U.S. patent application number 12/997689 was filed with the patent office on 2011-06-30 for biomaterials and implants for enhanced cartilage formation, and methods for making and using them.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Karla Brammer, Sungho JIN, Seunghan Oh.
Application Number | 20110159070 12/997689 |
Document ID | / |
Family ID | 41466608 |
Filed Date | 2011-06-30 |
United States Patent
Application |
20110159070 |
Kind Code |
A1 |
JIN; Sungho ; et
al. |
June 30, 2011 |
BIOMATERIALS AND IMPLANTS FOR ENHANCED CARTILAGE FORMATION, AND
METHODS FOR MAKING AND USING THEM
Abstract
The invention provides products of manufacture, e.g.,
biomaterials and implants, for cartilage maintenance and/or
formation in-vivo, in-vitro, and ex-vivo, using nanotechnology,
e.g., using nanotube, nanowire, nanopillar and/or nanodepots
configured on surface structures of the products of
manufacture.
Inventors: |
JIN; Sungho; (San Diego,
CA) ; Oh; Seunghan; (San Diego, CA) ; Brammer;
Karla; (La Jolla, CA) |
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
|
Family ID: |
41466608 |
Appl. No.: |
12/997689 |
Filed: |
July 2, 2009 |
PCT Filed: |
July 2, 2009 |
PCT NO: |
PCT/US2009/049523 |
371 Date: |
March 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61078141 |
Jul 3, 2008 |
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61087957 |
Aug 11, 2008 |
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Current U.S.
Class: |
424/423 ; 216/41;
219/121.14; 219/121.64; 219/635; 228/174; 424/400; 424/443;
424/450; 424/93.7; 435/395; 514/1.1; 514/44R; 514/8.1; 514/8.8;
514/8.9; 514/9.1; 514/9.6; 977/762; 977/810; 977/888; 977/900;
977/901; 977/906; 977/915 |
Current CPC
Class: |
A61L 2300/602 20130101;
A61L 27/06 20130101; A61P 43/00 20180101; C12N 5/0655 20130101;
A61K 9/127 20130101; A61K 31/7088 20130101; A61L 2300/64 20130101;
B23K 15/0046 20130101; H05B 6/02 20130101; A61K 38/1841 20130101;
A61L 2300/414 20130101; A61K 38/1875 20130101; A61L 27/3817
20130101; A61L 2300/626 20130101; A61K 38/1808 20130101; A61L 27/54
20130101; A61P 19/04 20180101; A61P 19/08 20180101; A61L 2430/06
20130101; A61K 35/32 20130101; A61K 38/1866 20130101; C12M 25/00
20130101; A61K 38/1825 20130101; A61L 27/025 20130101; A61L 2400/12
20130101; B23K 26/20 20130101; A61L 27/3834 20130101; A61L 27/3843
20130101 |
Class at
Publication: |
424/423 ;
424/400; 424/443; 424/93.7; 424/450; 514/44.R; 514/1.1; 514/9.1;
514/9.6; 514/8.1; 514/8.9; 514/8.8; 435/395; 216/41; 228/174;
219/635; 219/121.14; 219/121.64; 977/762; 977/810; 977/915;
977/906; 977/888; 977/900; 977/901 |
International
Class: |
A61L 27/54 20060101
A61L027/54; A61L 27/38 20060101 A61L027/38; A61K 9/00 20060101
A61K009/00; A61K 9/70 20060101 A61K009/70; A61K 35/12 20060101
A61K035/12; A61K 9/127 20060101 A61K009/127; A61K 31/7088 20060101
A61K031/7088; A61K 48/00 20060101 A61K048/00; A61K 38/02 20060101
A61K038/02; A61K 38/18 20060101 A61K038/18; A61K 38/22 20060101
A61K038/22; C12N 5/077 20100101 C12N005/077; A61P 43/00 20060101
A61P043/00; A61P 19/04 20060101 A61P019/04; A61P 19/08 20060101
A61P019/08; C23F 1/04 20060101 C23F001/04; B23K 31/02 20060101
B23K031/02; H05B 6/02 20060101 H05B006/02; B23K 15/00 20060101
B23K015/00; B23K 26/20 20060101 B23K026/20 |
Claims
1. A product of manufacture comprising: wherein optionally the
product of manufacture is a cell-, cartilage- and/or bone
growth-enhancing or cell differentiation-enhancing product of
manufacture, or a bone- or cartilage-maintaining and/or bone or
cartilage growth-enhancing product of manufacture, or an implant,
(a) nanostructures comprising a nanotube, nanowire, nanopore,
nanoribbon and/or a nanopillar surface configuration on a Ti and/or
Ti-comprising alloy, or on a Ti-coated or Ti alloy-coated surface,
or on a TiO.sub.2 and/or TiO.sub.2 alloy surface or coating,
wherein the Ti and/or Ti-comprising alloy or the TiO.sub.2 and/or
TiO.sub.2 alloy surface or coating, or the Ti-coated or Ti
alloy-coated surface, comprises one or more surfaces (or a
subsurface or a partial surface) of the product of manufacture,
wherein optionally the nanostructures (nanotubes, nanowires,
nanopores, nanoribbons and/or nanopillars) comprise a metal and/or
a metal alloy comprising a Ti, a Zr, a Hf, a Nb, a Ta, a Mo and/or
a W, or an oxide of a Ti, a Zr, a Hf, a Nb, a Ta, a Mo and/or a W,
wherein optionally the nanostructures (nanotubes, nanowires,
nanopores, nanoribbons and/or nanopillars) are formed directly
and/or indirectly on and/or attached to a Ti surface and/or a
Ti-coated surface, or Ti oxide surface and/or a Ti oxide-coated
surface, wherein optionally the nanotubes have a diameter dimension
in the range of between about 30 to 600 nm outside diameter, or
between about 50 to 400 nm diameter, or between about 70 to 200 nm
diameter, and/or optionally a height dimension in the range of
between about 30 to 10,000 nm, and/or optionally between about 200
to 2,000 nm thickness, or between about 200 to 500 nm thickness,
wherein optionally the Ti surface and/or Ti-coated surface, or Ti
oxide surface and/or a Ti oxide-coated surface, comprises: the
surface of a wire or microwire; the surface of a springy and/or a
hairy wire or microwire; the surface of a mesh or mesh screen; the
surface of an implant; a "pre-patterned" and/or a "pre-etched"
surface made by machining or mask patterning and/or etching of the
surface of the product of manufacture structure, wherein optionally
the three-dimensional Ti wire or microwire is between about 10 to
100 .mu.m in diameter and/or the Ti wire or microwire is a springy
and compliant wire or microwire, wherein optionally the material
used for the three-dimensional springy, coil, wire, or mesh screen
scaffold comprises at least one of a metal or an alloy selected
from the group consisting of Ti, Zr, Hf, Nb, Ta, Mo and W, or an
alloy or an oxide or a mixture thereof, or stainless steel, or a
Co--Cr--Ni--Mo alloy (commonly known as MP35N alloy), wherein the
surface of the springy wire scaffold contains vertically configured
nanotube or nanopore arrays with about 30 to 600 nm diameter,
preferably 70 to 200 nm diameter, and about 200 to 2,000 nm
thickness, and preferably 200 to 500 nm thickness, wherein
optionally the Ti or Ti oxide alloy or Ti or Ti oxide on the
Ti-coated, or Ti oxide-coated or Ti alloy-coated surface is between
about 100 to 2000 .mu.m thick; and wherein optionally the product
of manufacture structure of (a) comprises (i) oxides of alloys
comprising Ti or a Ti oxide or a TiO.sub.2 by at least about 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more in weight %,
or (ii) oxides of alloys comprising Zr, Hf, Nb, Ta, Mo, W, by at
least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or
more weight %, or (ii) a ceramic, a polymer, a plastic, a
Si-comprising composition, a Au-comprising composition, a
Pd-comprising composition, a Pt-comprising composition, or a
stainless steel; (b) the product of manufacture of (a), and further
comprising a chondrocyte, a stem cell, a totipotent cell, a
multipotent progenitor cell and/or a pluripotent cell, wherein the
chondrocyte functionality, as indicated by the degree of
extracellular matrix formation, is increased by at least about 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more, as compared
with the identical material but without the TiO.sub.2 nanotube or
nanopillar surface configuration; (c) the product of manufacture of
(a) or (b), further comprising a chondrocyte, a colony-forming
unit-fibroblast (CFU-F), a marrow stromal cell or mesenchymal stem
cell (MSC), a stem cell, a totipotent cell, a multipotent
progenitor cell and/or a pluripotent cell, wherein optionally the
cell is implanted in, seeded in or placed in the product of
manufacture in-vivo, in-vitro, and/or ex-vivo; (d) the product of
manufacture of (b) or (c), wherein the stem cell is a mesenchymal
stem cell (MSC), an adult stem cell, an induced pluripotent stem
cell (abbreviated as iPS cell or iPSC) and/or an embryonic stem
cell; (e) the product of manufacture of any of (b) to (d), wherein
the chondrocyte is an autologous chondrocyte, a hypertrophic
chondrocyte, or a human chondrocyte; (f) the product of manufacture
of any of (a) to (d), further comprising on the surface of the
product of manufacture a nano-depot, a microcavity and/or a
macrocavity comprising a cell, a drug and/or a biological agent,
wherein optionally the nanotube or a nanopillar, or microcavity
and/or a macrocavity, acts as a depot or storage area comprising a
cell, a drug and/or a biological agent, wherein optionally the
microcavity has an entrance dimension of between about 1 to 100
micrometer, or a macrocavity having an entrance dimension of
between about 100 to 1,000 micrometer; or (g) the product of
manufacture of any of (a) to (f), having a structure as illustrated
in any one of FIGS. 16 to 29.
2. The product of manufacture of claim 1, wherein the product of
manufacture comprises (a) a thin coating of a metal, a metal oxide,
and/or an alloy at least about 1, 2, 3, 4, 5, 10, 15 nm, 20 nm, 30
nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100 nm or more nm
in thickness at the surface, and/or (b) at least a portion of the
surface underneath comprises a vertically aligned and adhering
nanotube, nanoribbon, nanowire and/or nanopillar array structure or
structures, and/or a plurality of recessed nanopore structures.
3. The product of manufacture of claim 1, wherein the entrance
dimension of the nano-depot, nanotube and/or nanopore is reduced
(constricted or impeded) by a selective deposition of a metal or an
alloy, a metal oxide and/or alloy oxide, and/or another compound,
to induce a partial bottlenecking (constricting) configuration to
slow down or impede the release rate of a compound or a substance
stored in the nano-depot, nanotube and/or nanopore, wherein
optionally the compound or substance comprises a drug and/or a
biological agent stored in the nano-depot, nanotube and/or
nanopore, wherein optionally the slowing down or impeding of the
release rate of the compound or a substance stored in the
nano-depot, nanotube and/or nanopore is at least by a factor of 2
or 3 or slower, or at least by a factor of about 10 or slower, than
the case of non-bottlenecked (non-constricted) structure, wherein
optionally the other compound used to partially bottleneck
(constrict or impeded) the nano-depot and/or nanopore comprises a
nitride, a fluoride, a carbide and/or a polymer material, wherein
optionally the product of manufacture surface has a multiplex
and/or a duplex distribution of nanostructure structures with
different dimensions such that the product of manufacture comprises
both one or more nano-depot, nanotube and/or nanopore structures
having bottle-necked (constricted or impeded) pore structures
together with nano-depot, nanotube and/or nanopore structures which
do not have the bottleneck diameter (constricted or impeded
opening) reductions, wherein optionally the relative area fraction
of bottle necked (constricted or impeded opening) nano-depot,
nanotube and/or nanopore structures in the product of manufacture
is in the range of about 2% to 50% of the total available surface
area of the product of manufacture, or in the range of about 2% to
50% of the total available surface area available for stimulating
cell growth, cartilage growth and/or bone deposition.
4. The product of manufacture of claim 1, wherein the product of
manufacture further comprises a chemical, a drug and/or a
biological agent, and optionally the chemical, drug and/or
biological agent comprises a small molecule, a growth factor, a
collagen, a protein, a biomolecule, a gene, a nucleic acid, an RNA
or a DNA, a nucleic acid expression vector, an antibiotic, a
hormone, a therapeutic drug, a functional particle, a liposome, or
a magnetic, metallic, ceramic or a polymer particle; or, a
differentiation-inducing chemical, drug and/or biomolecule, and
optionally the chemical, drug and/or biological agent is attached
to or coated on the product of manufacture, or is stored in a
nanopore, nanodepot and/or nanotube, or the chemical, drug and/or
biological agent is attached to, coated on or stored between
nanostructures comprising a plurality of nanopillars, nanotubes,
nanowires and/or nanoribbons, and optionally the chemical, drug
and/or biological agent comprises (are) a fibroblast growth factor
(FGF), an epidermal growth factor (EGF), a vascular endothelial
growth factor (VEGF), a transforming growth factor beta-1
(TGF-.beta.1) or a transforming growth factor beta-2 (TGF-.beta.2),
a bone morphogenic protein (BMP), an agent that stimulates
chondrocyte growth, maintenance and/or differentiation, a chemical
or biomolecule osteogenic-inducing agent, a fibroblast growth
factor and/or a vascular endothelial growth factor, a
bisphosphonate, a chemical agent that suppresses the bone loss by
suppressing osteoclasts (the type of bone cell that breaks down
bone tissue), wherein optionally the chemical, drug and/or
biological agent are positioned on the side of an implant surface
intended for cartilage growth and comprise (are) chondrogenic
inducing agents, and/or a chemical or a biomolecule-comprising
agent that stimulates chondrocyte growth, maintenance and/or
differentiation; and optionally a biological agent positioned on
another or opposite side of the implant surface is intended for
bone growth and optionally comprises a chemical, drug and/or
biological agent that stimulates or maintains bone growth; or (c)
the product of manufacture of (b), wherein the bone morphogenic
protein (BMP) is (or comprises) bone morphogenetic protein 2
(BMP-2), bone morphogenetic protein 3 (BMP-3), bone morphogenetic
protein 4 (BMP-5), bone morphogenetic protein 5 (BMP-5), bone
morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7
(BMP-7), bone morphogenetic protein 8 (BMP-8a), bone morphogenetic
protein 10 (BMP-10), bone morphogenetic protein 15 (BMP-15).
