U.S. patent application number 13/347491 was filed with the patent office on 2012-05-17 for biocomposite for artificial tissue design.
This patent application is currently assigned to UNIVERSITY OF SOUTH FLORIDA. Invention is credited to Kerriann Robyn Greenhalgh, Thomas J. Koob, EDWARD TUROS.
Application Number | 20120121531 13/347491 |
Document ID | / |
Family ID | 39184100 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120121531 |
Kind Code |
A1 |
TUROS; EDWARD ; et
al. |
May 17, 2012 |
BIOCOMPOSITE FOR ARTIFICIAL TISSUE DESIGN
Abstract
The present invention concerns a biocomposite useful in
artificial tissue replacement, methods for its production, and
methods of use. The biocomposite can be implanted into humans or
animals as an artificial tissue for treatment of a tissue defect.
The biocomposite can be formed or manipulated into a desired shape
for implantation.
Inventors: |
TUROS; EDWARD; (Wesley
Chapel, FL) ; Koob; Thomas J.; (Tampa, FL) ;
Greenhalgh; Kerriann Robyn; (Tampa, FL) |
Assignee: |
UNIVERSITY OF SOUTH FLORIDA
TAMPA
FL
|
Family ID: |
39184100 |
Appl. No.: |
13/347491 |
Filed: |
January 10, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11901209 |
Sep 13, 2007 |
8093027 |
|
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13347491 |
|
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|
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60844290 |
Sep 13, 2006 |
|
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Current U.S.
Class: |
424/78.18 ;
525/54.1 |
Current CPC
Class: |
A61L 2300/64 20130101;
A61L 27/48 20130101; A61L 27/54 20130101; A61L 27/48 20130101; C08L
89/06 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/78.18 ;
525/54.1 |
International
Class: |
A61K 31/785 20060101
A61K031/785; A61P 43/00 20060101 A61P043/00; C08H 1/00 20060101
C08H001/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
number DGE 0221681 awarded by the National Science Foundation. The
government has certain rights in the invention.
Claims
1. A synthetic biocomposite comprising a biotic material and an
abiotic material.
2. The biocomposite of claim 1, wherein the biotic material is
collagen and the abiotic material is a polymer.
3. The biocomposite of claim 1, wherein the biocomposite is a fiber
comprising an intertwined polymer emulsion and fibrils.
4. The biocomposite of claim 3, wherein the fibrils are collagen
fibrils.
5. The biocomposite of claim 1, wherein the abiotic material is a
polymer emulsion.
6. The biocomposite of claim 1, wherein the abiotic material
comprises a copolymer emulsion.
7. The biocomposite of claim 6, wherein the copolymer is ethyl
acrylate-methyl methacrylate.
8. The biocomposite of claim 1, further comprising a biologically
active agent.
9. The biocomposite of claim 8, wherein the biologically active
agent is a cell.
10. A device comprising a surface with a synthetic biocomposite
comprising a biotic material and an abiotic material.
11. The device of claim 10, wherein the device is a medical
implant.
12. A method for producing a synthetic biocomposite, comprising
polymerizing a biotic material and an abiotic material by emulsion
polymerization, thereby forming the synthetic biocomposite.
13. The method of claim 12, wherein the biotic material is collagen
and the abiotic material is a polymer.
14. The method of claim 12, wherein the biocomposite is a fiber
comprising an intertwined polymer emulsion and fibrils.
15. The method of claim 14, wherein the fibrils are collagen
fibrils.
16. The method of claim 12, wherein the abiotic material is a
polymer emulsion.
17. The method of claim 12, wherein the abiotic material comprises
a copolymer emulsion.
18. The method of claim 17, wherein the copolymer is ethyl
acrylate-methyl methacrylate.
19. The method of claim 12, wherein the biocomposite further
comprises a biologically active agent.
20. The method of claim 19, wherein the biologically active agent
is a cell.
21. A method for treating a tissue defect, comprising applying a
synthetic biocomposite at the site of the defect, wherein the
biocomposite comprises a biotic material and an abiotic material.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 11/901,209, filed Sep. 13, 2007, now U.S. Pat.
No. 8,093,027, which claims the benefit of U.S. Provisional
Application Ser. No. 60/844,290, filed Sep. 13, 2006, each of which
is hereby incorporated by reference herein in its entirety,
including any figures, tables, nucleic acid sequences, amino acid
sequences, and drawings.
BACKGROUND OF THE INVENTION
[0003] Fibrous vertebrate tissues are generally composed of two
types of biopolymers that are responsible for the mechanical
properties of the tissues: collagen for strength and stiffness, and
elastin for extensibility. It would be advantageous to have
available a biocomposite material that addresses the problems
associated with the current medical practices involving prosthetic
materials. Such technology would greatly improve the overall
efficiency and affordability of numerous surgeries which involve
prosthetics by decreasing the risk of autoimmune response to the
prosthesis, increasing the structural soundness of the prosthesis,
and by decreasing the risk of post-operative infection at the site
of the implant.
BRIEF SUMMARY OF THE INVENTION
[0004] The invention concerns a composite material (a
"biocomposite") comprising a biotic material and an abiotic
material, useful in artificial tissue replacement surgeries. In one
embodiment, the biotic material of the biocomposite comprises
collagen and the abiotic material comprises a polymer.
[0005] In one embodiment, the polymer is a co-polymer such as ethyl
acrylate-methyl methacrylate copolymer. In another embodiment, the
polymer is either polyacrylate or poly(acrylate-styrene)
co-polymer.
[0006] The biocomposite can be produced in a variety of regular or
irregular forms. In one embodiment, the biocomposite is one or more
fibers (also referred to herein as a fibril or fibrillar
biocomposite, or a biocomposite fiber).
[0007] In an exemplified embodiment described herein, sea cucumber
collagen fibrils were combined with polymeric nanoparticles in a
water-based solution to form emulsified collagen fibers. These new
fibers have improved biomechanical properties over both
constituents alone, and display unique features that allow them to
have the potential to be utilized in numerous areas of prosthetics,
including but not limited to heart valve replacements, artificial
skin replacement, and ligament and tendon replacements. Various
polymeric nanoparticles and ratios of polymer-to-biotic material
(e.g., polymer-to-collagen) can be utilized in order to alter the
mechanical properties of the biocomposite (e.g., composite fiber),
and mammalian biotic material (e.g., fibers and/or fibrils) can
also be incorporated into the system in order to provide a more
biocompatible prosthesis.
[0008] Another aspect of the invention concerns a method for
producing a biocomposite comprising a biotic material and an
abiotic material. In one embodiment, the production method
comprises polymerizing a biotic material and an abiotic material by
radical emulsion polymerization, thereby forming the synthetic
biocomposite. The degree of stiffness and elasticity can be easily
controlled by varying the ratios of abiotic material to biotic
material (e.g., varying the ratios polymer to collagen). In one
embodiment, the polymer in the polymer emulsion is a co-polymer
such as ethyl acrylate-methyl methacrylate copolymer. In another
embodiment, polymer in the polymer emulsion is either polyacrylate
or poly(acrylate-styrene) co-polymer. Optionally, the polymers can
be synthesized as nanoparticles in an aqueous emulsion.
