncoated, chemically modified or unmodified polyanionic polysaccharides
compositions: improved mechanical properties in both the dry and wet states, making
the products stronger and easier to handle and resulting in a longer in vivo
residence time; slower hydration of the polyanionic polysaccharide component to
maintain the adhesive properties and placement of the compositions; and improved
efficacy in preventing post-surgical adhesions due to the addition of the hydrophobic
bioabsorbable polymer component. The compositions can be processed with a hydrophilic
side that adheres to tissue and one non-adhesive, hydrophobic side. The hydrophobic
side will slow hydration of the hydrophilic side, which will adhere to tissue while
the hydrophobic side will prevent other tissue, surgical instruments and gloves
from adhering to the composition.
Polyanionic polysaccharides and their salts may be obtained
from a variety of standard commercial sources. Water-insoluble polyanionic polysaccharide
gels, films and foams can be prepared by any method for use in this invention. The
gels may be generated via the formation of covalent intra- and inter-chain crosslinks
as previously described (e.g., see Sparer et al., supra; DeBelder et al.,
supra; Balazs et al., supra; Mälson et al., supra; and Prestwich
et al., EP Publication No. 0416250A2, 1991). Alternatively, water-insoluble gels
which do not contain covalent cross-links between the polyanionic polysaccharide
molecules may be formed using the methods described in Hamilton et al., U.S. Patent
No. 4,937,270; Burns et al., U.S. Patent No. 5,017,229, see, in particular, column
As disclosed in the last-mentioned reference, polyanionic
polysaccharide-modified HA gels and films are prepared generally by mixing HA with
a polyanionic polysaccharide and an activating agent from a water-insoluble precipitate.
Foams and films of compositions containing soluble polyanionic polysaccharides and
their derivatives can be generated by lyophilizing or freeze drying the solution.
Compositions containing water-insoluble polyanionic polysaccharide composition can
also be treated to generate the desired film, foam, powder or fibers. For example,
to obtain films, the reaction mixture is typically poured into a vessel, e.g., a
tray, having the desired size and shape and allowed to air dry.
Alternatively a film can be formed by compressing a water-insoluble
gel under conditions that permit escape of water, as, for example, by compressing
the water-insoluble gel between two surfaces, at least one of which is porous, as
described, for example, in EP 0 193 510.
Another alternative method of producing sheets of the material
is to subject it to freeze drying. The pore size of the final product can be controlled
by adjusting the initial freezing temperature and drying conditions. Curved surfaces
and other shapes can be produced in a similar manner by initially casting the water-insoluble
gel onto a negative image surface and then processing as described. The dried sheet
can be processed further, if desired, by pressing to a defined thickness, e.g.,
in a Carver laboratory press. This is particularly useful for applications requiring
placement of a thin film between anatomical structures where space is limited, and
for imparting additional mechanical strength.
The formation of foams, fibers and other shapes or articles
can also be accomplished using techniques well-known in the plastics and textile
For instance, foams of the water-insoluble polysaccharide
derivatives can be generated by freeze drying procedures that are well known in
the art, e.g., Yannas et al., (U.S. Patent No. 4,280,954) and Dagalakis et al.,
(1980, J. Biomed. Mater. Res., vol. 14, p. 511-528), describe methods
of freeze drying collagen-mucopolysaccharide composites and controlling pore structure.
Typical conditions are temperatures below -20°C and a vacuum below 250 mTorr.
Fibers of the water-insoluble polysaccharide derivatives
can be made by wet spinning procedures that are well known in the art. For example,
Rupprecht (1979, Acta Chem. Scand., vol. 33, p. 779-780) describes the wet
spinning of aqueous hyaluronic acid solutions into an ethanol coagulation bath to
form fibers. Alternatively, fibers of the hydrophobic bioabsorbable polymers can
be made by more conventional melt spinning techniques that are well known in the
art. For example, Wasserman et al. (U.S. Patent No. 3,792,010 and 3,839,297) describe
the manufacture of monofilament and braided polyester sutures of lactide-glycolide
copolymers. The fibers can be made into fabrics of knitting and weaving techniques
well known in the art.
The film and foam derivatives of polyanionic polysaccharide
compositions can be strengthened by dehydrothermal treatment (DHT: 95-105°C
at 200-760mm Hg for 6-24 hours) and combined with hydrophobic bioabsorbable polymers.