5. The product of manufacture of any of claim 4, wherein the
functional particles comprise magnetic oxide particles or metallic
particles utilized for remotely actuated RF heating and creation of
temperature gradient for accelerated or switch-on, or switch-off
release of the chemical, drug and/or biological agent stored in the
nanodepot space.
6. A method of fabricating a chondrocyte attachment-enhancing
and/or chondrocyte growth-enhancing product of manufacture
comprising a nanotube, nanowire, nanopore and/or nanopillar
configuration comprising: (a) use of anodization, formation and
selective phase removal of a two-phase mask layer using diblock
copolymer layer, spinodally decomposing alloy layer, or two-phased
alloy film, followed by selective etching of a biomaterial surface
to produce a nanotube or nanopillar surface configuration on a
surface of the product of manufacture; (b) spot-welding, or
induction melting-bonding, or electron-beam ("e-beam") bonding, or
laser bonding, or braze-bonding, a plurality of nanotubes or
nanowires onto a TiO.sub.2 base on a surface of the product of
manufacture, wherein the base comprises: a Ti, Zr, Hf, Nb, Ta, Mo
or W; or a Ti alloy or oxide, a TiO.sub.2, an Au or Au oxide, a Pt
or Pt oxide, a Pd or a Pd oxide; a mixture comprising an alloy or
an oxide of one or more of Ti, Au, Pt or Pd; or, an oxide of an
alloy comprising Zr, Hf, Nb, Ta, Mo or W; (c) fabricating on the
base surface of the product of manufacture a nanotube, nanopore
and/or nanopillar configuration, wherein the base comprises a bulk
metal or alloy, deposited thin film or deposited thick layer
selected from: a Ti, Zr, Hf, Nb, Ta, Mo or W; or a Ti alloy
comprising Ti, Al, and V, or Ti alloy one or more of Ti, Au, Pt or
Pd; or, an alloy comprising Zr, Hf, Nb, Ta, Mo or W; (d) the method
of any of (a) to (c), wherein the product of manufacture comprises
a surface having a duplex distribution of the nanostructure
dimensions such that a nanopore or nano-depot has an intentionally
bottle-necked or constricted pore structure or opening, wherein
optionally the bottle-necked or constricted pore structure or
opening results in a slower release of a stored agent or
composition, and optionally the agent or composition comprises a
drug and/or a biological agent, and optionally nanostructure with
bottle-necked or constricted pore structures or openings are mixed
and distributed together with regular (non-bottle-necked or
non-constricted pore structures or openings) nanotubes or nanopores
(which do not have the bottleneck diameter reduction), and
optionally the relative area fraction of the bottle necked agent or
composition release region is in the range of about 2% to 50% of
the total available surface area of the product of manufacture, or
in the range of about 2% to 50% of the total available surface area
available for cartilage and/or bone growth or attachments, wherein
optionally the product of manufacture comprises a configuration as
illustrated in FIG. 13; or (e) the method of any of (a) to (d),
wherein the product of manufacture comprises a product of
manufacture composition of claim 1, or the product of manufacture
has a structure as illustrated in any one of FIGS. 5, 13 and 16 to
29.
7. A method of fabricating a product of manufacture comprising a
macroscale added-on scaffold structure for 3-dimensional cartilage
construction using protruding springy wires, mesh screens, vertical
pillar array columns, comprising (i) (a) attaching or forming a
plurality of space-containing and/or springy protruding surface
scaffold structures for three-dimensional chondrocyte assembly and
cartilage growth, wherein optionally space-containing and/or
springy protruding surface scaffold structures are attached or
formed on a surface of the product of manufacture by spot-welding,
or induction melting-bonding, or electron-beam ("e-beam") bonding,
or laser bonding, or braze-bonding of a plurality of wires coils,
mesh screens onto a surface of the product of manufacture, wherein
optionally the material used for the three-dimensional springy,
coil, wire, or mesh screen scaffold comprises a metal or an alloy
or an oxide thereof selected from Ti, Zr, Hf, Nb, Ta, Mo or W, or
an alloy or an oxide comprising at least one of these elements, or
a stainless steel, or a Co- or Cr-comprising alloy, or a
Co--Cr--Ni--Mo alloy, wherein optionally the wire diameter in the
range of between about 10 to 100 um, wherein optionally the wires
in the attached three-dimensional scaffold have a surface structure
of either nanotubes or nanopores having diameter in the range of
between about 30 to 600 nm, or between about 70 to 200 nm, wherein
optionally the wires in the attached three-dimensional scaffold
have a thickness of between about 300 to 400 nm, between about 200
to 500 nm, to between about 100 to 600 nm, wherein the material
used for the three-dimensional springy, coil, wire, or mesh screen
scaffold comprises a metal or alloy selected from Ti, Zr, Hf, Nb,
Ta, Mo or W, or alloys containing at least one of these elements,
or stainless steel, or Co--Cr--Ni--Mo alloy (commonly known as
MP35N alloy), wherein the surface of the springy wire scaffold
contains vertically configured nanotube or nanopore arrays with
about 30 to 600 nm diameter, preferably 70 to 200 nm diameter, and
about 200 to 2,000 nm thickness, and preferably 200 to 500 nm
thickness, wherein a base material onto which the springy
three-dimensional metal scaffold is attached comprises a bulk metal
or alloy, deposited thin film or deposited thick layer selected
from: a Ti, Zr, Hf, Nb, Ta, Mo or W; or a Ti alloy comprising Ti,
Al, and V, or Ti alloy one or more of Ti, Au, Pt or Pd; or, an
alloy comprising Zr, Hf, Nb, Ta, Mo or W, (ii) the method of (i),
further comprising introducing a chondrocyte-growth-enhancing
nanostructure on the surface of the three-dimensional scaffold wire
or pillar surface by use of anodization, formation and selective
phase removal of a two-phase mask layer using diblock copolymer
layer, spinodally decomposing alloy layer, or two-phased alloy
film, followed by selective etching of a biomaterial surface to
produce a nanotube or nanopillar surface configuration; or (iii)
the method of (i) or (ii), wherein the product of manufacture
comprises a composition of claim 1, or the product of manufacture
has a structure as illustrated in any one of FIGS. 5, 13 and 16 to
29.
8-10. (canceled)
11. A patch bone implant piece comprising a product of manufacture
of claim 1, wherein optionally the product of manufacture serves a
dual purpose of comprising at least one exposed surface that
enhances a chondrocyte growth and cartilage formation while
comprising another surface (e.g., an opposing surface or a bottom
surface) facing the existing bone to induce a strong
osseo-integration.
12. (canceled)
13. An implant, or a bone implant, or a patch implant comprising a
product of manufacture of claim 1, optionally comprising Ti or
TiO.sub.2.
14-15. (canceled)
16. A chondrocyte cell culture substrate for enhanced or new
chondrocyte, stem cell and/or extracellular matrix production
comprising the product of manufacture claim 1.
17. A method for making an implant comprising a cell, comprising:
(a) (i) providing a chondrocyte, a colony-forming unit-fibroblast
(CFU-F), a marrow stromal cell (MSC), a stem cell, a totipotent
cell, a multipotent progenitor cell and/or a pluripotent cell; (ii)
providing a product of manufacture of claim 1, wherein optionally
the product of manufacture comprises a TiO.sub.2-comprising
nanotube, nanowire and/or nanopore; and (iii) adding the cell of
(a) to the product of manufacture of (b) under cell culture
conditions; (b) the method of (a), wherein the stem cell is a
mesenchymal stem cell, an adult stem cell, an induced pluripotent
stem cell (abbreviated as iPS cell or iPSC) and/or an embryonic
stem cell; (c) the method of (a), wherein the product of
manufacture is fabricated as a bone implant, or a patch implant, or
patch bone implant piece; (d) the method of any of (a) to (c),
wherein the cell culture conditions comprise use of cell growth
and/or cell differentiation factors; (e) the method of (d), wherein
the cell growth and/or cell differentiation factors comprise a drug
or chemical or biological agent that promotes the growth,
maintenance and/or regeneration of a cell; (f) the method of (e)
wherein the drug or chemical or biological agent that promotes the
growth, maintenance and/or regeneration of a cell promotes the
differentiation growth, maintenance and/or regeneration of a
chondrocyte, a stem cell, a totipotent cell, a multipotent
progenitor cell and/or a pluripotent cell; (g) the method of (d),
(e) or (f), wherein the cell growth, cell differentiation factor or
biological agent comprises a chondrogenic agent or a bone
morphogenic protein (BMP) or an agent, drug or chemical that
stimulates chondrocyte growth, maintenance and/or differentiation,
or a fibroblast growth factor and/or a vascular endothelial growth
factor, and optionally the chondrogenic agent is placed on an
implant surface region intended for cartilage growth, and
optionally bone morphogenic protein (BMP) is placed on an implant
surface region intended for bone growth, and optionally fibroblast
growth factor and/or vascular endothelial growth factor are placed
on an implant surface region intended for bone growth for
osseointegration attachment to the existing bone structure; (h) the
method of (g), wherein the bone morphogenic protein (BMP) is (or
comprises) a bone morphogenetic protein 2 (BMP-2), a bone
morphogenetic protein 3 (BMP-3), a bone morphogenetic protein 4
(BMP-5), a bone morphogenetic protein 5 (BMP-5), a bone
morphogenetic protein 6 (BMP-6), a bone morphogenetic protein 7
(BMP-7), a bone morphogenetic protein 8 (BMP-8a), a bone
morphogenetic protein 10 (BMP-10), a bone morphogenetic protein 15
(BMP-15); (i) the method of (g), wherein the wherein the drug or
chemical or biological agent comprises a fibroblast growth factor
(FGF), an epidermal growth factor (EGF), a vascular endothelial
growth factor (VEGF), a transforming growth factor beta-1
(TGF-.beta.1) or a transforming growth factor beta-2 (TGF-.beta.2),
a bone morphogenic protein (BMP) (e.g., an agent that stimulates
chondrocyte growth, maintenance and/or differentiation), fibroblast
growth factors and/or vascular endothelial growth factors (j) the
method of any of (d) to (i), wherein cell growth, cell
differentiation factor or biological agent comprises a recombinant
protein, or an autologous protein, or a human protein; (k) the
method of any of (a) to (i), wherein the cell culture conditions
comprise a chondrogenic-inducing media; (l) the method of (k),
wherein the chondrogenic-inducing media comprises one, several of
all of: a serum-free DMEM, an ascorbate, a dexamethasone,
L-proline, sodium pyruvate, ITS-plus, an antibiotic and/or a
recombinant protein; or (m) the method of any of (a) to (l),
wherein the cell is implanted in, seeded in or placed in the
implant in-vivo, in-vitro, and/or ex-vivo.
Description
TECHNICAL FIELD
[0001] This invention relates to the fields of nanotechnology,
tissue engineering and regenerative medicine, and in alternative
embodiments, the present invention provides biomaterials and
implants for cartilage maintenance and/or formation in-vivo,
in-vitro, and ex-vivo, using nanotechnology, e.g., using nanotube
or nanopillar configured surface structures.
BACKGROUND
[0002] Cartilage defects are a main issue in orthopedics to which
there is no known physiological treatment to restore the identical
tissue. Because of the ever increasing elderly population with
osteoarthritic disease and an estimated 1 million or more total
joint arthroplasties performed annually in the United States,
cartilage defects remain a major concern in orthopedics and there
is a strong need to resolve this cartilage repair problem.
[0003] Cartilage tissue engineering remains a challenge because
cartilage does not heal itself spontaneously as it does not contain
blood vessels. The main cells of cartilage, chondrocytes, are
limited in self repair. The low density of cells, which do not
replicate, hide among a dense isolating extracellular matrix. This
leads to a lack of regeneration ability because the cells are cut
off from each other, other body tissues, and nutritive sources such
as blood supply, and the usual inflammation response is absent.
[0004] Currently, treatments for cartilage repair are less than
satisfactory, and rarely restore the necessary function nor return
the tissue to its native normal state. Artificial cartilage
prepared from cultured chondrocytes offers promise as a treatment
for cartilage defects, but connecting this artificial soft tissue
to bone in the attempts to restore the defected cartilage is
difficult.
[0005] In the attempts to replace cartilage, most research involves
the combination of in vitro expansion of chondrocytes with
three-dimensional (3-D) synthetic or natural polymer scaffolds.
Constructs have been comprised of materials such as polyglycolic
acid, polylactic acid, various co-polymers, as well as natural
materials of polysaccharides and collagen, and extracellular matrix
proteins and hyaluronic acid constructs to mimic the natural in
vivo environment.
[0006] Although the response of chondrocytes to these polymeric
chemical constructs provided valuable information to advance the
repair of chondral lesions, there are still complications to
overcome. Problems have occurred such as acidic by product
accumulation, local or systemic inflammatory reaction during in
vivo degradation, and the degradation time is too short to allow
neocartilage formation, leaving polymers less promising in clinical
application. Furthermore, most of the polymers are lacking the dual
functionality of osseointegration and cartilage growth, and do not
always provide suitable mechanical properties needed to fully
integrate with native bone tissue.
SUMMARY
[0007] The invention provides compositions and methods for
maintaining and/or replacing damaged, injured or degenerated
cartilage injury, lesions and defects with new cartilage, and to
reduce the pain and dysfunction associated with cartilage injury,
lesions and defects. The compositions and methods of the invention
can be used as (or with) osteochondral autografts, osteocyte
allografts, and autologous or allogenic chondrocyte implantations,
including any techniques used for replacing or repairing
cartilage.
[0008] The compositions and methods of the invention can be used
with any type of metallic or ceramic biocompatible constructs,
configuration and/or biomaterial design to optimize chondrocyte
culture to successfully reproduce cartilage. In alternative
aspects, the chondrocytes used to practice this invention include
any cell that can produce and maintain the cartilaginous matrix,
including osteochondrogenic cells and immature chondrocytes, e.g.,
chondroblasts, and more mature and/or differentiated forms, e.g.,
hypertrophic chondrocytes.