[0009] Another aspect of the invention concerns a method for
treating a tissue defect within a human or animal subject,
comprising applying an effective amount of a biocomposite of the
invention to the site of the tissue defect. The biocomposites of
the invention have the potential to improve the efficiency and
affordability of surgical procedures involving prosthetics by
decreasing the risk of autoimmune response to the prosthesis,
increasing the structural integrity of the prosthesis, and by
decreasing the risk of post-operative infection at the site of the
implant.
[0010] Another aspect of the invention concerns a method for
delivering a biologically active agent to a target anatomical site
in a human or animal subject, comprising applying an effective
amount of a biocomposite of the invention to the target site,
wherein the biocomposite includes a biologically active agent. For
example, the biologically active agent can comprise at least one
member selected from the group consisting of medicaments; vitamins;
mineral supplements; substances used for the treatment, prevention,
diagnosis, cure or mitigation of disease or illness; substances
affecting the structure or function of the body; drugs;
antimicrobial agents; antifungal agents; antibacterial agents;
antiviral agents; antiparasitic agents; growth factors; angiogenic
factors; anaesthetics; mucopolysaccharides; metals; cells; acid
mucopolysaccharides; proteins; enzymes, peptides; and wound healing
agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1A and 1B show, adjacent a control collagen fiber,
segments (approximately 15 mm in length) of embodiments of fibril
biocomposites of the invention, prepared by radical emulsion
polymerization to synthesize a liquid ethyl acrylate-methyl
methacrylate copolymer emulsion, which was then mixed in various
ratios with native sea cucumber collagen fibrils, from which the
fibril biocomposites were formed.
[0012] FIGS. 2A and 2B show TEM of a cross-section of control
collagen fiber (FIG. 2A) and KRG3-treated fiber (FIG. 2B). The
control fiber contains mainly linearly-arranged fibrils tightly
packed in the fiber in a longitudinal orientation. The
emulsion-treated fibrils do not appear to have a linear arrangement
and are also much more loosely arranged within the fiber. The
spatial difference in the composite fiber is due to the
intertwining of the emulsion with the fibrils during fiber
formation. The random orientation is most likely a factor of the
mixing performed when the emulsion is introduced.
[0013] FIGS. 3A-3D show micrographs of dry and wet collagen fibers
and dry and wet biocomposite fibers of the invention. Dry control
collagen fibers (FIG. 3C) are twice as strong as the dry
biocomposite fibers (FIG. 3A), but when hydrated (as would the
material would be in biological applications), the wet biocomposite
fibers (FIG. 3B) are up to 40 times stronger than the wet collagen
fibers (FIG. 3D). The mechanical properties of the collagen fibers
change drastically when hydrated, and a large degree of swelling is
observed (FIG. 3C to 3D), but there is little to no differences
observed for the mechanical properties of the biocomposite
materials, and very little swelling was observed for these fibers
(FIG. 3A to 3B). Thus, the biocomposite is able to retain its
mechanical properties in a moist environment.
[0014] FIG. 4 is a graph comparing stress and strain in wet versus
dry KRG3 coated sea cucumber collagen fibers.
[0015] FIGS. 5A and 5B are graphs showing cyclic analysis of sea
cucumber collagen fiber containing 100 .mu.L KRG3 (FIG. 5A) and
mechanical analysis of calcium treated kRG3 polymer/collagen
biocomposite fibers (FIG. 5B).
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention concerns a synthetic biocomposite
comprising a biotic material and an abiotic material, which is
useful in artificial tissue replacement; methods for its
production; and methods of use. In one embodiment, the biotic
material of the biocomposite comprises collagen and the abiotic
material comprises a polymer. The biocomposite can be implanted
into humans or animals as an artificial tissue. The biocomposite
can be formed or manipulated into a desired shape for
implantation.
[0017] As described in the Examples section, radical emulsion
polymerization was employed to synthesize an ethyl acrylate-methyl
methacrylate copolymer, which, when in solid film form, displays
mechanical properties comparable to elastin. The polymerization was
performed under a nitrogen environment at 70.degree. C. and
utilizes a two-step synthesis. First, the acrylate monomers were
emulsified in 80% water and 3% surfactant, then the acrylates are
co-polymerized through the use of a radical initiator. The
resulting nanoparticle-based emulsion was then mixed in various
ratios with native sea cucumber collagen fibrils suspended in water
and placed in dialysis bags. The dialysis bags were then placed in
3% acetic acid overnight for fiber formation, then the fibers are
removed from the bags and allowed to air dry. The dry fibers are
then cut into pieces of approximately 15 mm in length for
mechanical analysis. The composite fibers displayed the most
advantageous property of each constituent: they preserve the high
tensile strength of collagen while sustaining the long range
elasticity of the polymer films. The degree of fiber stiffness and
elasticity is easily controlled by varying the ratios of polymer to
collagen. Biocompatibility of the polymer was established in vitro
against human dermal fibroblasts. Therefore, the polymer/collagen
composite fibers may be used as biocompatible materials for
artificial tissue. In one embodiment, the biocomposite is doped
with an inorganic mineral, such as calcium.
[0018] In one embodiment, the biocomposite is intercalated, e.g.,
with the abiotic material intercalated into at least a portion of
the biotic material, or vice-versa. As shown by embodiments in
FIGS. 2A and 2B, the polymer and the collagen fibrils are
intertwined, with the fibrils in a random (non-linear orientation),
representative of intercalation of the abiotic material (polymer)
into the biotic material (collagen). As shown in FIGS. 3A-3D, the
biocomposites can be produced and maintained in hydrated or
dehydrated form. The biocomposite is capable of retaining its
mechanical properties in a moist environment.
[0019] One aspect of the invention concerns a method for producing
a synthetic biocomposite, comprising polymerizing a biotic material
and an abiotic material by emulsion polymerization (e.g., radical
emulsion polymerization), thereby forming the synthetic
biocomposite. Emulsion polymerization is a popular and well-known
polymerization method (Esumi, K., Polymer Interfaces and Emulsions,
Marcel Dekker, New York, 1999; Gilbert, R. G. Emulsion
Polymerization: a Mechanistic Approach Academic Press, London,
1996; Anderson C. D. and Daniels E. S., Emulsion Polymerization and
Latex Applications, Rapra Review Reports, volume 14, report 160;
Odian, Principles of Polymerization, 3.sup.rd Ed., John Wiley and
Sons, Inc., New York, 1991; Rosen, S. L., Fundamental Principles of
Polymeric Materials", 2.sup.nd Ed., John Wiley and Sons, Inc., New
York, 1993, which are each incorporated herein by reference in
their entirety). Reactants that are necessary or useful in carrying
out emulsion polymerization reactions, such as monomers,
comonomers, initiators, surfactants (emulsifiers), non-surfactant
stabilizers, chain transfer agents, buffering agents, inert salts,
and preservatives (e.g., to retard bacterial growth) are known in
the art and can be employed in carrying out the production method
of the invention. The degree of stiffness and elasticity can be
easily controlled by varying the ratios of abiotic material to
biotic material (e.g., varying the ratios polymer to collagen). In
one embodiment, the polymer in the polymer emulsion is a co-polymer
such as ethyl acrylate-methyl methacrylate copolymer. In another
embodiment, polymer in the polymer emulsion is either polyacrylate
or poly(acrylate-styrene) co-polymer. Optionally, the polymers can
be synthesized as nanoparticles in an aqueous emulsion.