For example, bioabsorbable polymers such as polyglycolide (PGA), polylactide (PLA)
and copolymers of PGA/PLA are dissolved in volatile solvents such as methylene chloride,
acetone, ethylacetate, tetrahydrofuran, n-methylpyrrolidone at concentrations of
0.5-50.0% w/w with a preferred range of 1%-3% (w/w). Various ratios of PGA and PLA
can be used including 100% PGA, 85% PGA:15% PLA, 50% PGA:50% PLA and 100% PLA; 1:1
PGA:PLA is preferred. Additionally, other hydrophobic bioabsorbable polymers such
as polydioxanones, polyorthoesters, polyestercarbonates, polylactones (especially
polycaprolactone) and polyhydroxybutyrate/valerate can be used alone or as copolymers,
especially copolymers of PLA and polycaprolactone. These solutions are then sprayed
onto the polyanionic polysaccharide based device using spraying devices such as
a small chromatography sprayer with compressed air or argon gas at 2-20 psi to achieve
a 5-100% weight gain. Coated foams can be pressed into thin membranes at 1.0-5-0
metric tons employing a Carver laboratory press with 1-50 mm spacers or left unpressed
as thick foams.
In one alternative method, the polysaccharide-based materials
and hydrophobic bioabsorbable polymers are laminated together by heat-pressing a
form of the polymer (film, foam, mesh etc.) onto a polyanionic polysaccharide foam
or film. The preferred conditions of lamination depend on the thermal properties
of the various hydrophobic polymers but generally fall within the following ranges:
40-230°C at 0-8 metric tons of compression for 0-5 minutes. In addition, the
hydrophobic polymer can be rendered more hydrophilic following lamination by plasma
In a second alternative method, bioabsorbable polymer fibers
are incorporated into the polysaccharide-based materials by cutting or chopping
the fibers to specific sizes and dispersing them into polysaccharide-based solutions
before casting or lyophilizing into films or foams. The bioabsorbable-polymer fibers
can also be laid onto a substrate as a mesh or matte and then polysaccharide-based
solutions can be cast on top.
In a third method, the polysaccharide-based films and foams
are coated with hydrophobic polymers by means other than the spray-coating method
described above. For example, bioabsorbable polymers such as PGA, PLA and copolymers
of PGA/PLA, PLA/polycaprolactone, and PGA/polycaprolactone can be dissolved in organic
solvents at concentrations of 0.5-50%, preferably 1.0-3.0%. The polymer solution
can then be spread with a drawdown knife or cast on the surface of a polysaccharide-based
film or foam and then dried. Alternatively, the water-insoluble polysaccharide-based
devices can be dipped or soaked in the polymer solution and then allowed to air
dry to achieve incorporation.
In still another method, composite fibers can be made which
contain a water-insoluble polysaccharide derivative core and a hydrophobic bioabsorbable
polymer coating. Aqueous solutions containing polysaccharide derivatives are extruded
through a spinneret or syringe needle into a coagulation bath containing a bioabsorbable
polymer solution, such as PGA/PLA, PLA/polycaprolactone, or PGA/polycaprolactone
dissolved in organic solvent. The water-insoluble polysaccharide-based material
precipitates in the coagulation bath and is simultaneously coated with bioabsorbable
polymer. Alternatively, the water-insoluble polysaccharide-based fiber can be coated
with bioabsorbable hydrophobic polymer after the coagulation stage of the wet-spinning
process by drawing the polysaccharide derivative fiber through a solution of bioabsorbable
The invention is described in more detail in the following
examples. These examples are given by way of illustration and are not intended to
limit the invention except as set forth in the claims.
A solution of HA (5.5 g, 13.7 moles, MW 2,350,000) and
CMC (2.5 g, 9.7 moles, MW 250,000) in water (1 L) was pH adjusted to 4.74 with 0.1M
HCl, after which 1-(-3-dimethylaminopropyl)-3-ethylcarbodiimide (10.6 g, 55.5 moles)
was added. The pH was maintained between 4.6-5.1 for 1 hour by the addition of 0.1
M HCl. The reacted solution was dialyzed in membrane tubing (MW cut off 12-14,000)
for 24 hours against deionized water, pH 4.0. The purified chemically modified HA/CMC
solution was poured into stainless steel trays and lyophilized into solid foam sheets.
Specifically, the temperature of the product was lowered at a ra te of 0.1°C/min
to -20°C. Then the drying cycle was executed with vacuum set at 150 mTorr and
shelf temperature raised at 0.1°C/min to 0°C. The temperature was held
at 0°C for 900 minutes and then raised at 0.1°C/min to 27°C. The
foams were then strengthened by dehydrothermal treatment (105°C at 200 µm
Hg for 24 hours). The foams were then weighed and placed in a polypropylene frame
prior to coating.
Lactide/Glycolide copolymer (2.0 g, 50% PGA: 50% PLA Medisorb
Corporation) was dissolved in methylene chloride (100 ml) . This coating solution
was then sprayed at 5 psi onto the foams using a small chromatography sprayer equipped
with compressed air. A weight gain of 10-15% was achieved by varying the duration
of spraying time based on the size of the foam and the calculated flow rate of spray.