[0009] This invention provides novel nanostructured biomaterials,
devices comprising such biomaterials, and fabrication methods for
efficient reproduction of cartilage in human and animal body. The
biomaterials of the invention can be used to initiate and/or
accelerate chondrocyte cell growth and cartilage formation; and in
alternative embodiments, release growth factors and other chemical
or biological materials, e.g., as materials stored in a nano-depot
of the nanostructured biomaterial surfaces of this invention. In
alternative embodiments, by releasing growth factors and other
chemical or biological materials, compositions and methods of the
invention can be used to ameliorate local or systemic inflammatory
reaction that slow or inhibit cartilage regeneration or repair,
e.g., slow or inhibit local or systemic inflammatory reactions
causing in vivo cartilage degradation.
[0010] This invention provides compositions and methods for
building a cartilage, including e.g., a cartilage construction
technique which in alternatively embodiments can be enhanced by
incorporating (comprising) a stem cell, e.g., a mesenchymal stem
cell, an adult stem cell and/or an embryonic stem cell, a
pluripotent stem cell, an induced pluripotent stem cell
(abbreviated as iPS cell or iPSC), a multipotent progenitor cell
and/or a totipotent cell. In one embodiment, these stem cells are
mammalian, e.g., human, stem cells. In alternative embodiments,
nanostructures of the invention are utilized to improve the
differentiation of stem cells (e.g., adult stem cells, iPS cells
and/or embryonic stem cells) towards formation, adhesion and growth
of chondrocytes, which is important for cartilage growth.
[0011] The invention provides products of manufacture
comprising:
[0012] wherein optionally the product of manufacture is a cell-,
cartilage- and/or bone growth-enhancing or cell
differentiation-enhancing product of manufacture, or a bone- or
cartilage-maintaining and/or bone or cartilage growth-enhancing
product of manufacture, or an implant,
[0013] (a) nanostructures comprising a nanotube, nanowire,
nanopore, nanoribbon and/or a nanopillar surface configuration on a
Ti and/or Ti-comprising alloy, or on a Ti-coated or Ti alloy-coated
surface, or on a TiO.sub.2 and/or TiO.sub.2 alloy surface or
coating, wherein the Ti and/or Ti-comprising alloy or the TiO.sub.2
and/or TiO.sub.2 alloy surface or coating, or the Ti-coated or Ti
alloy-coated surface, comprises one or more surfaces (or a
subsurface or a partial surface) of the product of manufacture,
[0014] wherein optionally the nanostructures (nanotubes, nanowires,
nanopores, nanoribbons and/or nanopillars) comprise a metal and/or
a metal alloy comprising a Ti, a Zr, a Hf, a Nb, a Ta, a Mo and/or
a W, or an oxide of a Ti, a Zr, a Hf, a Nb, a Ta, a Mo and/or a
W,
[0015] wherein optionally the nanostructures (nanotubes, nanowires,
nanopores, nanoribbons and/or nanopillars) are formed directly
and/or indirectly on and/or attached to a Ti surface and/or a
Ti-coated surface, or Ti oxide surface and/or a Ti oxide-coated
surface,
[0016] wherein optionally the nanotubes have a diameter dimension
in the range of between about 30 to 600 nm outside diameter, or
between about 50 to 400 nm diameter, or between about 70 to 200 nm
diameter, and/or optionally a height dimension in the range of
between about 30 to 10,000 nm, and/or optionally between about 200
to 2,000 nm thickness, or between about 200 to 500 nm
thickness,
[0017] wherein optionally the Ti surface and/or Ti-coated surface,
or Ti oxide surface and/or a Ti oxide-coated surface, comprises:
the surface of a wire or microwire; the surface of a springy and/or
a hairy wire or microwire; the surface of a mesh or mesh screen;
the surface of an implant; a "pre-patterned" and/or a "pre-etched"
surface made by machining or mask patterning and/or etching of the
surface of the product of manufacture structure,
[0018] wherein optionally the three-dimensional Ti wire or
microwire is between about 10 to 100 .mu.m in diameter and/or the
Ti wire or microwire is a springy and compliant wire or
microwire,
[0019] wherein optionally the material used for the
three-dimensional springy, coil, wire, or mesh screen scaffold
comprises at least one of a metal or an alloy selected from the
group consisting of Ti, Zr, Hf, Nb, Ta, Mo and W, or an alloy or an
oxide or a mixture thereof, or stainless steel, or a Co--Cr--Ni--Mo
alloy (commonly known as MP35N alloy),
[0020] wherein the surface of the springy wire scaffold contains
vertically configured nanotube or nanopore arrays with about 30 to
600 nm diameter, preferably 70 to 200 nm diameter, and about 200 to
2,000 nm thickness, and preferably 200 to 500 nm thickness,
[0021] wherein optionally the Ti or Ti oxide alloy or Ti or Ti
oxide on the Ti-coated, or Ti oxide-coated or Ti alloy-coated
surface is between about 100 to 2000 .mu.m thick; and
[0022] wherein optionally the product of manufacture structure of
(a) comprises (i) oxides of alloys comprising Ti or a Ti oxide or a
TiO.sub.2 by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80% or 90% or more in weight %, or (ii) oxides of alloys comprising
Zr, Hf, Nb, Ta, Mo, W, by at least about 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80% or 90% or more weight %, or (ii) a ceramic, a
polymer, a plastic, a Si-comprising composition, a Au-comprising
composition, a Pd-comprising composition, a Pt-comprising
composition, or a stainless steel;
[0023] (b) the product of manufacture of (a), and further
comprising a chondrocyte, a stem cell, a totipotent cell, a
multipotent progenitor cell and/or a pluripotent cell, wherein the
chondrocyte functionality, as indicated by the degree of
extracellular matrix formation, is increased by at least about 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more, as compared
with the identical material but without the TiO.sub.2 nanotube or
nanopillar surface configuration;
[0024] (c) the product of manufacture of (a) or (b), further
comprising a chondrocyte, a colony-forming unit-fibroblast (CFU-F),
a marrow stromal cell or mesenchymal stem cell (MSC), a stem cell,
a totipotent cell, a multipotent progenitor cell and/or a
pluripotent cell, wherein optionally the cell is implanted in,
seeded in or placed in the product of manufacture in-vivo,
in-vitro, and/or ex-vivo;
[0025] (d) the product of manufacture of (b) or (c), wherein the
stem cell is a mesenchymal stem cell (MSC), an adult stem cell, an
induced pluripotent stem cell (abbreviated as iPS cell or iPSC)
and/or an embryonic stem cell;
[0026] (e) the product of manufacture of any of (b) to (d), wherein
the chondrocyte is an autologous chondrocyte, a hypertrophic
chondrocyte, or a human chondrocyte;
[0027] (f) the product of manufacture of any of (a) to (d), further
comprising on the surface of the product of manufacture a
nano-depot, a microcavity and/or a macrocavity comprising a cell, a
drug and/or a biological agent,
[0028] wherein optionally the nanotube or a nanopillar, or
microcavity and/or a macrocavity, acts as a depot or storage area
comprising a cell, a drug and/or a biological agent,
[0029] wherein optionally the microcavity has an entrance dimension
of between about 1 to 100 micrometer, or a macrocavity having an
entrance dimension of between about 100 to 1,000 micrometer; or
[0030] (g) the product of manufacture of any of (a) to (f), having
a structure as illustrated in any one of FIGS. 16 to 29.
[0031] In alternative embodiments of the products of manufacture of
the invention, the product of manufacture comprises (a) a thin
coating of a metal, a metal oxide, and/or an alloy at least about
1, 2, 3, 4, 5, 10, 15 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm,
80 nm, 90 nm or 100 nm or more nm in thickness at the surface,
and/or (b) at least a portion of the surface underneath comprises a
vertically aligned and adhering nanotube, nanoribbon, nanowire
and/or nanopillar array structure or structures, and/or a plurality
of recessed nanopore structures.
[0032] In alternative embodiments of the products of manufacture of
the invention, the entrance dimension of the nano-depot, nanotube
and/or nanopore is reduced (constricted or impeded) by a selective
deposition of a metal or an alloy, a metal oxide and/or alloy
oxide, and/or another compound, to induce a partial bottlenecking
(constricting) configuration to slow down or impede the release
rate of a compound or a substance stored in the nano-depot,
nanotube and/or nanopore,
[0033] wherein optionally the compound or substance comprises a
drug and/or a biological agent stored in the nano-depot, nanotube
and/or nanopore,
[0034] wherein optionally the slowing down or impeding of the
release rate of the compound or a substance stored in the
nano-depot, nanotube and/or nanopore is at least by a factor of 2
or 3 or slower, or at least by a factor of about 10 or slower, than
the case of non-bottlenecked (non-constricted) structure,
[0035] wherein optionally the other compound used to partially
bottleneck (constrict or impeded) the nano-depot and/or nanopore
comprises a nitride, a fluoride, a carbide and/or a polymer
material,
[0036] wherein optionally the product of manufacture surface has a
multiplex and/or a duplex distribution of nanostructure structures
with different dimensions such that the product of manufacture
comprises both one or more nano-depot, nanotube and/or nanopore
structures having bottle-necked (constricted or impeded) pore
structures together with nano-depot, nanotube and/or nanopore
structures which do not have the bottleneck diameter (constricted
or impeded opening) reductions,
[0037] wherein optionally the relative area fraction of bottle
necked (constricted or impeded opening) nano-depot, nanotube and/or
nanopore structures in the product of manufacture is in the range
of about 2% to 50% of the total available surface area of the
product of manufacture, or in the range of about 2% to 50% of the
total available surface area available for stimulating cell growth,
cartilage growth and/or bone deposition.
[0038] In alternative embodiments of the products of manufacture of
the invention, the product of manufacture further comprises a
chemical, a drug and/or a biological agent,
[0039] and optionally the chemical, drug and/or biological agent
comprises a small molecule, a growth factor, a collagen, a protein,
a biomolecule, a gene, a nucleic acid, an RNA or a DNA, a nucleic
acid expression vector, an antibiotic, a hormone, a therapeutic
drug, a functional particle, a liposome, or a magnetic, metallic,
ceramic or a polymer particle; or, a differentiation-inducing
chemical, drug and/or biomolecule,
[0040] and optionally the chemical, drug and/or biological agent is
attached to or coated on the product of manufacture, or is stored
in a nanopore, nanodepot and/or nanotube, or the chemical, drug
and/or biological agent is attached to, coated on or stored between
nanostructures comprising a plurality of nanopillars, nanotubes,
nanowires and/or nanoribbons,
[0041] and optionally the chemical, drug and/or biological agent
comprises (are) a fibroblast growth factor (FGF), an epidermal
growth factor (EGF), a vascular endothelial growth factor (VEGF), a
transforming growth factor beta-1 (TGF-.beta.1) or a transforming
growth factor beta-2 (TGF-.beta.2), a bone morphogenic protein
(BMP), an agent that stimulates chondrocyte growth, maintenance
and/or differentiation, a chemical or biomolecule
osteogenic-inducing agent, a fibroblast growth factor and/or a
vascular endothelial growth factor, a bisphosphonate, a chemical
agent that suppresses the bone loss by suppressing osteoclasts (the
type of bone cell that breaks down bone tissue),
[0042] wherein optionally the chemical, drug and/or biological
agent are positioned on the side of an implant surface intended for
cartilage growth and comprise (are) chondrogenic inducing agents,
and/or a chemical or a biomolecule-comprising agent that stimulates
chondrocyte growth, maintenance and/or differentiation;
[0043] and optionally a biological agent positioned on another or
opposite side of the implant surface is intended for bone growth
and optionally comprises a chemical, drug and/or biological agent
that stimulates or maintains bone growth; or
[0044] (c) the product of manufacture of (b), wherein the bone
morphogenic protein (BMP) is (or comprises) bone morphogenetic
protein 2 (BMP-2), bone morphogenetic protein 3 (BMP-3), bone
morphogenetic protein 4 (BMP-5), bone morphogenetic protein 5
(BMP-5), bone morphogenetic protein 6 (BMP-6), bone morphogenetic
protein 7 (BMP-7), bone morphogenetic protein 8 (BMP-8a), bone
morphogenetic protein 10 (BMP-10), bone morphogenetic protein 15
(BMP-15).
[0045] In alternative embodiments, the products of manufacture of
the invention comprise functional particles comprising e.g.,
magnetic oxide particles or metallic particles utilized for
remotely actuated RF heating and creation of temperature gradient
for accelerated or switch-on, or switch-off release of the
chemical, drug and/or biological agent stored in the nanopore,
nanodepot and/or nanotube space.
[0046] The invention provides methods of fabricating a chondrocyte
attachment-enhancing and/or chondrocyte growth-enhancing product of
manufacture comprising a nanotube, nanowire, nanopore and/or
nanopillar configuration comprising:
[0047] (a) use of anodization, formation and selective phase
removal of a two-phase mask layer using diblock copolymer layer,
spinodally decomposing alloy layer, or two-phased alloy film,
followed by selective etching of a biomaterial surface to produce a
nanotube or nanopillar surface configuration on a surface of the
product of manufacture;
[0048] (b) spot-welding, or induction melting-bonding, or
electron-beam ("e-beam") bonding, or laser bonding, or
braze-bonding, a plurality of nanotubes or nanowires onto a
TiO.sub.2 base on a surface of the product of manufacture, wherein
the base comprises: a Ti, Zr, Hf, Nb, Ta, Mo or W; or a Ti alloy or
oxide, a TiO.sub.2, an Au or Au oxide, a Pt or Pt oxide, a Pd or a
Pd oxide; a mixture comprising an alloy or an oxide of one or more
of Ti, Au, Pt or Pd; or, an oxide of an alloy comprising Zr, Hf,
Nb, Ta, Mo or W;
[0049] (c) fabricating on the base surface of the product of
manufacture a nanotube, nanopore and/or nanopillar configuration,
wherein the base comprises a bulk metal or alloy, deposited thin
film or deposited thick layer selected from: a Ti, Zr, Hf, Nb, Ta,
Mo or W; or a Ti alloy comprising Ti, Al, and V, or Ti alloy one or
more of Ti, Au, Pt or Pd; or, an alloy comprising Zr, Hf, Nb, Ta,
Mo or W;
[0050] (d) the method of any of (a) to (c), wherein the product of
manufacture comprises a surface having a duplex distribution of the
nanostructure dimensions such that a nanopore or nano-depot has an
intentionally bottle-necked or constricted pore structure or
opening, wherein optionally the bottle-necked or constricted pore
structure or opening results in a slower release of a stored agent
or composition, and optionally the agent or composition comprises a
drug and/or a biological agent, and optionally nanostructure with
bottle-necked or constricted pore structures or openings are mixed
and distributed together with regular (non-bottle-necked or
non-constricted pore structures or openings) nanotubes or nanopores
(which do not have the bottleneck diameter reduction), and
optionally the relative area fraction of the bottle necked agent or
composition release region is in the range of about 2% to 50% of
the total available surface area of the product of manufacture, or
in the range of about 2% to 50% of the total available surface area
available for cartilage and/or bone growth or attachments, wherein
optionally the product of manufacture comprises a configuration as
illustrated in FIG. 13; or
[0051] (e) the method of any of (a) to (d), wherein the product of
manufacture comprises a product of manufacture composition of the
invention, or the product of manufacture has a structure as
illustrated in any one of FIGS. 5, 13 and 16 to 29.