[0020] Any monomer can be used for a polymer component (e.g., the
abiotic component) of the biocomposite and production methods of
the invention. Preferably, the monomer utilized is an acrylic
monomer, a vinyl monomer, or a modified resin of either. Monomers
utilized for the abiotic component in the subject invention can
include, but are not limited to, acrylonitrile, acrylic acid,
maleic acid, methyl acrylate, ethyl acrylate, butyl acrylate, butyl
methacrylate, 2-ethylhexyl acrylate, methoxyethyl acrylate,
dimethylamino acrylate, methacrylic acid, isobutyl methacrylate,
2-ethyl hexyl methacrylate, lauryl methacrylate, stearic
methacrylate, dimethyl amino methacrylate, allyl methacrylate,
2-hydroxyethyl acrylate, 2-hydroxy propyl acrylate, 2-hydroxy ethyl
methacrylate, modified acrylamide, modified methacrylamide glycidyl
acrylate, styrene, vinyl acetate, vinyl toluene, and synthetically
modified acrylics. Preferably, the monomer is ethyl acrylate. In
one embodiment, the polymer is a co-polymer such as ethyl
acrylate-methyl methacrylate copolymer. In another embodiment, the
polymer is either polyacrylate or poly(acrylate-styrene)
co-polymer.
[0021] The free-radicals that can be utilized in the production
methods of the subject invention include, but are not limited to,
peroxides; persulphates; alkyl hydroperoxides; sodium, ammonium,
and potassium salts of persulphate; thiosulphates; metabisulphites;
and hydrosulphides. The initiator must be water soluble, and the
free radicals may be generated thermally or by use of an
oxidation-reduction (or redox) couple. The major initiators used in
emulsion polymerization are persulphates. Even though initiating
efficiency and half life of persulphates vary, ammonium persulphate
is preferred in practice because of its better solubility.
Hydroperoxides are often used particularly as a post reaction
initiator to kill the unreacted monomers after emulsion
polymerization.
[0022] The rate of free radical generation increases with
temperature, and it is normal to employ reaction temperatures of
60-90.degree. C. when using thermal generation techniques. However,
when redox couples (thiosulphates, metabisulphites, and
hydrosulphides) are employed, the rate of free radical generation
is increased to that provided by thermal generation at the same
temperature. Therefore, when using redox couples, reaction
temperatures can be made as low as 30.degree. C., or even room
temperature. The free radicals can be added as an aqueous solution
repeatedly until a milky solution is formed.
[0023] The aqueous media utilized in any pre-emulsifying and
initiating steps can include de-ionized water or nano-pure water.
As known to those skilled in the art, a buffer solution may be
necessary depending on the surfactant and stabilization.
[0024] The type of reaction vessel or vessels utilized for
producing the biocomposites of the invention, and the sizes of the
vessels, are not critical. Any vessel or substrate capable of
holding or supporting the reactants so as to allow the reaction to
take place can be used. It should be understood that, unless
expressly indicated to the contrary, the terms "adding",
"contacting", "mixing", "reacting", "combining" and grammatical
variations thereof, are used interchangeable to refer to the
mixture of reactants of the process of the present invention (e.g.,
monomer, biotic material, polymerization initiator, surfactant, and
so forth), and the reciprocal mixture of those reactants, one with
the other (i.e., vice-versa).
[0025] The biocomposites can be made in the form of single fibers,
fibrous mats (fibers may be in various ordered (e.g., woven or
parallel) or random orientations with respect to one another),
particles, sheets, films, etc.
[0026] In one aspect, the invention concerns a synthetic
biocomposite comprising a biotic material and an abiotic material.
In one embodiment, the biotic material is collagen and the abiotic
material is a polymer. In another embodiment, the biocomposite is
an abiotic fiber comprising intertwined polymer emulsion and
fibrils, such as collagen fibrils. In another embodiment, the
abiotic material is a polymer emulsion.
[0027] In one embodiment, the biotic material of the biocomposite
is fibrous. As used herein, the term "fibrous" is intended to refer
to matrices having at least some fiber (high aspect ratio)
component. The fibers can be of various lengths, diameters, and
orientations (e.g., parallel, mesh, random). The terms "fibers" and
"fibrils" are used interchangeably herein to refer to filamentous
or thread-like matter and any sub-fibers that contribute to a
larger fiber. Fibrils may also represent sub-fibers of a larger
fiber. The fibers can be produced by self-assembly or directed
assembly of molecules through synthesis procedures. Other fibrous
matrices can be found, for example, in Vincent, J. (Structural
Biomaterials, Princeton University Press, Princeton, N.J., 1990,
Chapter 3, pp. 73-91).
[0028] Another aspect of the invention is a device comprising a
surface with the synthetic biocomposite of the invention. In one
embodiment, the device is a medical device intended for
implantation in to a human or animal subject. In another
embodiment, the device is fibrous scaffold for cell or tissue
engineering.
[0029] Another aspect of the invention is a method for treating a
tissue defect, comprising applying a synthetic biocomposite of the
invention to the site of the defect.
[0030] Another aspect of the invention is a method for producing a
synthetic biocomposite, comprising polymerizing a biotic material
and an abiotic material by radical emulsion polymerization, thereby
forming the synthetic biocomposite. The type of reaction vessel or
vessels utilized for producing the biocomposites of the invention,
and the sizes of the vessels, are not critical. Any vessel or
substrate capable of holding or supporting the reactants so as to
allow the reaction to take place can be used. It should be
understood that, unless expressly indicated to the contrary, the
terms "adding", "contacting", "mixing", "reacting", "combining" and
grammatical variations thereof, are used interchangeable to refer
to the mixture of reactants of the process of the present invention
(e.g., biotic material, abiotic material, biologically active
agent, and so forth), and the reciprocal mixture of those
reactants, one with the other (i.e., vice-versa).
[0031] An aspect of the invention concerns a method for treating a
tissue defect within a subject, comprising applying an effective
amount of a biocomposite of the invention to the site of the tissue
defect. As used herein, the term "subject" refers to any human or
non-human animal (e.g., mammal), such as one suffering from a
tissue defect. According to the method of the subject invention, a
therapeutically effective amount of the biocomposite can be applied
at the site of a tissue defect to partially or fully restore
structural integrity to the tissue. Once applied, the biocomposite
of the subject invention can function as a filler (or partial
filler) or plug, to mend the tissue defect. The amount to be
applied will depend upon the size and nature of the tissue defect,
and the clinical outcome that is sought. The biocomposite can be
applied in a malleable form, for example, as a paste or putty, such
that the administered composition takes the shape of the bone
defect. Alternatively, the biocomposite can be molded pre-cast into
a desired shape (such as the shape of the defect) using polymer
composition molding methods known to those of ordinary skill in the
art, and the molded composition can be administered as a solid or
semi-solid article. Thus, the size, volume, thickness, and shape of
the molded article can be controlled, as desired. The biocomposite
can be applied in particulate form. According to the method of the
subject invention, the biocomposite can be applied so that it
directly contacts existing tissue adjacent to, or defining, the
tissue defect site, or the biocomposite can be contacting another
implant, or both.