Evaporation of the methylene chloride solvent was slowed by covering the foam immediately
after spraying. After drying, the foams were pressed (1 metric ton, 15 sec, 0.25
mm spacer) into thin films, cut, packaged, and gamma-irradiated at 2.5 Mrad.
Material provided by this method was then evaluated for
prevention of post-surgical adhesions in a rat cecal abrasion model (Goldberg et
al., In Gynecologic Surgery and Adhesion Prevention. Willey-Liss, pp. 191-204,
1993). HA/CMC membranes or foams, Interceed TC7 membranes (Johnson & Johnson), and
HA/CMC films or foams which were coated with PGA: PLA polymer, were placed around
surgically abraded rat ceca, and compared to non-treated controls (animals whose
ceca were abraded but did not receive any treatment). The results from two studies
are shown in Table 1.
TABLE 1 EVALUATION OF HA-BASED DEVICES AND INTERCEED TC7 FOR POSTOPERATIVE
% of Animals with
Adhesions ≥ Grade 2
Control (No Treatment)
HA/CMC Foam w/PGA:PLA
Control (No Treatment)
HA/CMC Foam w/PGA:
These results demonstrate that films and foams coated with
the PGA: PLA polymer consistently reduced adhesion formation compared to the control
group, to animals that received Interceed TC7, and to animals that received either.
HA/CMC films or foams.
In this example, modified HA/CMC powder made according
to the methods of U.S. Patent No. 4,937,270 (4.5 g) was suspended in distilled water
(450 ml) using a high speed blender (20 minutes at 1000 rpm). The resuspended solution
was poured into Teflon coated stainless steel trays and lyophilized into solid foam
sheets. Lyophilization was performed as described in Example 1.
A thin film of polylactide copolymer (90% PLA-L:10% PLA-DL)
was obtained from Medisorb Corporation. The HA/CMC foam and polylactide film were
then heat-pressed together into thin sheets (155-165°C, 15-30 seconds, 1 metric
ton, 0.30 mm spacer). The wet tensile properties of the compositions were evaluated
with an Instron™ Universal Testing System Model 4201 equipped with a 500
g load cell. A test chamber was specifically designed for measuring the mechanical
properties of the samples while immersed in a physiological environment. Results,
shown in Table 2, demonstrate that the load at break under wet conditions was significantly
improved for the HA/CMC foams that were laminated with PLA. In this experiment,
the samples were tested in a specially designed environmental chamber containing
in a physiological environment (buffered saline at pH 7 at 25°C). The initial
grip separation was 25 mm and the crosshead speed was 5 mm/min.
TABLE 2 WET MECHANICAL PROPERTIES OF HA/CMC:PGA/PLA COMPOSITIONS
Wet Load (N)
Wet Elong. (%)
F30719 HA/CMC Foam
0.2 ± 0.06
38.6 ± 3.4
F30719-3 HA/CMC Foam
28.2 ± 20.6
112.2 ± 34.9
F30719-4 HA/CMC Foam:PLA
12.7 ± 3.7
4.1 ± 1/5
In this example, modified HA/CMC powder (4.5 g) was suspended
in distilled water (450 ml) using a high speed blender (20 min at 1000 rpm). A piece
of 100% PGA mesh was placed in a Teflon coated stainless steel tray. The resuspended
HA/CMC solution was poured into the tray and lyophilized into a solid foam sheet
according to the procedure described in Example 1. The foam and mesh composition
was pressed (1 metric ton, 15 seconds, 0.25 mm spacer) into thin sheets and strengthened
by dehydrothermal treatment (100°C for 6 hrs). The wet tensile properties of
the composition were evaluated and are shown in Table 2. The wet strength of the
composition was much greater than the strength of the initial HA/CMC foam.
The results from Examples 2 and 3 indicate that the composite
of the HA/CMC foam and the hydrophobic bioabsorbable polymers have much greater
strength under hydrated conditions (wet load) than HA/CMC foam without the hydrophobic
Hyaluronic acid (5.5 g, 13.7 moles, MW 2,350,000) and carboxymethylcellulose
(2.5 g, 9.7 moles, MW 250,000) were dissolved in one liter of water, and the pH
of the solution was adjusted to 4.75 with 0.1 M HCl. 1-(-3-dimethylaminopropyl)-3-ethylcarbodiimide
(10.6 g, 55.5 moles) was then added and the solution was maintained at a pH between
4.6-5.1 for 1 hour by the addition of 0.1 M HCl. The reacted solution was then dialyzed
(MW cut off 12-14,000) against deionized water, pH 4.0. The purified reaction mixture
was then poured into a polystyrene tray at a casting density of 2.2 g HA/CMC/ft2.
Polyglycolic acid fibers were prepared by cutting Dexon
sutures. The fibers were sonicated in water to produce a mat-like material with
a high degree of fiber entanglement. This material was then hydrated in methylene
chloride to allow the fibers to coalesce after which the fibers were air dried.