[0052] The invention provides methods of fabricating a product of
manufacture comprising a macroscale added-on scaffold structure for
3-dimensional cartilage construction using protruding springy
wires, mesh screens, vertical pillar array columns, comprising
[0053] (i) (a) attaching or forming a plurality of space-containing
and/or springy protruding surface scaffold structures for
three-dimensional chondrocyte assembly and cartilage growth,
[0054] wherein optionally space-containing and/or springy
protruding surface scaffold structures are attached or formed on a
surface of the product of manufacture by spot-welding, or induction
melting-bonding, or electron-beam ("e-beam") bonding, or laser
bonding, or braze-bonding of a plurality of wires coils, mesh
screens onto a surface of the product of manufacture,
[0055] wherein optionally the material used for the
three-dimensional springy, coil, wire, or mesh screen scaffold
comprises a metal or an alloy or an oxide thereof selected from Ti,
Zr, Hf, Nb, Ta, Mo or W, or an alloy or an oxide comprising at
least one of these elements, or a stainless steel, or a Co- or
Cr-comprising alloy, or a Co--Cr--Ni--Mo alloy,
[0056] wherein optionally the wire diameter in the range of between
about 10 to 100 um,
[0057] wherein optionally the wires in the attached
three-dimensional scaffold have a surface structure of either
nanotubes or nanopores having diameter in the range of between
about 30 to 600 nm, or between about 70 to 200 nm,
[0058] wherein optionally the wires in the attached
three-dimensional scaffold have a thickness of between about 300 to
400 nm, between about 200 to 500 nm, to between about 100 to 600
nm,
[0059] wherein the material used for the three-dimensional springy,
coil, wire, or mesh screen scaffold comprises a metal or alloy
selected from Ti, Zr, Hf, Nb, Ta, Mo or W, or alloys containing at
least one of these elements, or stainless steel, or Co--Cr--Ni--Mo
alloy (commonly known as MP35N alloy),
[0060] wherein the surface of the springy wire scaffold contains
vertically configured nanotube or nanopore arrays with about 30 to
600 nm diameter, preferably 70 to 200 nm diameter, and about 200 to
2,000 nm thickness, and preferably 200 to 500 nm thickness,
[0061] wherein a base material onto which the springy
three-dimensional metal scaffold is attached comprises a bulk metal
or alloy, deposited thin film or deposited thick layer selected
from: a Ti, Zr, Hf, Nb, Ta, Mo or W; or a Ti alloy comprising Ti,
Al, and V, or Ti alloy one or more of Ti, Au, Pt or Pd; or, an
alloy comprising Zr, Hf, Nb, Ta, Mo or W,
[0062] (ii) the method of (i), further comprising introducing a
chondrocyte-growth-enhancing nanostructure on the surface of the
three-dimensional scaffold wire or pillar surface by use of
anodization, formation and selective phase removal of a two-phase
mask layer using diblock copolymer layer, spinodally decomposing
alloy layer, or two-phased alloy film, followed by selective
etching of a biomaterial surface to produce a nanotube or
nanopillar surface configuration; or
[0063] (iii) the method of (i) or (ii), wherein the product of
manufacture comprises a composition of the invention, and/or a
product of manufacture made by a method of the invention, or the
product of manufacture has a structure as illustrated in any one of
FIGS. 5, 13 and 16 to 29.
[0064] The invention provides uses of a product of manufacture of
the invention, or a product of manufacture made by a method of the
invention, wherein the use comprises restoration, restructuring or
repair of cartilage tissue in a thumb, a fingers, a wrist, an
elbow, a shoulder, a hip, a knee, an ankle, a foot, a toe, an
inter-vertebral disc of the spinal cord or a rib cage, or a nose or
an ear, or a method of restoration or restructuring or repair of
cartilage tissue in a thumb, a fingers, a wrist, an elbow, a
shoulder, a hip, a knee, an ankle, a foot, a toe, an
inter-vertebral disc of the spinal cord or a rib cage, or a nose or
an ear, comprising use of the product of manufacture of the
invention, wherein optionally the method or use comprises in vivo
implantation of the product of manufacture of the invention.
[0065] The invention provides uses of a product of manufacture of
the invention, or a product of manufacture made by a method of the
invention, wherein the product of manufacture is used to enable
joint movement while providing the structural support and chemical
environment for new cartilage tissue to grow and fill defect, or to
replace damaged, infected, aged, or diseased cartilage caused by
various diseases such as arthritis, osteoarthritis, isolated
femropatellar osteoarthritis, rheumatoid arthritis, chronic or
systemic autoimmune disorder, lupus, or other autoimmune diseases,
osteonecrosis of the joint, or septic arthritis caused by joint
infection, or a method to enable joint movement while providing the
structural support and chemical environment for new cartilage
tissue to grow and fill defect, or to replace damaged, infected,
aged, or diseased cartilage caused by various diseases such as
arthritis, osteoarthritis, isolated femropatellar osteoarthritis,
rheumatoid arthritis, chronic or systemic autoimmune disorder,
lupus, or other autoimmune diseases, osteonecrosis of the joint, or
septic arthritis caused by joint infection, comprising use of the
product of manufacture of the invention, or a product of
manufacture made by a method of the invention, wherein optionally
the method or use comprises in vivo implantation of the product of
manufacture of the invention, or a product of manufacture made by a
method of the invention.
[0066] The invention provides in vivo uses of a product of
manufacture of the invention, or a product of manufacture made by a
method of the invention, wherein the product of manufacture is
applied in vivo as a patch bone implant piece, wherein optionally
the product of manufacture serves a dual purpose of comprising at
least one exposed surface that enhances a chondrocyte growth and
cartilage formation while comprising another surface (e.g., an
opposing surface or a bottom surface) facing the existing bone to
induce a strong osseo-integration.
[0067] The invention provides implants, e.g., patch bone implant
pieces, comprising a product of manufacture of the invention, or a
product of manufacture made by a method of the invention, wherein
optionally the product of manufacture serves a dual purpose of
comprising at least one exposed surface that enhances a chondrocyte
growth and cartilage formation while comprising another surface
(e.g., an opposing surface or a bottom surface) facing the existing
bone to induce a strong osseo-integration.
[0068] The invention provides in vivo uses of a product of
manufacture of the invention, or a product of manufacture made by a
method of the invention, wherein the product of manufacture
comprises a patch implant comprising Ti, or a Ti alloy or Ti oxide
or a mixture thereof, and optionally the patch implant is
permanently screwed onto or into an existing bone or temporarily
fixed onto or into an existing bone with strings or straps.
[0069] The invention provides implants, or bone implants, or patch
implants, comprising a product of manufacture of the invention, or
a product of manufacture made by a method of the invention,
optionally comprising a Ti, a Ti alloy or TiO.sub.2 or a mixture
thereof.
[0070] The invention provides in vivo uses of a product of
manufacture of the invention, or a product of manufacture made by a
method of the invention, wherein the product of manufacture is
utilized as a chondrocyte cell culture substrate for enhanced or
new chondrocyte, stem cell and/or extracellular matrix production,
or a method for enhancing chondrocyte, stem cell and/or
extracellular matrix production or stimulating new chondrocyte,
stem cell and/or extracellular matrix production comprising in vivo
or ex vivo use of the product of manufacture of the invention. In
alternative embodiments of uses or methods of the invention,
wherein an individual's own chondrocyte cells are used (autologous
cells are used), and the method optionally comprises implanting the
product of manufacture into the individual (a human or an animal)
near a cartilage damage regions or a tissue in need of repair
and/or reconstruction.
[0071] The invention provides chondrocyte cell culture substrates
for enhanced or new chondrocyte, stem cell and/or extracellular
matrix production comprising a product of manufacture of the
invention, or a product of manufacture made by a method of the
invention.
[0072] The invention provides methods for making an implant
comprising a cell, comprising:
[0073] (a) (i) providing a chondrocyte, a colony-forming
unit-fibroblast (CFU-F), a marrow stromal cell (MSC), a stem cell,
a totipotent cell, a multipotent progenitor cell and/or a
pluripotent cell;
[0074] (ii) providing a product of manufacture of the invention, or
a product of manufacture made by a method of the invention, wherein
optionally the product of manufacture comprises a
TiO.sub.2-comprising nanotube, nanowire and/or nanopore; and
[0075] (iii) adding the cell of (a) to the product of manufacture
of (b) under cell culture conditions;
[0076] (b) the method of (a), wherein the stem cell is a
mesenchymal stem cell, an adult stem cell, an induced pluripotent
stem cell (abbreviated as iPS cell or iPSC) and/or an embryonic
stem cell;
[0077] (c) the method of (a), wherein the product of manufacture is
fabricated as a bone implant, or a patch implant, or patch bone
implant piece;
[0078] (d) the method of any of (a) to (c), wherein the cell
culture conditions comprise use of cell growth and/or cell
differentiation factors;
[0079] (e) the method of (d), wherein the cell growth and/or cell
differentiation factors comprise a drug or chemical or biological
agent that promotes the growth, maintenance and/or regeneration of
a cell;
[0080] (f) the method of (e) wherein the drug or chemical or
biological agent that promotes the growth, maintenance and/or
regeneration of a cell promotes the differentiation growth,
maintenance and/or regeneration of a chondrocyte, a stem cell, a
totipotent cell, a multipotent progenitor cell and/or a pluripotent
cell;
[0081] (g) the method of (d), (e) or (f), wherein the cell growth,
cell differentiation factor or biological agent comprises a
chondrogenic agent or a bone morphogenic protein (BMP) or an agent,
drug or chemical that stimulates chondrocyte growth, maintenance
and/or differentiation, or a fibroblast growth factor and/or a
vascular endothelial growth factor,
[0082] and optionally the chondrogenic agent is placed on an
implant surface region intended for cartilage growth, and
optionally bone morphogenic protein (BMP) is placed on an implant
surface region intended for bone growth, and optionally fibroblast
growth factor and/or vascular endothelial growth factor are placed
on an implant surface region intended for bone growth for
osseointegration attachment to the existing bone structure;
[0083] (h) the method of (g), wherein the bone morphogenic protein
(BMP) is (or comprises) a bone morphogenetic protein 2 (BMP-2), a
bone morphogenetic protein 3 (BMP-3), a bone morphogenetic protein
4 (BMP-5), a bone morphogenetic protein 5 (BMP-5), a bone
morphogenetic protein 6 (BMP-6), a bone morphogenetic protein 7
(BMP-7), a bone morphogenetic protein 8 (BMP-8a), a bone
morphogenetic protein 10 (BMP-10), a bone morphogenetic protein 15
(BMP-15);
[0084] (i) the method of (g), wherein the wherein the drug or
chemical or biological agent comprises a fibroblast growth factor
(FGF), an epidermal growth factor (EGF), a vascular endothelial
growth factor (VEGF), a transforming growth factor beta-1
(TGF-.beta.1) or a transforming growth factor beta-2 (TGF-.beta.2),
a bone morphogenic protein (BMP) (e.g., an agent that stimulates
chondrocyte growth, maintenance and/or differentiation), fibroblast
growth factors and/or vascular endothelial growth factors
[0085] (j) the method of any of (d) to (i), wherein cell growth,
cell differentiation factor or biological agent comprises a
recombinant protein, or an autologous protein, or a human
protein;
[0086] (k) the method of any of (a) to (j), wherein the cell
culture conditions comprise a chondrogenic-inducing media;
[0087] (l) the method of (k), wherein the chondrogenic-inducing
media comprises one, several of all of: a serum-free DMEM, an
ascorbate, a dexamethasone, L-proline, sodium pyruvate, ITS-plus,
an antibiotic and/or a recombinant protein; or
[0088] (m) the method of any of (a) to (l), wherein the cell is
implanted in, seeded in or placed in the implant in-vivo, in-vitro,
and/or ex-vivo.
[0089] The invention provides methods of fabricating
chondrocyte-enhancing nanotube or nanopillar configurations using
anodization, formation and selective phase removal of a two-phase
mask layer using diblock copolymer layer, spinodally decomposing
alloy layer, or two-phased alloy film, followed by selective
etching of the biomaterial surface to produce nanotube or
nanopillar surface configurations.
[0090] The invention provides uses of the product of manufacture of
the invention, wherein the use comprises restoration, restructuring
or repair of cartilage tissue in a thumb, a fingers, a wrist, an
elbow, a shoulder, a hip, a knee, an ankle, a foot, a toe, an
inter-vertebral disc of the spinal cord or a rib cage, or a nose or
an ear. The invention provides methods of restoration or
restructuring or repair of cartilage tissue in a thumb, a fingers,
a wrist, an elbow, a shoulder, a hip, a knee, an ankle, a foot, a
toe, an inter-vertebral disc of the spinal cord or a rib cage, or a
nose or an ear, comprising use of the product of manufacture of
this invention, wherein in one embodiment the use comprises in vivo
implantation of a product of manufacture of this invention.