[0032] The biocomposites of the subject invention can be applied to
the tissue defect site as a in any physical state (solid, liquid,
semi-solid, etc.). Preferably, the biocomposite is applied as a
liquid. Once applied, with a syringe for example, the liquid
composition can coagulate or cure ("set") shortly after application
to form a solid.
[0033] The biocomposite of the subject invention (and compositions
comprising the biocomposite) can be used as a vehicle for the in
situ delivery of biologically active agents. The biologically
active agents incorporated into, or included as an additive within,
the biocomposite of the invention can include, without limitation,
medicaments, growth factors, vitamins, mineral supplements,
substances used for the treatment, prevention, diagnosis, cure or
mitigation of disease or illness, substances which affect the
structure or function of the body, or drugs. The biologically
active agent can take several forms, such as polypeptides, nucleic
acid molecules (DNA, RNA, etc.), small chemical molecules,
carbohydrates, etc. The biologically active agents can be used, for
example, to facilitate implantation of the biocomposite into a
patient and to promote subsequent integration and healing
processes. The active agents include, but are not limited to,
antifungal agents, antibacterial agents, anti-viral agents,
anti-parasitic agents, growth factors, angiogenic factors,
anaesthetics, mucopolysaccharides, metals, cells, and other wound
healing agents. Because the processing conditions can be relatively
benign (physiological temperature and pH), live cells can be
incorporated into fiber constructs during their formation, or
subsequently allowed to infiltrate the fibers or compositions
through tissue engineering techniques.
[0034] As indicated above, cells can be seeded on to and/or within
the biocomposites of the present invention. The cells can be in any
state of differentiation, such as undifferentiated adult or
embryonic stem cells, progenitor cells, or differentiated cells.
Likewise, tissues such as cartilage can be associated with the
biocomposite prior to implantation within a subject. Examples of
such cells include, but are not limited to, bone cells (such as
osteoclasts, osteoblasts, and osteocytes), blood cells, epithelial
cells, neural cells (e.g., neurons, astrocytes, and
oligodendrocytes), and dental cells (odontoblasts and ameloblasts).
Seeded cells can be autogenic, allogenic, or xenogenic to the
biotic material of the biocomposite and/or the subject to which the
biocomposite is to be administered. Seeded cells can be
encapsulated or non-encapsulated.
[0035] Examples of antimicrobial agents that can be incorporated
into and/or on the biocomposite of the invention (or in
compositions comprising the biocomposite) include, but are not
limited to, isoniazid, ethambutol, pyrazinamide, streptomycin,
clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin,
rifampin, azithromycin, clarithromycin, dapsone, tetracycline,
erythromycin, ciprofloxacin, doxycycline, ampicillin, amphotericine
B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine,
clindamycin, lincomycin, pentamidine, atovaquone, paromomycin,
diclarazaril, acyclovir, trifluorouridine, foscarnet, penicillin,
gentamicin, ganciclovir, iatroconazole, miconazole, Zn-pyrithione,
and silver salts, such as chloride, bromide, iodide, and
periodate.
[0036] Growth factors that can be incorporated into and/or on the
biocomposite of the invention (or in compositions comprising the
biocomposite) include, but are not limited to, bone growth factors
(e.g., BMP, OP-1) basic fibroblast growth factor (bFGF), acidic
fibroblast growth factor (aFGF), nerve growth factor (NGF),
epidermal growth factor (EGF), insulin-like growth factors 1 and 2
(IGF-1 and IGF-2), platelet-derived growth factor (PDGF), tumor
angiogenesis factor (TAF), vascular endothelial growth factor
(VEGF), corticotropin releasing factor (CRF), transforming growth
factors alpha and beta (TGF-.alpha. and TGF-.beta.), interleukin-8
(IL-8), granulocyte-macrophage colony stimulating factor (GM-CSF),
the interleukins, and the interferons.
[0037] Other agents that can be incorporated into the biocomposites
or compositions containing them include acid mucopolysaccharides
including, but not limited to, heparin, heparin sulfate,
heparinoids, dermatan sulfate, pentosan polysulfate, chondroitin
sulfate, hyaluronic acid, cellulose, agarose, chitin, dextran,
carrageenin, linoleic acid, and allantoin.
[0038] Proteins that can be incorporated into, or included as an
additive within, the biocomposite or compositions comprising the
biocomposite include, but are not limited to, collagen (including
cross-linked collagen), fibronectin, laminin, elastin (including
cross-linked elastin), osteopontin, osteonectin, bone sialoproteins
(Bsp), alpha-2HS-glycoproteins, bone Gla-protein (Bgp), matrix
Gla-protein, bone phosphoglycoprotein, bone phosphoprotein, bone
proteoglycan, protolipids, bone morphogenetic protein, cartilage
induction factor, platelet derived growth factor and skeletal
growth factor, enzymes, or combinations and biologically active
fragments thereof Other proteins associated with other parts of
human or other mammalian anatomy can be incorporated or included as
an additive, including proteins associated with cartilage, such as
chondrocalcining protein, proteins associated with dentin, such as
phosphoryin, glycoproteins and other Gla proteins, or proteins
associated with enamel, such as amelogenin and enamelin. Agents
incorporated into the biocomposites or compositions of the subject
invention may or may not facilitate or enhance osteoinduction.
Adjuvants that diminish an immune response can also be used in
conjunction with the fibers or compositions of the subject
invention.
[0039] The bioactive molecule need not be a therapeutic agent. For
example, the bioactive molecule may be cytotoxic to the local cells
to which it is delivered but have an overall beneficial effect on
the subject. Further, the bioactive molecule may be a diagnostic
agent with no direct therapeutic activity per se, such as a
contrast agent for bioimaging.
[0040] A description of these classes of drugs and diagnostic
agents and a listing of species within each class can be found, for
example, in Martindale, The Extra Pharmacopoeia, Twenty-ninth
Edition (The Pharmaceutical Press, London, 1989), which is
incorporated herein by reference in its entirety. Drugs or
diagnostic agents are commercially available and/or can be prepared
by techniques known in the art.
[0041] The biologically active agents can first be encapsulated
into microcapsules, microspheres, microparticles, microfibers,
reinforcing fibers and the like to facilitate mixing and achieving
controlled, extended, delayed and/or sustained release.
Encapsulating the biologically active agent can also protect the
agent against degradation during formation of the biocomposites or
compositions of the invention.
[0042] Additionally, the biologically active agents can be
pendantly attached to the biocomposite. The attachment can be
facilitated through covalently linking the agent to a component of
the biocomposite, or through the use of hydrogen bonding.
[0043] In preferred embodiments of the invention, the biologically
active agent is controllably released into a human or animal
subject when the biocomposite or composition of the invention is
implanted into the subject, either through degradation in the
aqueous environment, or more preferably, due to bioresorption
relying on the time scale resulting from cellular remodeling. The
biocomposites of the subject invention can be used to replace an
area of discontinuity in the tissue in the subject's body. The area
of discontinuity in the tissue can be as a result of trauma,
disease, genetic defect, tumor, or surgery, for example.