The resulting mat-like material was placed on top of the cast HA/CMC reaction mixture
at a density of 0.1 g/ft2. The entire composition was then air dried
to form a bilayer of PGA fibers and modified HA/CMC.
Procedures are well known in the art for seeding and growing
mammalian cells on physical matrices. The purpose of the matrix is to give support
to the cells, to allow the cells to migrate through the matrix, to allow easy handling
of the cells for implantation and to help keep the cells in place once implanted.
The novel PAS composites of the present invention can be used as a matrix for this
purpose. In one example, the PAS derivative hydrophobic bioabsorbable matrix formed
as described in Example 2 is cut to size and shape of a cell culture dish. Mammalian
fibroblasts, isolated from skin by trypsinization, or obtained from a standard cell
line (e.g., available from the ATCC), are cultured at 37°C in a 5% CO2
atmosphere and approximately 95% to 100% relative humidity. Once they are grown,
these fibroblasts are removed from the culture flask by trypsinization and washed
with culture medium containing fetal calf serum. The cell density is adjusted to
approximately 104 to 106 cells/ml.
The matrix is placed in the culture dish with the hydrophobic
side down; the cell suspension is placed on the matrix in the cell culture dish,
ensuring complete coverage of the matrix; and the admixture is incubated at 37°C
and 5% CO2. The cells are grown on the matrix until cell proliferation
throughout the matrix has occurred. The matrix infiltrated with fibroblasts can
then be placed on dermal ulcers, burns and wounds to aid in wound healing or to
act as a skin substitute. The preferred source of the fibroblasts is autologous
tissue. However, in cases where the use of autologous tissue is not convenient,
or the tissue is not readily available, allogeneic or even xenogeneic fibroblasts
can be used. Biocompatible matrices containing xenogeneic or allogeneic cells are
useful for providing extracellular scaffolding to aid in the migration and establishment
of autologous cells during the healing process. Biocompatible matrices which contain
non-autologous cells can also be co-administered (e.g., at the same time, or immediately
following placement of the matrix) with standard immunosuppressive therapies (e.g.,
steroids, azathioprine, cyclosporine) if desired.
Further, the biocompatible matrices can also be impregnated
with drugs or growth factors to prevent infection at the placement site and to enhance
the growth of the cells, respectively. For example, fibroblast infiltrated matrices
containing TGF&bgr;2 are expected to be especially useful in promoting
growth of epidermal tissues.
We have shown that these devices have improved handling
properties and reduce the incidence of post-surgical adhesions in experimental animal
models more successfully than existing products. In these experiments, HA/CMC:PGA/PLA
compositions reduced adhesion formation when compared to animals that received HA/CMC
devices, Interceed TC7 film (marketed by Johnson & Johnson for adhesion prevention),
or untreated control animals.
The water-insoluble compositions of the invention can be
used in abdominal operations, operations of the urogenital tracts, nerve surgery,
joint operations and ophthalmological operations for purposes requiring maintenance
of placement of tissues without adhesion formation. They can also be of use as sealing
agents in anastomotic sites for catheters, bowel anastomoses, endoscopic surgical
procedures, vascular grafts and any prosthetic device requiring gluing together
or sealing of potential leakage sites; as a new biocompatible fiber for processing
into thread, braids, woven and non-woven webs, weaves and mats and sutures for wound
closure; sclerosing agents for varicose vein removal, tumors and aneurisms; artificial
extracellular matrix materials for cell and tissue replacement for skin, tendon,
ligament, bone, cartilage and other tissues and organs.
The time period required to effectively prevent adhesion
will vary according to the type of surgery or injury involved. Generally, the tissues
should remain separated for at least 48 hours, and preferably, for a period of at
least 7 days. Accordingly, the rate of diffusion of the composition used in any
particular situation can be varied, for example, by altering the extent of the composition's
solubility or insolubility, by varying the density of the polyanionic polysaccharide
used, or by varying the thickness of the film, foam, gel or fiber used. These characteristics
can be altered by routine procedures and the properties desired for any type of
surgery or trauma can be determined by routine experimentation using the guidance
of the examples described herein.
Films, foams or gels of the invention can further be used
for drug delivery. For example, in the case where rapid, localized delivery is desirable,
water-soluble compositions within the invention can be used. Alternatively, compositions
containing water-insoluble polyanionic polysaccharides are useful for sustained
release drug delivery. The drug to be delivered can be dispersed within the composition,
or can be covalently bonded to the foam, film or gel as described, for example,
in R.V. Sparer et al., 1983, Chapter 6, pages 107-119, in T.J. Roseman et al.,
Controlled Release Delivery Systems, Marcel Dekker, Inc., New York; and the
foam, film or gel can then be implanted or injected at the locus where delivery