[0091] The invention provides uses of the product of manufacture of
the invention, wherein the product of manufacture is used to enable
joint movement while providing the structural support and chemical
environment for new cartilage tissue to grow and fill defect, or to
replace damaged, infected, aged, or diseased cartilage caused by
various diseases such as arthritis, osteoarthritis, isolated
femropatellar osteoarthritis, rheumatoid arthritis, chronic or
systemic autoimmune disorder, lupus, or other autoimmune diseases,
osteonecrosis of the joint, or septic arthritis caused by joint
infection, or a method to enable joint movement while providing the
structural support and chemical environment for new cartilage
tissue to grow and fill defect, or to replace damaged, infected,
aged, or diseased cartilage caused by various diseases such as
arthritis, osteoarthritis, isolated femropatellar osteoarthritis,
rheumatoid arthritis, chronic or systemic autoimmune disorder,
lupus, or other autoimmune diseases, osteonecrosis of the joint, or
septic arthritis caused by joint infection, comprising use of the
product of manufacture of any of this invention, wherein optionally
the method or use comprises in vivo implantation of the product of
manufacture of any of this invention.
[0092] The invention provides uses of the product of manufacture of
the invention, wherein the product of manufacture is applied as an
implant, or a bone implant, or a patch implant patch bone, wherein
the implant surfaces serve dual/multiple purposes; for example
having more than one (e.g., two or more) surfaces with multiple
purposes, e.g., one surface for cartilage growth and another
surface of the implant for bone growth. For example, implant
surfaces can serve dual/multiple purposes--where one (e.g., can be
described as "exposed") implant surface enhances chondrocyte growth
and cartilage formation while another surface, e.g., the opposite
(e.g., non-exposed) surface of the implant, or one or more implant
surfaces intended for bone growth, or the one or more implant
surfaces facing the existing bone, induce (or are designed to
induce or stimulate) bone growth and/or strong
osseo-integration.
[0093] The invention provides an in-vivo use of the product of
manufacture of the product of manufacture of the invention, wherein
the product of manufacture is applied as an implant, or a bone
implant, or a patch implant, or patch bone implant piece, wherein
in alternative embodiments the product of manufacture serves a dual
or multiple purposes, e.g., comprising one or more exposed
surface(s) that enhance(s) chondrocyte growth and cartilage
formation while another (one or more) surface(s) facing the
existing bone to induce bone growth and/or a strong
osseo-integration. For example, in alternative embodiments, the
invention provides an implant, or a bone implant, or a patch
implant, or patch bone implant piece comprising any product of
manufacture of the invention, wherein in alternative embodiments
the product of manufacture serves a dual or multiple purposes
comprising enhancing chondrocyte growth and cartilage formation on
one or more surfaces (e.g., exposed surfaces) and also comprising
another surface, e.g., the opposite (e.g., non-exposed) surface of
the implant, or one or more implant surfaces intended for bone
growth, or one or more implant surfaces facing the existing bone to
induce bone growth and/or a strong osseo-integration.
[0094] The invention provides an in-vivo use of the product of
manufacture of the invention, wherein the product of manufacture
comprises a patch implant is made of Ti, and is permanently screwed
onto the existing bone or temporarily fixed with strings or straps.
The invention provides a patch implant comprising a product of
manufacture of the invention, optionally comprising Ti or
TiO.sub.2.
[0095] The invention provides in-vivo use of the product of
manufacture of the invention, wherein the product of manufacture is
utilized as a chondrocyte cell culture substrate for enhanced or
new chondrocyte, stem cell and/or extracellular matrix production,
or a method for enhancing chondrocyte, stem cell and/or
extracellular matrix production or stimulating new chondrocyte,
stem cell and/or extracellular matrix production comprising in-vivo
use of the product of manufacture of the invention. In alternative
embodiments of the use or method an individual's own chondrocyte
cells are used (autologous cells are used), optionally followed by
implanting the product of manufacture into the individual (a human
or an animal) near a cartilage damage regions or a tissue in need
of repair and/or reconstruction.
[0096] In one aspect of uses of compositions and methods of the
invention, an individual's own chondrocyte or other cells are used
(e.g., chondrocytes and fibroblasts, osteoclasts or osteoblasts),
followed by implanting into a human or an animal near a cartilage
damage regions or a tissue in need of repair and/or
reconstruction.
[0097] The invention provides methods for making an implant (an
implant for an individual, e.g., human or animal) comprising a cell
(or a plurality of cells), comprising:
[0098] (a) (i) providing a chondrocyte, a colony-forming
unit-fibroblast (CFU-F), a marrow stromal cell or mesenchymal stem
cell (MSC), a stem cell, a totipotent cell, a multipotent
progenitor cell and/or a pluripotent cell, wherein optionally the
cell is implanted in, seeded in or placed in the implant in-vivo,
in-vitro, and/or ex-vivo;
[0099] (ii) providing a product of manufacture of any of claims 1
to 5, wherein optionally the product of manufacture comprises a
TiO.sub.2-comprising nanotube, nanowire and/or nanopore; and
[0100] (iii) adding the cell of (a) to the product of manufacture
of (b) under cell culture conditions;
[0101] (b) the method of (a), wherein the stem cell is a
mesenchymal stem cell, an adult stem cell, an induced pluripotent
stem cell (abbreviated as iPS cell or iPSC) and/or an embryonic
stem cell;
[0102] (c) the method of (a), wherein the product of manufacture is
fabricated as a bone implant, or a patch implant, or patch bone
implant piece;
[0103] (d) the method of any of (a) to (c), wherein the cell
culture conditions comprise use of cell growth and/or cell
differentiation factors;
[0104] (e) the method of (d), wherein the cell growth and/or cell
differentiation factors comprise a biological agent that promotes
the growth, maintenance and/or regeneration of a cell;
[0105] (f) the method of (e) wherein the biological agent that
promotes the growth, maintenance and/or regeneration of a cell
promotes the differentiation growth, maintenance and/or
regeneration of a chondrocyte, a stem cell, a totipotent cell, a
multipotent progenitor cell and/or a pluripotent cell;
[0106] (g) the method of (d), (e) or (f), wherein the cell growth,
cell differentiation factor or biological agent comprises a bone
morphogenic protein (BMP) (e.g., an agent that stimulates
chondrocyte growth, maintenance and/or differentiation), fibroblast
growth factors and/or vascular endothelial growth factors;
[0107] (h) the method of (g), wherein the bone morphogenic protein
(BMP) is (or comprises) a bone morphogenetic protein 2 (BMP-2), a
bone morphogenetic protein 3 (BMP-3), a bone morphogenetic protein
4 (BMP-5), a bone morphogenetic protein 5 (BMP-5), a bone
morphogenetic protein 6 (BMP-6), a bone morphogenetic protein 7
(BMP-7), a bone morphogenetic protein 8 (BMP-8a), a bone
morphogenetic protein 10 (BMP-10), a bone morphogenetic protein 15
(BMP-15);
[0108] (i) the method of (g), wherein the wherein the biological
agent comprises a fibroblast growth factor (FGF), an epidermal
growth factor (EGF), a vascular endothelial growth factor (VEGF), a
transforming growth factor beta-1 (TGF-.beta.1) or a transforming
growth factor beta-2 (TGF-.beta.2), a bone morphogenic protein
(BMP) (e.g., an agent that stimulates chondrocyte growth,
maintenance and/or differentiation), fibroblast growth factors
and/or vascular endothelial growth factors
[0109] (j) the method of any of (d) to (i), wherein cell growth,
cell differentiation factor or biological agent comprises a
recombinant protein, or an autologous protein, or a human
protein;
[0110] (k) the method of any of (a) to (i), wherein the cell
culture conditions comprise a chondrogenic-inducing media;
[0111] (k) the method of (j), wherein the chondrogenic-inducing
media comprises one, several of all of: a serum-free DMEM, an
ascorbate, a dexamethasone, L-proline, sodium pyruvate,
Insulin-Transferrin-Selenium (ITS) or ITS-PLUS.TM.
(Gibco-Invitrogen, Carlsbad, Calif.), an antibiotic and/or a
recombinant protein.
[0112] Also provided herein are kits comprising compositions of the
invention including instructions for practicing the methods
provided herein.
[0113] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
[0114] All publications, patents, patent applications, GenBank
sequences and ATCC deposits, cited herein are hereby expressly
incorporated by reference for all purposes.
DESCRIPTION OF DRAWINGS
[0115] FIG. 1(a)-(c) schematically illustrates exemplary devices
comprising self-organized TiO.sub.2 nanotube arrays formed on
titanium substrate to accelerate chondrocyte proliferation and
cartilage formation as well as osteoblast cell proliferation and
bone osseointegration according to the invention; as described in
detail, below.
[0116] FIG. 2's four panels (at 30, 50, 70 and 100 nm diameter as
indicated) illustrate SEM micrographs of exemplary self-aligned
TiO.sub.2 nanotubes with different diameters; scale bars are 200
nm; as described in detail, below.
[0117] FIG. 3 illustrates structure of exemplary vertically aligned
TiO.sub.2 nanotubes on a titanium substrate, FIG. 3 (a) illustrates
a scanning electron microscope (SEM) micrograph image at 200 nm,
FIG. 3 (b) illustrates a longitudinal view transmission electron
microscope (TEM) micrograph at 200 nm, FIG. 3 (c) illustrates a
cross-sectional TEM at 100 nm; as described in detail, below.
[0118] FIG. 4(a) and FIG. 4(b) schematically illustrate exemplary
devices comprising nano-pillar configured TiO.sub.2 arrays formed
on titanium substrate to accelerate cell, e.g., stem cell or
chondrocyte, proliferation and cartilage formation; which also can
include osteoblast cell proliferation and bone osseointegration; as
described in detail, below.
[0119] FIG. 5 illustrates an exemplary nano-imprint stamping
process to fabricate a nanopillar array of Ti oxide or related
metal and alloy oxide nanotubes: FIG. 5(a) illustrates an exemplary
nano-imprinting of masking resist, FIG. 5(b) illustrates an
exemplary chemical or RIE etch to form Ti nanopillar array, which
can be converted to TiO.sub.2 nanopillars or Ti nanopillars with
surface TiO.sub.2 layer, FIG. 5(c) illustrates how this exemplary
nanopillar construction can act as a guided and vertically aligned
adhesion and growth matrix for cells and cartilage using e.g., Ti
wires, pillars or columns, e.g., optionally having surface
nanotubes; as described in detail, below.
[0120] FIG. 6(a) to (f) illustrates an exemplary method for
fabricating nanopillar or nanotube array on implant surface by
guided etching using a vertically two-phase decomposed coating; as
described in detail, below.
[0121] FIG. 7 illustrates fifteen panel images of SEM micrographs
of bovine cartilage chondrocytes (BCCs) on flat Ti and 30, 50, 70,
100 nm diameter TiO.sub.2 nanotube surfaces of this invention after
2 hours (top row), 24 hours (middle row), and 5 days (lower row) of
culture. Arrows indicate difference in ECM fibril formation and
cell clustering on the nanotube substrates, seen in larger
diameters (70 and 100 nm), compared to flat Ti; as described in
detail, below.
[0122] FIG. 8(a) and FIG. 8(b) illustrate higher magnification SEM
observations of bovine cartilage chondrocytes (BCCs) reveal a
striking difference in the formation of ECM (see arrow) between the
flat Ti surfaces, as illustrated in FIG. 8(a) versus surfaces of an
exemplary nanotube structure as illustrated in FIG. 8(b), after 24
hours of culture; as described in detail, below.
[0123] FIG. 9 schematically illustrates six panels of
immunofluorescent images of cells grown on exemplary compositions
of this invention (having nanotube surfaces) to show the viability
of bovine cartilage chondrocytes (BCCs) using fluorescein diacetate
(FDA) cytoplasmic staining (a viability-staining technique); the
cells were cultured for 5 days on control polystyrene culture
dishes, flat Ti and 30, 50, 70, 100 nm diameter TiO.sub.2 nanotube
surfaces of this invention; viability-staining demonstrates that
practically all the cells were alive on all surfaces; as described
in detail, below.
[0124] FIG. 10 schematically illustrates in bar graph form a
summary of data of bovine cartilage chondrocyte (BCC) cell shape
analysis based on fluorescein diacetate (FDA) viability staining;
the bar graph shows the average percentage of round cells
.+-.standard error bars; larger diameter exemplary nanotubes show
significantly larger percentages of round cells over polystyrene
and flat Ti controls; this demonstrates that the nanotubes of this
invention preserve the spherical phenotypic shape of the
chondrocytes more efficiently; as described in detail, below.
[0125] FIG. 11 schematically illustrates in bar graph form a
summary of data of glycosaminoglycan (GAG) secretion (relative to
that measured in control culture dish) in the media in contact with
flat control surface (polystyrene) and exemplary 30, 50, 70, 100 nm
diameter nanotube substrates (nanotube substrata .+-.SEM); as
described in detail, below.
[0126] FIG. 12 illustrates immunofluorescent images of collagen
type II (red) ECM fibrils produced by bovine cartilage chondrocytes
(BCCs); FIG. 12(a): illustrates a low magnification comparing
collagen shape on exemplary flat Ti vs 100 nm diameter nanotubes of
the invention, FIG. 12(b): illustrates a higher magnification
showing immunofluorescent images of collagen type II (red) and DAPI
(blue) nuclear staining of BCCs on polystyrene, flat Ti, and 30,
50, 70, 100 nm TiO.sub.2 surfaces after 5 days of culture; as
described in detail, below.
[0127] FIG. 13 schematically illustrates an embodiment of TiO.sub.2
nanotube based implants comprising nanotubes with slow-releasing
drugs and/or biological agents stored in the vertically aligned
nanotube pores: FIG. 13(a) illustrates an exemplary embodiment
including as-made TiO.sub.2 nanotubes, FIG. 13(b) illustrates an
exemplary embodiment where cells, drugs and/or biological agents
stored in the nano-depots, FIG. 13(c) illustrates an exemplary
embodiment comprising a diameter-reduced nano-depot entrance for
slower release of stored cells, drugs and/or biological agents,
FIG. 13(d) illustrates an exemplary embodiment comprising locally
distributed bottlenecked regions within the regular nanotube
region; as described in detail, below.