[0044] The biocomposites of the invention can be incorporated into
medical devices. As used herein, a "medical device" may be defined
as a device that has surfaces that contact blood or other bodily
tissues in the course of their operation. This can include, for
example, extracorporeal devices for use in surgery such as blood
oxygenators, blood pumps, blood sensors, tubing used to carry blood
and the like which contact blood which is then returned to the
patient. This can also include implantable devices such as
artificial skin (skin grafts), vascular grafts, stents, electrical
stimulation leads, heart valves, bladder products (e.g., for
augmentation or replacement of a malfunctioning bladder due to
congenital deficiencies, resections following cancer, trauma, or
other genitourinary conditions), orthopedic devices, catheters,
shunts, sensors, replacement devices for nucleus pulposus, cochlear
or middle ear implants, intraocular lenses, and the like.
[0045] The biocomposite of the subject invention can be formulated
into a variety of shapes suitable for its function as a medical
device, such as a plate, pin, rod, tube, coil, screw, anchor, tack,
arrow, staple, button, or other regular or irregular shape. The
biocomposite of the present invention can be formulated as a
three-dimensional scaffold and, optionally, seeded with one or more
cell types for implantation within a human or animal subject. In
embodiments of a prosthesis in which the biotic material is
collagen, for example the invention allows formation of a
prosthesis in any shape or size. The bulk collagen (e.g., bovine
tendon collagen) can be solubilized by digestion with a protease
(e.g., papain). The soluble collagen is then extruded into a
synthetic fiber or otherwise shaped, where spontaneous formation of
fibrils lends weak integrity to the prosthesis.
[0046] As indicated above, the abiotic material can be a polymer.
The biotic material can also be a polymer (a polymer of biological
origin, e.g., a protein such as collagen). In those embodiments in
which the biotic material of the biocomposite comprises collagen,
various types of collagens from various sources may be utilized.
Collagen type 1, collagen type 2, collagen type 3, collagen type 4,
collagen type 5, collagen type 6, collagen type 7, collagen type 8,
collagen type 9, collagen type 10, collagen type 11, collagen type
12, collagen type 13, collagen type 14, collagen type 15, collagen
type 16, collagen type 17, collagen type 18, collagen type 19, and
collagen type 20, or combinations thereof can be utilized, for
example (Kadler et al., Biochem. J., 1996, 316:1-11; Badylak, Cell
& Developmental Biology, 2002, 13:377-383; and Gelse et al.,
Advanced Drug Delivery Reviews, 2003, 55:1531-1546). Other polymers
of biological origin that may be utilized include, for example,
gelatin (including denatured gelatin), alginates, chitosan, silk,
and cellulose. Other examples of biotic and abiotic polymeric
materials that may be utilized for the biocomposite of the
invention are described in U.S. Pat. No. 6,565,960 (Koob et al.),
which is incorporated herein by reference in its entirety.
[0047] In embodiments in which the biotic material is a collagen,
suitable collagens can be any form of a collagen polypeptide. For
example, the species origin of the collagen polypeptide can be from
any eukaryotic organism, including invertebrates (e.g., sea
cucumbers, starfish, sea urchins, worms, and sponges) and
vertebrates (e.g., hagfish, sharks, skates and rays, bony fish, and
mammals such as monkeys, chickens, pigs, and cows), and from any
tissue, including skin, tendon, cartilage, or ligament. Also
suitable are collagen polypeptides produced recombinantly (e.g., in
a human cell line, bacterium, or yeast). In addition, the collagen
can be, but need not be, free of any tertiary or quaternary
structure, such as in gelatin, a denatured form of collagen.
[0048] In another embodiment, if collagen is to be utilized, the
collagen can be in the form of a complex biological structure, such
as in a freshly harvested tendon fiber. Where the collagen is
already formed into a complex structure, it may be necessary to
incubate the collagen in the presence of the compound before
oxidation and polymerization to allow the compound to infiltrate
the collagen.
[0049] The biocomposites are useful in a variety of methods for
making materials for biomedical and other applications. The
biocomposite can be applied as a bandage, or incorporated in to a
bandage, for application to an acute or chronic wound.
[0050] Other aspects of the invention relate to the polymer
compositions, methods of making them, and methods of improving
characteristics of the polymeric materials relevant for biomedical
applications (e.g., tensile strength, biocompatibility,
biodegradation (or lack thereof), porosity (e.g., for cell
infiltration and tissue engineering), elasticity, compressive
strength, thermal stability, moldability, or non-antigenicity,
etc.) using the compounds and methods herein.
[0051] The abiotic material of the composite can be a gel. Gelatin
gels are particularly useful for in vivo applications. Hydrogels
can be used alone or in combination with other materials such as
glycosaminoglycans and collagen fibrils for applications including,
for example, as a space filling biomaterial for reconstructive
surgery (e.g., cranial and facial reconstruction), a biomimetic
cartilage for repair of focal lesions, a resorbable carrier for
phased release of cytokines such as bone morphogenetic proteins
(BMPs) for bone repair, or a patterning scaffold for
musculoskeletal regeneration.
[0052] Gelatin in liquid form can be used as well. Typically,
gelatin remains a viscous fluid at 37.degree. C. Thus, use of
liquid gelatin for biomedical applications in vivo is not possible
when the application requires compressive load capacity or other
strength property (e.g., tensile strength or elasticity) of the
material. For example, it can be used as an injectable space
filling material for reconstructive surgery (e.g., cranial or
facial), injected to fill voids without surgical intervention, it
can be molded to irregular shapes for tissue augmentation during
reconstructive surgery, or it can form a polymerizable scaffold in
which to deliver and then immobilize cells, cytokines or
therapeutic agents.
[0053] Gelatin foams (e.g., GELFOAM, available from Pharmacia,
Peapack, N.J.) are an additional area for application of the
biocomposite and methods of the invention. Gelatin foams are useful
as delivery systems for therapeutic agents, cytokines, and
musculoskeletal cells, particularly mesenchymal stem cells. The
potential use of the gelatin foam in tissue engineering is suspect
because it is extremely friable and lacks compressive stiffness and
resilience.
[0054] In addition to their therapeutic applications, the
biocomposites of the present invention may be used as scaffolds on
which to grow cells in vitro, or as filler in plastics, paint,
coatings, and paper production.
[0055] U.S. Pat. No. 6,565,960 (Koob et al.) is incorporated herein
by reference in their entirety. U.S. Patent Application Publication
No. US2007/019160A1 (Turos et al.) and PCT Publication No. WO
2005/020933 (Turos et al.) are incorporated herein by reference in
their entirety.
Measuring Beneficial Properties
[0056] If the biocomposites of the invention are to be implanted or
otherwise applied or administered in the body of a subject, the
composition should be biocompatible. To assess biocompatibility,
cells (e.g., a fibroblast cell line) can be seeded onto the
composition in a culture dish. If the fibroblasts are able to
replicate and attach to the composition, the composition is likely
to be biocompatible. Alternatively, the composition can be
implanted into the body of a subject (e.g., a mouse, rat, dog, pig,
or monkey) for a specified time, then removed to evaluate the
number and/or health of the cells attached to the composition. The
ability of the biocomposite to support growth of fibroblasts is
particularly important when infiltration of cells and deposition of
an extracellular matrix on the composition are desired in vivo.
[0057] Biocompatibility can also be assessed by evaluating the
immune response, or lack thereof, against the composition. A
suitable in vitro test is to contact human monocyte/macrophages to
the biocomposite, which can be in the form of small particles of
about 1 to 10 .mu.m in diameter for testing. The
monocyte/macrophage culture is then examined for signs of an
inflammatory response, including (1) phagocytosis of the
biocomposite particles, (2) increased lysozomal activity, and/or
(3) increased prostaglandin E.sub.2 in the culture. For in vivo
testing, the biocomposite can be implanted into an animal (e.g., a
rabbit), and the animal monitored for signs of, for example,
increased lymphocyte, neutrophil, or macrophage cell number locally
or systemically; increased concentrations of cytokines locally or
systemically; fever; and immune cell infiltration of the
composition. The quantitation of specific cell types, such as
monocytes, helper T cells, and B cells, is accomplished using
standard methods such as fluorescence-assisted cell sorting. In
addition, B cell activation can be evaluated by culturing B cells
from the animal in which the biocomposite had been implanted,
contacting the B cells with the biocomposite, and determining
whether the B cells proliferate in response to the biocomposite.
Other standard methods for evaluating an immune response in an
animal can be used.
[0058] Another measure of biocompatibility that can be utilized is
the susceptibility of the biocomposite to proteolytic digestion in
vivo. The desired level of protease resistance depends on the
intended use of the composition. For measuring the level of
protease resistance of the biocomposite, the biocomposite can be
incubated with a protease (e.g., collagenase or papain) for a
specified amount of time, then evaluated for any decrease in a
physical property (e.g., tensile strength) of the biocomposite or
the presence of any degradation product of the biotic material
(e.g., collagen) or abiotic material (e.g., polymer).
Physical Properties
[0059] The biocomposites of the invention can exhibit beneficial
physical properties, such as high tensile strength, high stiffness
(or elastic modulus), and low strain at failure. A high tensile
strength is desirable in, e.g., a prosthetic tendon, to avoid
rupture of the prosthesis under stress in vivo, such as when the
muscle to which the prosthesis is attached is contracted.
Similarly, a high stiffness is necessary to stabilize the relative
positions of the parts (e.g., muscle to bone) to which the
prosthesis is attached. A high stiffness means that a synthetic
fiber, for example does not stretch much under applied loads, which
is important for a tendon because nearly all of the force from a
muscle is transferred to the insertion site with little loss of
energy in extending the tendon. Moreover, the proper full excursion
of the limb as the muscle contracts requires that the tendon does
not extend. A low strain at failure indicates that the fiber does
not extend under load before failure. High tensile strength with no
fiber stretching will provide the most efficient transfer of energy
from the muscle to the bone insertion. To measure these physical
properties, any suitable apparatus having (1) two clamps for
attaching to the prosthesis, (2) a force transducer attached to one
of the clamps for measuring the force applied to the prosthesis,
(3) a means for applying the force, and (4) a means for measuring
the distance between the clamps, is suitable. Tensiometers are
commercially available. To calculate the tensile strength, the
force at failure is divided by the cross-sectional area of the
prosthesis through which the force is applied, resulting in a value
that can be expressed in force (e.g., Newtons) per area. The
stiffness is the slope of the linear portion of the stress/strain
curve. Strain is the real-time change in length during the test
divided by the initial length of the specimen before the test
begins. The strain at failure is the final length of the specimen
when it fails minus the initial specimen length, divided by the
initial length.
[0060] An additional physical property that is associated with the
extent of cross-linking in a composition is the shrinkage
temperature. In general, the higher the temperature at which a
collagenous composition begins to shrink, the higher the level of
cross-linking. The shrinkage temperature of a fiber can be
determined by immersing the fiber in a water bath, raising the
temperature of the water bath, and observing the temperature of the
water bath at which the fiber shrinks. Tension on the fiber may be
required for observing the shrinkage. The shrinking temperature for
the compositions of the invention can be at least about 60.degree.
C. (e.g., at least 65 or 70.degree. C.).
[0061] For biocomposites or compositions comprising them that are
not elongated in shape, such as in a disk, the fracture pressure
can be an indication of physical strength. The fracture pressure is
the minimum force per area at which a material cracks.
Exemplified Embodiments
[0062] The invention includes, but is not limited to, the following
embodiments: [0063] Embodiment 1. A synthetic biocomposite
comprising a biotic material and an abiotic material. [0064]
Embodiment 2. The biocomposite of embodiment 1, wherein the biotic
material is collagen and the abiotic material is a polymer. [0065]
Embodiment 3. The biocomposite of embodiment 1, wherein the
biocomposite is a fiber comprising an intertwined polymer emulsion
and fibrils. [0066] Embodiment 4. The biocomposite of embodiment 3,
wherein the fibrils are collagen fibrils. [0067] Embodiment 5. The
biocomposite of embodiment of any of embodiments 1-3, wherein the
abiotic material is intercalated into the biotic material, or
vice-versa. [0068] Embodiment 6. The biocomposite of any preceding
embodiment, wherein the abiotic material is a polymer emulsion.
[0069] Embodiment 7. The biocomposite of any preceding embodiment,
wherein the abiotic material comprises a copolymer emulsion. [0070]
Embodiment 8. The biocomposite of embodiment 7, wherein the
copolymer is ethyl acrylate-methyl methacrylate. [0071] Embodiment
9. The biocomposite of embodiment 1, wherein the abiotic material
is polyacrylate or poly(acrylate-styrene) copolymer. [0072]
Embodiment 10. The biocomposite of any preceding embodiment,
further comprising a biologically active agent. [0073] Embodiment
11. The biocomposite of embodiment 10, wherein the biologically
active agent is a cell. [0074] Embodiment 12. A device comprising a
surface with the synthetic biocomposite of any of embodiments 1-11
thereon. [0075] Embodiment 13. The device of embodiment 12, wherein
the device is a medical implant. [0076] Embodiment 14. A method for
producing a synthetic biocomposite, comprising polymerizing a
biotic material and an abiotic material by radical emulsion
polymerization, thereby forming the synthetic biocomposite. [0077]
Embodiment 15, wherein the biocomposite is one according to any of
embodiments 1-11. [0078] Embodiment 16. The method of embodiment
14, wherein the biotic material is collagen and the abiotic
material is a polymer. [0079] Embodiment 17. The method of
embodiment 14, wherein the biocomposite is an abiotic fiber
comprising intertwined polymer emulsion and fibrils. [0080]
Embodiment 18. The method of embodiment 17, wherein the fibrils are
collagen fibrils. [0081] Embodiment 19. The method of any of
embodiments 14-18, wherein the abiotic material is a polymer
emulsion. [0082] Embodiment 20. The method of any of embodiments
14-19, wherein the abiotic material comprises a copolymer emulsion.
[0083] Embodiment 21. The method of embodiment 20, wherein the
copolymer is ethyl acrylate-methyl methacrylate. [0084] Embodiment
22. The method of any of embodiments 14-21, further comprising a
biologically active agent. [0085] Embodiment 23. The method of
embodiment 22, wherein the biologically active agent is a cell.
[0086] Embodiment 24. A method for treating a tissue defect,
comprising applying a synthetic biocomposite at the site of the
defect, wherein the biocomposite comprises a biotic material and an
abiotic material. [0087] Embodiment 25. The method of embodiment
24, wherein the biocomposite is one of embodiments 1-11.