[0128] FIG. 14 schematically illustrates an embodiment of TiO.sub.2
nano-pillar configured implants comprising slow-releasing drugs
and/or biological agents stored in the gap between vertically
aligned nanopillars; FIG. 14(a) illustrates an exemplary embodiment
comprising a TiO.sub.2 nano-pillar, FIG. 14(b) illustrates an
exemplary embodiment comprising cells, drugs and/or biological
agents stored in the gap between nanopillars, FIG. 14(c)
illustrates an exemplary embodiment comprising a dimension reduced
entrance for slower release of stored cells, drugs and/or
biological agents from the nanopillar gap; as described in detail,
below.
[0129] FIG. 15 schematically illustrates: FIG. 15(a) an articular
cartilage defect (the arrows illustrating the example cartilage
defect and particular anatomical details of the illustrated knee,
including the femur, articular cartilage, fibula, knee cap, the
example cartilage defect and the tibia), FIG. 15(b) accelerated in
vitro chondrocyte culture using an exemplary TiO.sub.2 nanotube
substrate of this invention (which in this example is used as a
"scaffold" to repair the cartilage defect), FIG. 15(c) an injection
of cultured chondrocytes to an in vivo implanted exemplary
TiO.sub.2 nanotube substrate "scaffold" to correct the cartilage
defect (see arrow); as described in detail, below.
[0130] FIG. 16 schematically illustrates an embodiment of the
invention comprising a Ti patch implant with TiO.sub.2 nanotubes on
two surfaces, e.g., a "top" surface for chondrocyte growth and an
opposing (e.g., bottom) surface for strong osseointegration with
existing bone surface, and enhanced chondrocyte and cartilage
formation on the top surface; as described in detail, below.
[0131] FIG. 17(a) illustrates an embodiment of the invention
comprising a three-dimension Ti implant surface; extended and
protruding wire-like biocompatible structures are shown. FIG. 17(b)
schematically illustrates three-dimensional and geometrically
secured cartilage growth around compliant, hairy or spring-shaped
Ti having TiO2 nanotubes or other nanostructures; as described in
detail, below.
[0132] FIG. 18(a) illustrates an embodiment of the invention
comprising exemplary TiO.sub.2 nanotube or nanopore arrays;
hairy-shaped or mesh-screen shaped surface structures; FIG. 18(b)
schematically illustrates an embodiment of three-dimensional and
geometrically secured cartilage growth around compliant
spring-shaped Ti wires having TiO2 nanotubes or other
nanostructures; as described in detail, below.
[0133] FIG. 19(a) to (d) illustrate an embodiment of the invention
comprising diffusional bonding of Ti hairy-shaped or Ti mesh-screen
shaped surface structures onto an exemplary composition of the
invention, e.g., a Ti implant; as described in detail, below.
[0134] FIG. 20(a) to (d) illustrate an embodiment of the invention
comprising melt-bonding of Ti or stainless steel hairy-shaped or
mesh-screen shaped surface structures onto an exemplary composition
of the invention, e.g., a Ti implant; as described in detail,
below.
[0135] FIG. 21(a) to (d) illustrate an embodiment of the invention
comprising spot-welding of Ti or stainless steel hairy-shaped or
mesh-screen shaped surface structures onto an exemplary composition
of the invention, e.g., a Ti implant; as described in detail,
below.
[0136] FIG. 22(a) and FIG. 22(b) illustrate an embodiment of the
invention comprising exemplary TiO.sub.2 nanotube or nanopore
arrays; a side view of exemplary hairy-shaped or mesh-screen shaped
surface structures are shown; as described in detail, below.
[0137] FIG. 23(a), FIG. 23(b) and FIG. 23(c) illustrate embodiments
of the invention comprising exemplary flat or dual-structured Ti
implants having compliant, three-dimensional wire assembly
structure; as described in detail, below.
[0138] FIG. 24(a), FIG. 24(b) and FIG. 24(c) illustrate embodiments
of the invention comprising exemplary TiO.sub.2 compositions of the
invention comprising Ti or Ti alloy particles or fibers; as
described in detail, below.
[0139] FIG. 25 illustrates an embodiment of the invention
comprising sintering exemplary Ti or Ti alloy particles or fibers
onto an exemplary composition of the invention, e.g., a Ti
implant.
[0140] FIG. 26(a), FIG. 26(b) and FIG. 26(c) illustrate embodiments
of the invention comprising exemplary compositions of the invention
comprising Ti or Ti alloy wire or ribbon arrays unidirectionally or
vertically aligned, with the surface of wires or ribbons anodized
to form TiO2 nanotubes; in this exemplary embodiment the
chondrocyte or cartilage growth is guided somewhat vertically along
the Ti wire or ribbon direction, as described in detail, below.
[0141] FIG. 27(a) and FIG. 27(b) illustrate embodiments comprising
exemplary compositions of the invention comprising compliant,
springy or bent Ti or Ti alloy wires, mesh screens or ribbon
arrays, as described in detail, below.
[0142] FIG. 28(a), FIG. 28(b) and FIG. 28(c) illustrate exemplary
compositions of the invention comprising macroscopically enhanced
Ti surfaces, as described in detail, below.
[0143] FIG. 29(a) and FIG. 29(b) illustrate embodiments of the
invention comprising exemplary compositions comprising nanotubes
and/or nanopores on non-Ti surfaces such as ceramics, polymers,
plastics and other non-Ti metals (e.g., Si, Au, Pt, Al) deposited
or made by e.g., anodization, as described in detail, below.
[0144] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0145] The invention provides products of manufacture, e.g.,
biomaterials and implants, for cartilage formation in-vivo,
in-vitro, and ex-vivo, using nanotechnology, e.g., using nanotube
or nanopillar configured surface structures.
[0146] In one embodiment, the invention provides products of
manufacture comprising a dually functional substrate that supports
the growth and attachment of cartilage tissue on one extremity and
encourages osseointegration--a direct structural and functional
connection to living bone--on the other. In one embodiment, the
invention provides products of manufacture that provide an
engineered interface between artificial cartilage and native
bone.
[0147] In alternative embodiments, the compositions of the
invention comprise cartilage-inducing substrate materials with the
novel surface configurations of nanotubes and nanopillars of this
invention. In one aspect, nanostructure, e.g., nanotubes and
nanopillars, of this invention comprise Ti and Ti oxide (e.g.,
TiO.sub.2) as well as alloys containing Ti or Ti oxide (e.g.,
TiO.sub.2), e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80% or 90% or more weight %. In alternative embodiments, Ti
oxide alloys and TiO.sub.2 alloys used to practice this invention
are oxides of an alloy comprising Ti and other metal(s), e.g., the
oxide of the well known implant alloy like Ti-6% Al-4% V. For
example, a Ti oxide alloy and/or an TiO.sub.2 alloy used to
practice this invention either have only (e.g., consist essentially
of) Ti as a metal or have other (e.g., comprise) metal or metals,
or another material, e.g., a ceramic or a carbon-based
material.
[0148] In alternative embodiments, other related materials are
used, e.g., such as Zr, Hf, Nb, Ta, Mo, W, and their oxides, or
alloys of these metals and oxides, e.g., by at least 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80% or 90% or more weight %. Other
materials such as stainless steels, Si, Si oxide, carbon, diamond,
noble metals (such as Au, Ag, Pt and their alloys), polymer or
plastic materials, or composite metals, ceramics or polymers can
also be utilized to produce and use similar desired surface
configurations for bio implant and cell growth applications;
alternative embodiments have a coating of nano-structured Ti and Ti
oxide, Zr, Hf, Nb, Ta, Mo, W and/or their oxides, or their alloys,
with a thickness of at least about 1, 2, 3, 4, 5, 10, 15, 20, 30,
40, 50, 60, 70, 80, 90 or 100 nm, and/or have a coating coverage of
at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more
of the total surfaces.
[0149] The invention provides materials, fabrication methods, and
therapeutic applications of cartilage-inducing biomaterials
substrate based on nanostructured surfaces, in particular, with Ti
oxide based nanotube or nanopillar configurations. In alternative
embodiments, the novel biomaterials are fabricated by anodization
or nanomasked etching techniques to enable accelerated chondrocyte
cell growth and cartilage formation, and to allow release of growth
factors and other chemical or biological materials stored in the
nano-depot of the nanostructured biomaterial surfaces. Other
materials such as Ti alloy based oxides or containing Zr, Hf, Nb,
Ta, Mo, W based oxides, or stainless steel based alloys are also
utilized.
[0150] The chondrocyte growth enhancing nanotube or nanopillar
configuration materials can also be in the form of thin coating of
other metals or alloys, at least about 1, 2, 3, 4, 5, 10, 15, 20,
30, 40, 50, 60, 70, 80, 90 or 100 or more nm thick surface
portions, which can be converted into a vertically aligned and
adhering nanotube or nanopillar array structures.
[0151] The novel inventive cartilage-inducing biomaterials can be
utilized for repair of articular cartilage of knee or finger bones,
vertebral disks, and other cartilages, in the form of bone implant
surface coatings to induce osseo-integration to existing bone on
the contact side while inducing enhanced chondrocyte culture and
cartilage formation on the exposed implant surface.
[0152] The inventive cartilage-inducing biomaterials can also be
utilized as in vitro or ex vivo cell culture substrate for enhanced
chondrocyte and extracellular matrix, followed by implanting into
human or animal body.
[0153] Nano-depot configurations of the inventive biomaterials can
also be utilized as a reservoir to store and slowly and
continuously deliver growth factors, antibiotics, and other drugs
and biochemicals for further therapeutic benefits for patients.
[0154] The invention provides improved biomaterials implants and
substrates for enhanced cartilage formation, and novel techniques
for fabricating such novel biomaterials, and various biological and
therapeutic applications using such materials are disclosed.
Referring to the drawings, FIGS. 1(a)-(c) schematically illustrate
exemplary devices comprising self-organized TiO.sub.2 based
nanotube arrays grown on titanium metal or alloy substrate to
accelerate chondrocyte cell proliferation according to the
invention. In alternative embodiments, TiO.sub.2 nanotubes or any
other biocompatible nanotubes used in devices of the invention have
dimensions of anywhere between about 10 to 1000 nm in diameter, or
30 to 300 nm, or 60 to 200 nm in diameter. In alternative
embodiments, heights of the tubules are determined in part by the
desired aspect ratio as relatively short height with an aspect
ratio of less than 10, or less than 5, for reduced tendency for
ease of storing and eventual dispensing of drugs or biological
agents intentionally placed within the tubule cavity, as well as to
reduce a possibility of long tubules in thick nanotube layers
delaminating or breaking off and floating around in the human body.
In alternative embodiments, heights can be between about 40 to 2000
nm, or 100 to 600 nm.
[0155] For some embodiments, a vertical alignment with an open top
pore is crucial for bio implant and related applications; for
example, FIG. 1(a) illustrates an exemplary nanotube construction
of the invention having an open top, arrows illustrate and
emphasize the spacing between nanotubes for fluid flow, and the
parallel aligned three-dimensionally configured nanotube array,
which in some embodiments comprise TiO.sub.2 nanotubes; FIG. 1(b)
illustrates how this exemplary nanotube construction of the
invention allows the penetration of the cells into a nanopore
cavity for good adhesion, as illustrated in FIG. 1(c), where the
arrows emphasize how cells, e.g., chondrocyte cells, can adhere and
grow on the surface of these exemplary "open-topped" nanotubes, and
how in some embodiment the surface of the array is coated with an
extracellular matrix composition, e.g., collagen, proteoglycans or
any mixture thereof.
[0156] In alternative embodiments, the desirable diameter range for
nanotubes used in products of manufacture of this invention can be
for the purpose of optimal cell adhesion and growth, while in
alternative embodiments a desired height range can be for the
purpose of minimizing the accumulated stress and delamination often
associated with thick layer of TiO.sub.2 or related nanotubes.
Delamination can be a serious problem when nanotubes, e.g., when
TiO.sub.2 nanotube or ZrO.sub.2 nanotubes, are more than a few
micrometer thick; thus, in alternative embodiments, thinner layer
nanotubes (e.g., TiO.sub.2 nanotube or ZrO.sub.2 nanotubes) are
used to practice this invention.
[0157] In alternative embodiments, nanotubes used to practice this
invention have a diameter dimension in the range of between about
30 to 600 nm outside diameter, or between about 50 to 400 nm
diameter, or between about 70 to 200 nm diameter, and/or can have a
height dimension in the range of between about 30 to 10,000 nm,
and/or can have a thickness of between about 200 to 2,000 nm
thickness, or between about 200 to 500 nm thickness. In alternative
embodiments, longer and/or thicker nanotubes are used to practice
this invention; however, in some embodiments, to give more room to
store biological or other agents in a "nanodepot" (depending on the
intended use of the product of manufacture of the invention), thick
layer nanotubes, e.g., for implants, are less desirable.
[0158] In some embodiments, structures of the invention allow cells
to adhere well to a surface to stay healthy and grow fast (e.g., by
coating with an extracellular matrix composition, e.g., collagen,
proteoglycans or any mixture thereof); the cells that may not
adhere exhibit reduced or minimal growth. In the exemplary vertical
nanotube structures of this invention, an examples of which are
illustrated in FIG. 2 and FIG. 3, such an exemplary configuration
is illustrated. This exemplary design of the invention allows
desired accelerated chondrocyte growth and extracellular matrix
formation. FIG. 2 and FIG. 3 illustrate scanning electron
micrograph images (SEM micrographs) of self-aligned TiO.sub.2
nanotubes with different diameters, the scale bars are 200 nm.
[0159] In alternative embodiments, titanium nanotubes are formed by
electrolytic anodization, for example using 5% hydrofluoric acid
and applying .about.10-20 volts of potential, and allowing several
minutes to a few hours depending on the temperature and other
electrochemical process parameters. The resultant TiO.sub.2
nanotube diameter is dependent on the anodization voltage.
TiO.sub.2 nanotubes can be prepared by various anodization
processes: see e.g., Gong (2001) J. of Materials Res.