Definitions
[0088] The term "tissue defect", as used herein refers to any
tissue deficient region, such as a void, gap, recess, or other
discontinuity in the hard or soft tissues. The tissue defect can be
artificially or naturally established, and can occur due to disease
or trauma, for example. Thus, the tissue defect can occur as a
consequence of pathologic, inflammatory, or tumor diseases,
surgical interventions, congenital defects, or bone fractures, and
the like. Typically, tissue defects in dental applications will be
on or within the gum or jaw of the patient's mouth. For example, in
the case of certain diseases, such as tumors, the tissue defect is
artificially established by removing the tumor tissue. Thus,
according to the method of the subject invention, the biocomposite
or compositions comprising the biocomposite can be applied, for
example, to repair periodontal defects, for craniofacial
reconstruction, joint reconstruction, fracture repair, to conduct
orthopedic surgical procedures, and spinal fusion, for example. The
term "tissue defect" is also intended to include anatomical sites
where augmentation to a tissue feature is desired by the patient in
the absence of disease or trauma, such as in elective cosmetic
surgery. Thus, the "defect" can be one that is subjectively
perceived by the patient, and where augmentation of the bone
deficient region is desired.
[0089] The terms "comprising", "consisting of', and "consisting
essentially of are defined according to their standard meaning and
may be substituted for one another throughout the instant
application in order to attach the specific meaning associated with
each term.
[0090] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural reference unless
the context clearly dictates otherwise. Thus, for example, a
reference to "a fiber" includes more than one such fiber, and the
like. Reference to "a polymer" includes more than one such polymer.
Reference to "a cell" includes more than one such cell. Reference
to "a biologically active agent" includes more than one such
agent.
[0091] As used herein, the term "drug" is interchangeable with the
term "biologically active agent" and refers to any agent capable of
having a physiologic effect (e.g., a therapeutic or prophylactic
effect) on a biosystem such as prokaryotic or eukaryotic cells or
organisms, in vivo or in vitro, including, but without limitation,
chemotherapeutics, toxins, radiotherapeutics, radiosensitizing
agents, gene therapy vectors, antisense nucleic acid constructs,
interfering RNA molecules (e.g., siRNA or shRNA), transcription
factor decoys, imaging agents, diagnostic agents, agents known to
interact with an intracellular protein, polypeptides, and
polynucleotides. Drugs that may be utilized in the biocomposites
include any type of compound including antibacterial, antiviral,
antifungal, or anti-cancer agents. The drug is preferably a
water-insoluble or water-soluble solid or a highly viscous
liquid.
[0092] The drug can be selected from a variety of known classes of
drugs, including, for example, analgesics, anesthetics,
anti-inflammatory agents, anthelmintics, anti-arrhythmic agents,
antiasthma agents, antibiotics (including penicillins), anticancer
agents (including Taxol), anticoagulants, antidepressants,
antidiabetic agents, antiepileptics, antihistamines, antitussives,
antihypertensive agents, antimuscarinic agents, antimycobacterial
agents, antineoplastic agents, antioxidant agents, antipyretics,
immunosuppressants, immunostimulants, antithyroid agents, antiviral
agents, anxiolytic sedatives (hypnotics and neuroleptics),
astringents, bacteriostatic agents, beta-adrenoceptor blocking
agents, blood products and substitutes, bronchodilators, buffering
agents, cardiac inotropic agents, chemotherapeutics, contrast
media, corticosteroids, cough suppressants (expectorants and
mucolytics), diagnostic agents, diagnostic imaging agents,
diuretics, dopaminergics (antiparkinsonian agents), free radical
scavenging agents, growth factors, haemostatics, immunological
agents, lipid regulating agents, muscle relaxants, proteins,
peptides and polypeptides, parasympathomimetics, parathyroid
calcitonin and biphosphonates, prostaglandins,
radio-pharmaceuticals, hormones, sex hormones (including steroids),
time release binders, anti-allergic agents, stimulants and
anoretics, steroids, sympathomimetics, thyroid agents, vaccines,
vasodilators, and xanthines.
[0093] The biologically active agent need not be a therapeutic
agent. For example, the agent may be cytotoxic to the local cells
to which it is delivered but have an overall beneficial effect on
the subject. Further, the biologically active agent may be a
diagnostic agent with no direct therapeutic activity per se, such
as a contrast agent for bioimaging.
[0094] A description of these classes of drugs and diagnostic
agents and a listing of species within each class can be found, for
instance, in Martindale, The Extra Pharmacopoeia, Twenty-ninth
Edition (The Pharmaceutical Press, London, 1989), which is
incorporated herein by reference in its entirety. The drugs or
diagnostic agents are commercially available and/or can be prepared
by techniques known in the art.
[0095] Poorly water soluble drugs which may be suitably used in the
practice of the subject invention include but are not limited to
alprazolam, amiodarone, amlodipine, astemizole, atenolol,
azathioprine, azelatine, beclomethasone, budesonide, buprenorphine,
butalbital, carbamazepine, carbidopa, cefotaxime, cephalexin,
cholestyramine, ciprofloxacin, cisapride, cisplatin,
clarithromycin, clonazepam, clozapine, cyclosporin, diazepam,
diclofenac sodium, digoxin, dipyridamole, divalproex, dobutamine,
doxazocin, enalapril, estradiol, etodolac, etoposide, famotidine,
felodipine, fentanyl citrate, fexofenadine, finasteride,
fluconazole, flunisolide, flurbiprofen, fluvoxamine, furosemide,
glipizide, gliburide, ibuprofen, isosorbide dinitrate,
isotretinoin, isradipine, itraconazole, ketoconazole, ketoprofen,
lamotrigine, lansoprazole, loperamide, loratadine, lorazepam,
lovastatin, medroxyprogesterone, mefenamic acid,
methylprednisolone, midazolam, mometasone, nabumetone, naproxen,
nicergoline, nifedipine, norfloxacin, omeprazole, paclitaxel,
phenytoin, piroxicam, quinapril, ramipril, risperidone, sertraline,
simvastatin, sulindac, terbinafine, terfenadine, triamcinolone,
valproic acid, zolpidem, or pharmaceutically acceptable salts of
any of the above-mentioned drugs.
[0096] The terms "biosystem", "host", "host biosystem", "patient",
"recipient", and "subject", are used interchangeably and, for the
purposes of the present invention, include both prokaryotic and
eukaryotic cells, such as human cells and non-human animal cells
(e.g., mammal cells). Biocomposites of the subject invention may be
applied, administered, or otherwise contacted to such cells in
vitro or in vivo. Thus, the methods of administration are
applicable to both human therapy and veterinary applications, as
well as research applications in vitro or within animal models.
[0097] As used herein, an "effective amount" of biocomposite or of
biologically active agent is that amount effective to bring about
the physiological changed desired in the biosystem to which the
biocomposite is administered. The term "therapeutically effective
amount" as used herein, means that amount of biocomposite or of
biologically active agent, alone or in combination with another
agent according to the particular aspect of the invention, that
elicits the biological or medicinal response in a biosystem that is
being sought by a researcher, veterinarian, medical doctor or other
clinician, which includes alleviation of the symptoms of the
disease or disorder being treated.
[0098] For example, if the biologically active agent is a
therapeutic agent, an effective amount of the biocomposite
comprising the biologically active agent can be that amount
sufficient to treat a pathological condition (e.g., a disease or
other disorder, a tissue defect) in the biosystem to which the
biocomposite is administered. For example, in the case of cancer,
the therapeutically effective amount of the biologically active
agent may reduce the number of cancer cells; reduce the tumor size;
inhibit (i.e., slow to some extent and preferably stop) cancer cell
infiltration into peripheral organs; inhibit (i.e., slow to some
extent and preferably stop) tumor metastasis; inhibit, to some
extent, tumor growth; and/or relieve, to some extent, one or more
of the symptoms associated with the cancer. To the extent the agent
may prevent growth and/or kill existing cancer cells, it may be
cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for
example, be measured by assessing the time to disease progression
(TTP) and/or determining the response rate (RR).
[0099] The terms "linked", "joined", "grafted", "tethered",
"associated", and "conjugated" in the context of the biocomposite
of the invention, are used interchangeably to refer to any method
known in the art for functionally connecting moieties (such as
biologically active agents), including, without limitation,
recombinant fusion, covalent bonding, disulfide bonding, ionic
bonding, hydrogen bonding, and electrostatic bonding.
[0100] The term "modified" refers to an alteration from an entity's
normally occurring state. An entity can be modified by removing
discrete chemical units or by adding discrete chemical units.
[0101] The term "polypeptides" refers to any polymer comprising any
number of amino acids, and is interchangeable with "protein", "gene
product", and "peptide".
[0102] The term "intercalates" or "interaction" is the immersion or
dispersal of one substance into at least a portion of another
substance.
[0103] The methods of the invention can utilize general techniques
known in the field of polymer chemistry. General polymer chemistry
concepts and methods that may be utilized are described in the
Polymer Handbook (4.sup.th Edition), eds., Brandup et al., New
York, John Wiley and Sons, 1999; and Polymer Synthesis and
Characterization: A Laboratory Manual, eds. Sandler et al.,
Academic Press, 1998. The practice of the subject invention can
employ, unless otherwise indicated, conventional techniques of
molecular biology, microbiology, recombinant DNA technology,
electrophysiology, and pharmacology, which are within the ordinary
skill in the art. Such techniques are explained fully in the
literature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular
Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning,
Vols. I and II (D. N. Glover ed. 1985); Perbal, B., A Practical
Guide to Molecular Cloning (1984); the series, Methods In
Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);
Transcription and Translation (Hames et al. eds. 1984); Gene
Transfer Vectors For Mammalian Cells (J. H. Miller et al. eds.
(1987) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.);
Scopes, Protein Purification: Principles and Practice (2nd ed.,
Springer-Verlag); and PCR: A Practical Approach (McPherson et al.
eds. (1991) IRL Press)).
[0104] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
EXAMPLE 1
Production of Synthetic Elastic Polymer/Collagen Fibril
Biocomposite
[0105] Radical emulsion polymerization was employed to synthesize
an ethyl acrylate-methyl methacrylate copolymer emulsion, which,
when in solid film form, displays mechanical properties comparable
to elastin. Briefly, the liquid emulsion was then mixed in various
ratios with native sea cucumber collagen fibrils, from which fibers
were formed. Mechanical analysis was then performed on the
fibers.
[0106] Production of the fibril biocomposites will now be described
in more detail. Emulsions are formed through radical emulsion
polymerization at 70.degree. C. under a nitrogen environment for
6-8 hours. Add 25-150 mL of concentrated (20% solid content) ethyl
acrylate-methyl methacrylate (4:1) emulsion (KRG3) to 10 mL of sea
cucumber collagen fibrils in water and place mixture in dialysis
tubing. Gently mix the solution for even distribution of polymer,
then place the tubing in 3% acetic acid overnight for fiber
formation. The fibers are then removed from the tubing and allowed
to air dry by slowly lifting them from the acetic acid solution.
The dry fibers were cut into pieces of approximately 15 mm in
length for mechanical analysis.
EXAMPLE 2
Characterization of Synthetic Elastic Polymer/Collagen Fibril
Biocomposite
[0107] FIGS. 2A and 2B are TEM of a cross-section of a control
collagen fiber (FIG. 2A) and KRG3-treated fiber (FIG. 2B). The
control fiber contains mainly linearly-arranged fibrils tightly
packed in the fiber in a longitudinal orientation. The
emulsion-treated fibrils do not appear to have a linear arrangement
and are also much more loosely arranged within the fiber. The
spatial difference in the composite fiber is due to the
intertwining of the emulsion with the fibrils during fiber
formation. The random orientation is most likely a factor of the
mixing performed when the emulsion is introduced.
[0108] Dry control collagen fibers (FIG. 3C) are twice as strong as
the dry biocomposite fibers (FIG. 3A), but when hydrated (as would
the material would be in biological applications), the wet
biocomposite fibers (FIG. 3B) are up to 40 times stronger than the
wet collagen fibers (FIG. 3D). The mechanical properties of the
collagen fibers change drastically when hydrated, and a large
degree of swelling is observed (FIG. 3C to 3D), but there is little
to no differences observed for the mechanical properties of the
biocomposite materials, and very little swelling was observed for
these fibers (FIG. 3A to 3B). Thus, the biocomposite is able to
retain its mechanical properties in a moist environment.
[0109] The maximum tensile strength after a 25-cycle analysis is in
the same range as the fiber un-fatigued; therefore, cyclic analysis
did not alter the overall strength of the fiber. There is a slight
decrease in strength between each cycle, but the overall max
strength remains constant. Little deformation was observed on the
fiber by the repeated cyclic displacement.
[0110] Both fibers appear to display relatively the same mechanical
properties. While the calcium-doped fibers appeared to be slightly
stiffer (less strain observed), the maximum tensile strength for
each set of fibers was equivalent, thus the calcium treatment had
no effect on the fiber's tensile strength.
TABLE-US-00001 TABLE 1 Control 25 uL 50 uL 100 uL 150 uL Wet Fibers
Force (N) 0.131144 2.295319 4.5605 4.230542 3.516884 Stress (MPa)
1.154574 7.458517 36.03989 41.75548 32.92559 Strain 0.187441
0.707732 0.490528 0.42103 0.370264 Young's Modulus (MPa) -9.47825
3.224868 67.08264 89.95072 87.40711 % Swell 102.9298 36.85137
56.77656 33.57825 23.21725 Dry Fibers Force (N) 5.36957 3.052235
5.047687 3.774777 4.890496 Stress (MPa) 222.9718 44.16573 172.4999
114.4796 59.32537 Strain 0.356441 0.378474 0.439481 0.372034
0.496644 Young's Modulus (MPa) 614.0175 120.3238 279.1168 224.6649
96.0726
[0111] The composite fibers displayed the most advantageous
property of each individual constituent: they preserve the high
tensile strength of collagen while sustaining the long range
elasticity of the polymer films. The degree of fiber stiffness and
elasticity is easily controlled by varying the ratios of polymer to
collagen. The ability to retard swelling of the fibers was also
achieved in the composite fibers. Biocompatibility of the polymer
has been established in vitro against human dermal fibroblasts;
therefore, the formation of polymer/collagen composite fibers is an
important step toward implementation of biocompatible materials for
artificial tissue.
[0112] All patents, patent applications, provisional applications,
and publications referred to or cited herein, supra or infra, are
incorporated by reference in their entirety, including all figures
and tables, to the extent they are not inconsistent with the
explicit teachings of this specification.
[0113] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application.
* * * * *