16(12):3331-3334; J. M. Macak (2005) Angew. Chem. Int. Ed.,
44:7463-7465; Electrochimica Acta 50 (2005) 3679-3684 (2005) Angew.
Chem. Int. Ed., Vol. 44, 2100-2102 (2005); Ghicov (2005)
Electrochemistry Communications 7:505-509; Oh (2005) Biomaterials,
Vol. 26, page 4938-4943; Oh (2006) Journal of Biomedical Materials
Research, Vol. 78A, page 97-103; Oh (2009) Stem cell fate dictated
solely by altered nanotube dimension, Proc. Natl. Acad. Sci.
106(7):2130-2135.
[0160] In alternative embodiments, titanium oxide nanotubes for
biological applications of this invention significantly enhance
bone growth; exemplary biological applications are described e.g.,
in the Oh et al. articles cited above.
[0161] In alternative embodiments, the structure of the anodized
TiO.sub.2 nanotube array, such as the diameter, spacing and height
of nanotubes, is controllable during the electrochemical
anodization process.
[0162] In alternative embodiments, the concentration of
electrolytes is chosen, e.g., as described in articles by Gong, et
al., Oh, et al, Macak, et al., and Ghicov, et al. cited above. Some
exemplary electrolytes and their concentrations are; 0.5 wt %
hydrofluoric acid (HF) in water, 0.5 wt. % ammonium fluoride
(NH.sub.4F) in 1 M ammonium sulphate ((NH.sub.4).sub.2SO.sub.4),
and 1 wt. % NaF in 1M Na.sub.2SO.sub.4 solution. In alternative
embodiments, various anodization processing parameters such as the
applied voltage, reaction time, the pH and the temperature of the
bath, etc. are controlled and optimized as well.
[0163] In alternative embodiments, the base material can be pure Ti
or can be an alloy based on Ti such as Ti--Al--V alloys or other
solid solution hardened or precipitation hardened alloys with
increased mechanical strength and durability. While accelerated
chondrocyte growth and extracellular matrix formation illustrated
herein are mostly using exemplary embodiments comprising substrate
material of Ti and Ti oxide, in alternative embodiments, alloys
used to make the products of manufacture of this invention can
comprise other elements. Products of manufacture of this invention
also can have Ti or Ti oxide by at least about 5%, 10%, 15%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90% or more weight %. The use of
other transition or refractory metals such as Zr, Hf, Nb, Ta, Mo,
W, and their oxides, or alloys of these metals and oxides also can
be used. Other materials such as stainless steels, Si, Si oxide,
carbon, diamond, noble metals (such as Au, Ag, Pt and their
alloys), polymer or plastic materials, or composite metals,
ceramics or polymers, engineered into specific nanotube or nanopore
array structure can also be utilized for products of manufacture,
e.g., bio implants of this invention, and accelerated cell growth
applications; and in alternative embodiments using a coating of Ti
and Ti oxide, Zr, Hf, Nb, Ta, Mo, W and their oxides, and/or their
alloys, with a thickness of at least about 1, 2, 3, 4, 5, 10, 15,
20, 30, 40, 50, 60, 70, 80, 90 or 100 or more nm and the coating
coverage of at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or more of the total surfaces.
[0164] In alternative embodiments, the chondrocyte growth enhancing
nanotube or nanopillar configuration materials are in the form of
thin coating of metals or alloys, and at least about 1, 2, 3, 4, 5,
10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more nm thick
surface portion of which is converted into a vertically aligned and
adhering nanotube or nanopillar array structure.
[0165] In alternative embodiments, for chondrocyte cell growth and
formation of extracellular matrix, compositions of the invention
are designed to allow a continuous supply of nutrients including
proteins, mineral ions, fluid, etc. is supplied to the cell through
the flow of body fluids. For example, the gap (spacing) between
adjacent TiO.sub.2 nanotubules in the exemplary compositions of the
invention as illustrated in FIGS. 1 to 3, serves the function of
allowing the body fluid to continuously pass through and supply
nutrients to a surface or side of a product of manufacture facing
or adjacent to growing cells (e.g., supply nutrients to an opposing
or "bottom" side of a product of manufacture facing growing cells).
Exemplary gaps between the nanotubules are in the range of about
between about 2 to 100 nm, or between about 5 to 30 nm.
Transmission electron microscope (TEM) photographs shown for an
exemplary TiO.sub.2 nanotubule array structure of the invention is
illustrated in FIGS. 3(b) and (c), to give an average of
approximately 15 nm spacing between the nanotubes.
[0166] Nanotube and nanopillar array configurations of the
invention can allow a continuous supply of cell grow nutrients,
e.g., an exemplary nanopillar array configuration of the invention
is illustrated in FIG. 4(a) and FIG. 4(b) can continuously supply
of cell grow nutrients, and its nano topography structure and the
gap between the nanopillars allows strong cell adhesion. The arrows
in FIG. 4(a) emphasize the vertically aligned pillar array of
TiO.sub.2 "nanopillars", and that the spacing between the
nanopillars allow for fluid flow. The arrows in FIG. 4(b) emphasize
how cell, e.g., chondrocyte or stem cells, can adhere and grow in
the nanopillars, and how an extracellular matrix (comprising e.g.,
collagen, proteoglycans, and the like) can be constructed as a
layer on the nanopillar surface. This exemplary nanopillar
structure, which cannot be produced by anodization technique
utilized for fabricating the structures of FIGS. 1 to 3, can be
formed on the surface of Ti, Zr, Hf, Nb, Ta, Mo, W, and/or their
alloys, and/or a thin coating of these metals and alloys, e.g., by
patterned masking and etching, or a combination of initial
patterned etching and subsequent anodization.
[0167] One exemplary way of fabricating a chondrocyte-enhancing
nanopillar structure of this invention is to utilize nano-imprint
technology; e.g., as illustrated in FIG. 5: nano stamping of
polymer mask resist such as PMMA (polymethyl-methacrylate) layer
can be carried out on the desired surface, in this exemplary
embodiment, as a Ti or related metal and alloy surface. The PMMA
can be first spin-coated into a thin layer, e.g., 20 to 200 nm
thick layer, then the nanostamp can be pressed onto this uncured
PMMA layer to pick up the resist islands, which is then transferred
and imprinted onto Ti or alloy surface to leave islands of PMMA
mask, as illustrated in FIG. 5(a). The Ti or alloy base is then
chemically etched or reactive ion etched (RIE) to form the desired
TiO.sub.2 nanopillar structure of FIG. 5(b). The pillars can be
wholly TiO.sub.2 or only the surface of the pillars can be
converted to TiO.sub.2 by oxidation heat treatment of anodization
treatment. Alternatively, the nanostamps can be made of patterned
Si, metal or elastomer (PDMS), with the mechanically compliant
elastomeric stamp allowing more reliable transfer of the masking
resist islands. Because of directional nature of the nanopillars,
cells, e.g., chondrocyte or stem cells, can adhere to the side of
the nanopillars and form a vertically aligned guided cartilage
column, FIG. 5(c). Such a guided vertical growth of cartilage
approximates the important natural tendency of initial stage of
human cartilage formation having a vertical geometrical
arrangement. The initial vertical alignment (called a Deep Zone) is
followed by more random Middle Zone, then by more horizontal
Superficial Zone structure. Such a zonal cartilage formation can be
made as described by Kim T K, et al. Experimental model for
cartilage tissue engineering to generate the zonal organization of
articular cartilage. Osteoarthritis Cartilage 2003; 11(9):653-664;
Sharma B, et al. Designing zonal organization into
tissue-engineered cartilage. Tissue Eng 2007; 13(2):405-414;
Woodfield T B, eta al. Design of porous scaffolds for cartilage
tissue engineering using a three-dimensional fiber-deposition
technique. Biomaterials 2004; 25(18):4149-4161.
[0168] An alternative processing route to utilize nano-imprinting
technology for formation of nanopillar or micropillar arrays as
illustrated in FIG. 5(b) on Ti and related implant metals and
alloys is to stamp a height-varying nanopattern of pillar or pore
geometry into a thermoplastic or UV-curable polymer resist layer,
with optional metal mask layer deposition and lift-off process,
followed by vertical reactive ion etch to enable a pattern transfer
to the underlying substrate.
[0169] Yet another exemplary technique of forming a location-guided
and diameter-guided uniform nanopillar array of the invention,
which in some embodiments is advantageous for fabricating exemplary
nanopillar structures on non-flat surface of Ti or related metals
and alloys, is to introduce guided etching using a vertically
two-phase decomposable coating as illustrated in FIG. 6(a) to (f).
In this exemplary technique: first, Ti implant or substrate for
chondrocyte culture and cartilage growth is coated with a material
which is then decomposed into a vertically aligned two-phase
structure. An example of such a decomposable material is a diblock
copolymer layer which, on heating, can decompose into vertically
aligned two phases. See e.g., M. Park et al., "Block copolymer
lithography: Periodic arrays of 10.sup.11 holes in 1 square
centimeter", Science, Vol. 272, page 1401 (1997).
[0170] Another exemplary composition of the invention comprises
decomposable material leading to a vertically aligned two-phase
structure, such as a spinodally decomposing alloy, e.g., as
described by N. Yasui et al, "Perpendicular recording media using
phase-separated AlSi films", Journal of Applied Physics, Vol. 97,
page 10N103 (2005). Either during the thin film deposition with
self-heating during the RF plasma sputter deposition process or
with post-deposition annealing .about.100-700.degree. C., a
desirable vertically aligned nano pore structure or nano island
structure can be obtained from a spinodal alloys in general. In the
case of Al--Si alloy films, with proper chemical etching, Al can be
selectively etched while Si oxidizes into SiO.sub.2 porous membrane
or SiO.sub.2 island array, thus creating a nanopore or nanopillar
structure depending on the relative volume fraction of the two
phases.
[0171] Yet another exemplary composition of the invention comprises
decomposable material leading to a vertically aligned two-phase
structure, is the employment of anodized aluminum oxide (AAO)
structure, for example, described by A. I. Gapin et al, J. Appl.
Phys. 99, 08G902 (2006). The nanopore arrays can be used as a mask
to chemically etch the substrate rods or wires (such as made of Ti,
Zr, Hf, Nb, Ta, Mo, W metals and related alloys) to form
nanopillars or nanopores.
[0172] In one embodiment, after an exemplary decomposable coating
is added and made to decompose into aligned two phase structure, as
illustrated in FIG. 6(b), one of the phases is removed from the two
phase structure via differential etching, e.g., by chemical etching
or ion etching to exhibit a nano island array FIG. 6(c). FIG. 6(c)
illustrates a nano-island coating left after preferential etching
away of one of the two phases. The coating of textured material can
be a co-sputtered layer, a decomposable diblock co-polymer, a
spinodally decomposing alloy, and the like. Etching of Ti or alloy
base through the masking islands can produce the exemplary
nanopillar array of FIG. 6(d). After the coating material is
removed, as illustrated in FIG. 6(e), optional additional etching
or guided anodization process may be utilized to further increase
the depth of the nanopillars, as illustrated in FIG. 6(f). FIG.
6(f) illustrates the optional additional etching or anodization to
produce a "deeper" or higher nanopillar or nanotube, which can act
as an implant surface.
[0173] In one embodiment, products of manufacture of the invention
comprise diblock copolymers, which can comprise two chemically
different polymer chains or blocks while they are joined by a
covalent bond. Because of this connectivity constraint yet chemical
incompatibility with each other, the diblock copolymers tend to
phase separate and self assemble into an ordered (often with a
hexagonal geometry), nanoscale, mixed-phase composites. Depending
on the chemistry and decomposition conditions, they can form an
ordered array with one of the polymer components taking a
nano-cylinder shape embedded in the other polymer component.
Examples of diblock copolymers used in products of manufacture of
the invention include a mixture of polystyrene-polybutadiene and
that of polystyrene-polyisoprene. The diblock copolymers can be
diluted with a solvent such as toluene, and can be dip coated,
brush coated or spray coated on a substrate. When the heat is
applied and drying proceeds and the copolymer concentration and
temperature reaches a critical point, the phase decomposition of
the diblock copolymer into an ordered structure takes place. The
desired temperature rise to nucleate and grow the ordered
decomposed diblock copolymer structure can be between about range
of between about 50.degree. C. to 350.degree. C., or between about
100.degree. C. to 250.degree. C.
[0174] The spinodal alloys can be spontaneously decomposed into a
uniform two phase structure by heating to a high temperature within
the spinodal phase stability range. Fe--Cr--Co, Al--Ni--Co--Fe,
Cu--Ni--Fe, Cu--Ni--Co, and Al--Si alloys are well known examples
of spinodally decomposing alloys. Due to the difference in chemical
etchability between the two decomposed phases, a nano-island mask
structure, e.g., as illustrated in FIG. 6(c) can be obtained over a
large area.
[0175] Another embodiment of the present invention uses nano-depot
spaces within nanotubes or in the space between nano-pillars; these
structures can be utilized to conveniently store drugs and/or
biological agents desirable for enhanced culture of chondrocytes,
like a growth factor, other biomolecules, antibiotics, etc. which
can be slowly released from the nano-depot, which can be a
TiO.sub.2 nanotube surface layer. In alternative embodiments, the
nanoscale space of the TiO.sub.2 nanotubes or spacing between the
nano-pillars, as compared to microsized pores, has an advantage of
being able to keep the stored drugs and/or biological agents much
longer and allow slower release over a longer period of time.
Controlled slow release of drugs and/or biological agents such as
growth factors, antibiotics, such as penicillin, streptomycin,
vancomycin, hormones and the like, can be incorporated; e.g.,
antibiotics can prevent infections near the chondrocyte-related
implant. Growth factors and/or drugs, etc. stored and slowly
released from the nano-depot space can also enhance cell growth
and/or differentiation, e.g., enhance chondrocyte, stem cell,
totipotent cell, multipotent progenitor cell and/or pluripotent
cell formation and/or differentiation over extended period of
time.
[0176] In alternative embodiments of the invention, the drugs
and/or biological agents that are stored in nano-depot spaces
(e.g., within nanotubes or in the space between nano-pillars)
include growth factors, collagens, various proteins/biomolecules,
genes, DNAs, antibiotics, hormones, therapeutical drugs, and/or
functional particles like magnetic, metallic, ceramic, polymer
particles. In alternative embodiments, biological agents can
comprise a fibroblast growth factor (FGF), an epidermal growth
factor (EGF), a vascular endothelial growth factor (VEGF), a
transforming growth factor beta-1 (TGF-.beta.1) or a transforming
growth factor beta-2 (TGF-.beta.2), a bone morphogenic protein
(BMP) (e.g., an agent that stimulates chondrocyte growth,
maintenance and/or differentiation), fibroblast growth factors
and/or vascular endothelial growth factors. In alternative
embodiments, a biological agent is or comprises an isolated
protein, an autologous protein, and/or a recombinantly generated
polypeptide.
[0177] The functional particles can be made of magnetic oxide
particles or metallic particles, and can be utilized for remotely
actuated RF heating and creation of temperature gradient for
accelerated or switch-on, switch-off release of the drugs and/or
biological agents stored in the nano-depots, e.g., within nanotubes
or in the space between nano-pillars.
[0178] Referring to the drawings, FIG. 13 schematically illustrates
an alternative embodiment of the invention comprising TiO.sub.2
nanotube based implants comprising nanotubes with slow-releasing
drugs and/or biological agents stored in the vertically aligned
nanotube pores: FIG. 13 (a) As-made TiO.sub.2 nanotubes, FIG. 13
(b) Drugs and/or Biological agents stored in the nano-depots, FIG.
13 (c) Diameter reduced nano-depot entrance for slower release of
stored drugs and/or biological agents.
[0179] For the purpose of storing more drugs and biological agents
in the nanodepot, the TiO.sub.2 nanotubes can be made taller, e.g.,
nanotubes between about 1 to 50 micrometers tall, or alternatively
nanotubes between about 1 to 10 micrometers tall. However, taller
or thicker nanotube layer tends to introduce accumulated stresses
and make the layer susceptible to delaminations. From this point of
view, shorter nanotubes between about 200 to 500 nm tall, or about
20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225 or 250
or more nm in height may be preferred, depending on its use and/or
method of manufacture. The balance between the desire to store more
biological agents and the need to keep the nanotube height short
may have to be carefully weighed depending on each specific
application. An alternative embodiment comprises taking a duplex
depth structures as illustrated in FIG. 23, in which a microcavity
is utilized to store a larger amount of biological agents, stem
cells, drugs, and chemicals while the nanotube height is maintained
short on the implant surface and associated spingy wire
surface.
[0180] An alternative embodiment comprises slowing down the release
of the stored drugs and/or biological agents by making the nanotube
entrance narrower by intentional sputtering or evaporation
deposition of TiO.sub.2 or Ti metal (to be oxidized later by either
anodization or by low temperature heat treatment in air or
oxygen-containing atmosphere). This is schematically illustrated in
FIG. 13(c). While regular vertical incident deposition also tends
to form such bottle necks, an optional oblique incident deposition
with rotating substrate during deposition makes it easier to form
the intentional bottle neck configuration.
[0181] In an alternative embodiment, diameter reduction is by
bottleneck addition, such as by sputter deposition of Ti,
TiO.sub.2, or other metals, alloys, oxides or polymers, can be
conducted to a diameter of less than 20 nm, or alternatively less
than 10 nm near the entrance to the pores to minimize unwanted
release of liquid agents such as dissolved antibiotics, small
molecule chemical growth factors, DNAs and genes. For larger
molecule biological agents such as proteins and polymers, the
bottle neck diameter has to be adjusted accordingly, e.g.,
approximately 40 nm diameter bottleneck to allow the release of
some of these larger diameter molecules from the nanotube nanodepot
space.
[0182] In alternative embodiments, a TiO.sub.2 nanotube structure
having the appropriate diameter may be essential for enhanced cell
growth, e.g., at least 50 nm diameter, for osteoblast and
chondrocyte cells. This objective may conflict with the exemplary
nanodepot embodiment comprising intentionally introducing the
bottleneck configuration at the top entrance to the nanotube pores
and the in-between gap regions so that the biological agents, e.g.,
liquid-based chemicals and/or drugs, do not get released too
rapidly. In order to resolve these conflicting requirements,
alternative embodiments comprise a duplex distribution of the
nanostructure dimensions such that the nano-depot regions having
intentionally bottle-necked pore structure are mixed and
distributed together with regular nanotube regions which do not
have the bottleneck diameter reduction. This exemplary embodiment
is schematically illustrated in FIG. 13(d). The former will serve
as a storage and slow release of biological agents while the latter
will serve to enhance cell adhesion and growth.
[0183] The average area of each of the distributed nano-depot
regions for drug- or biological agent release can be adjusted as
needed, for example in the range of 1 .mu.m-1,000 .mu.m. Each of
these regions contains many nanodepot reservoirs, and can have
circular, rectangular, or irregular shape. The distribution of
these regions on the implant surface can be periodic or random. In
alternative embodiments, the relative area fraction of the drug
release regions can be in the range of between about 2% to 50%, or
between about 10% to 30%, of the total available surface area for
cartilage or bone growth depending on the specific needs.
[0184] A similar nano-depot storage and slow release of the drugs
and/or biological agents as in the case nanotube array structure of
FIG. 13 can also be accomplished with the nano-pillar array
structure, according to the invention. This is schematically
illustrated in FIG. 14. With the intentionally induced bottleneck
configuration, the slow rate release is achieved.
[0185] In one embodiment of the invention, the methods of the
invention utilize vertically aligned TiO.sub.2 or Ti metal and/or
related materials in nanotube or nano-pillar array configurations;
and in one aspect incorporating nano-depot based reservoirs for the
slow release of drugs and/or biological agents. These
configurations can substantially enhance the kinetics and quantity
of chondrocyte functionality and extracellular matrix formation, as
well as cartilage growth rate, by e.g., at least about 1%, 2%, 3%,
4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more, as
compared with the same bio implant material or bio substrate
material without these nanotube or nanopillar structures of this
invention.
[0186] In another embodiment of the invention, the products of
manufacture and methods of the invention incorporate bonded macro
or microscale scaffold structures comprising springy wires or mesh
wires, for example as illustrated in FIGS. 17 to 23. The surfaces
of these scaffold wires are also nanopatterned as nanotube or
nanopore structures to enhance chondrocyte adhesion and growth. In
alternative embodiments, "springy" and "compliant" are terms that
are quantitatively defined, e.g., in some embodiments as "a
coil-like or curved or bent metal wire array or forest in the
metallic state that can be elastically compressed (e.g., before
implanting), e.g., that can be elastically compressed by at least
about 5%, 10%, 15%, 20% or 25% or more reduction in height, and in
some embodiments upon releasing the applied load the original
geometry is restored without noticeable permanent plastic
deformation, e.g., less that about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2% or 1%, or less permanent compression left.
[0187] Yet in another embodiment of the invention, the products of
manufacture and methods of the invention utilize patterning and
etching of the implant surface to introduce vertically aligned
macro or micro pillars of Ti and related metals and alloys such as
Ti--Al--V and Zr, Hf, Nb, Ta, Mo, W. An example structure is
illustrated in FIG. 26. These metal pillars, according to the
invention, are processed in such a way that the surfaces of each of
these metal pillars are also nanopatterned to provide nanotube or
nanopore structures for enhanced chondrocyte adhesion and growth.
These vertical pillar metal scaffolds with chondrocyte adherent
surfaces support a three-dimensional, vertical-direction-guided
growth of chondrocytes and cartilages approximating the initial
stage of natural cartilage growth in human.
[0188] Products of manufacture of this invention can be useful for
a variety therapeutic applications for human and animals, including
use for enhanced cartilage growth, initiation of cartilage growth
and/or cartilage repair. The compositions and methods or the
invention provide supportive scaffolding for new cartilage growth,
enhanced cartilage growth, initiation of cartilage growth and/or
cartilage repair at any moveable, cartilaginous, and synovial joint
site including but not limited to: Intervertebral discs (or
intervertebral fibrocartilage), bronchial tubes, Thumb and fingers
(between the metacarpal and carpals); Wrist; Elbow (between the
humerus and the ulna and between the radius and the ulna);
Shoulder; Hip; Knee; Ankles; Feet and toes (between tarsals and
metatarsals); Intervertebral discs of the spinal cord; Rib cage
and/or ears or noses, e.g., for reconstructive or plastic surgery
purposes. The compositions and methods of the invention provide
supportive scaffolding for new cartilage growth, enhanced cartilage
growth, initiation of cartilage growth and/or cartilage repair for
any cartilaginous tissue, including elastic cartilage, hyaline
cartilage and fibrocartilage. Products of manufacture of this
invention can be useful new cartilage growth, enhanced cartilage
growth, initiation of cartilage growth and/or cartilage repair for
endochondral ossification, osteoid and/or periosteum formation and
calcification, as some forms of bone formation require a
pre-existing cartilage structure.
[0189] The compositions and methods of the invention enable joint
movement while providing the structural support and chemical
environment for new or repaired cartilage tissue to grow, e.g., to
fill defects or injuries, e.g., to replace damaged, infected, aged,
or diseased cartilage caused by various diseases such as:
Arthritis; Osteoarthritis (e.g., due to sports injuries, extreme
trauma, impact injury, or repeated micro trauma); Isolated
femoropatellar osteoarthritis (e.g., kneecap osteoarthritis);
Rheumatoid arthritis (a chronic, systemic autoimmune disorder that
causes the immune system to attack the joints) lupus or any other
autoimmune disease where the immune system attacks directly or
indirectly the body's cells and tissue resulting in inflammation
and tissue damage, particularly in osteonecrosis of the joint;
Septic arthritis caused by joint infection; a previously infected
or injured knee or other joint.
[0190] At least two different embodiments of the inventions are
described in FIG. 15 and FIG. 16. One embodiment to repair damaged
cartilage, for example to repair and restore an articular cartilage
defect illustrated in FIG. 15(a), is to utilizes the chondrocyte
enhancing characteristics of the TiO.sub.2 nanotubes or nanopillars
and prepare in-vitro culture of patient's own chondrocytes, FIG.
15(b), which is then injection transplanted into the cartilage
defect regions in human or animal body, FIG. 15(c).
[0191] Another embodiment is to utilize TiO.sub.2 nanotube or
nanopillar material as a surface coating implant material on a
patch bone implant Ti piece to be inserted into the area of
cartilage defect, as illustrated in FIG. 16.
[0192] An exemplary process steps are as follows. [0193] i) The
defective or injured area of cartilage, see an example shown in
FIG. 15(a), is first cleaned by removing the damaged tissues.
[0194] ii) An appropriately shaped Ti implant piece is fabricated
by machining, casting, stamping, or other means so that a desirable
shape resembling the removed tissue and bone area as illustrated in
FIG. 16. The Ti patch implant material is processed (e.g., as
described herein) by e.g. anodization or nano-mask pattered
etching, or combination of these two process approaches, to possess
the TiO.sub.2 nanotube or nanopillar structure on both top and
bottom (or opposing) surfaces (more generally speaking, on all
outside surfaces of the Ti implants). As illustrated in FIG. 16,
the top surface of the TiO.sub.2 nanotubes of the top surface of
the implant act to enhance cell, e.g., stem cell or chondrocyte,
growth and subsequent cartilage formation; the bottom (or opposing)
surface of the implant, also having TiO.sub.2 nanotubes,
facilitates osseointegration of the implant with existing bone.
[0195] iii) The patch implant Ti (or a total joint replacement Ti
if needed) is then placed on the existing bone and fixed in
position by using one or more Ti or stainless screws, which can be
left permanently even after osseointegration of the Ti implant with
the existing bone, FIG. 16. Alternatively, a temporary fixture
consisting of removable or biodegradable strings or straps can be
used until the natural osseointegration at the bottom (or opposing)
side is essentially completed.
[0196] In alternative embodiments, the cartilage growth structures
are not limited to planar material configurations, but also
comprise additional variations and embodiments, including
comprising a TiO.sub.2 nanotube surface on a compliant 3-D
substrate, e.g., as illustrated in FIGS. 16 to 29. Other exemplary
scaffold structures, such as vertically aligned Ti metal pillar
arrays or nanoparticle arrays, used to practice this invention are
also described in these figures.
[0197] In one aspect, an advantage of nanotube or nanopillar
configurations of this invention is that a strong osseo-integration
of the implant with the existing bone occurs at the bottom (or
opposing) surface (at the interface between the implant and the
bone facing it), while a much enhanced chondrocyte growth and
cartilage formation occurs at the top surface of the implant.
[0198] Alternative embodiments of the invention comprise (e.g.,
incorporate) stem cells, e.g., a mesenchymal stem cell, an adult
stem cell, an induced pluripotent stem cell (abbreviated as iPS
cell or iPSC) and/or an embryonic stem cell, a human mesenchymal
stem cell or human embryonic stem cell, or an artificially created
stem cells through gene modification of somatic cells. In one
embodiment, when stem cells such as human mesenchymal stem cells
(hMSCs) are cultured without chondrogenic inducing media, the
TiO.sub.2 type nanotube, nanowire and/or nanopore and related
nanostructures, according to the invention, upregulate
differentiation into chondrocytes over cultures by nanotopography
alone. While the invention is not limited by any particular
mechanism of action, such a behavior is related to the recently
discovered phenomenon on the effect of nanotubes causing enhanced
and preferential hMSC differentiation to osteoblast cells by
nanotopography structure alone even in the absence of
differentiation-inducing agents; as described e.g., by Oh (2009)
Stem cell fate dictated solely by altered nanotube dimension, Proc.
Natl. Acad. Sci. USA 106(7):2130-2135; also cited above.
[0199] In one embodiment, when stem cells such as human mesenchymal
stem cells (hMSCs) are cultured with chondrogenic inducing media,
the TiO.sub.2 type nanotube, nanopore and related nanostructures,
according to the invention, upregulate differentiation into
chondrocytes over cultures with the aid of the nanotopography of
the products of manufacture of this invention. Exemplary
chondrogenic inducing media comprise a chemically defined medium
comprising, for example, serum-free DMEM, ascorbate, dexamethasone,
L-proline, sodium pyruvate, ITS-plus, antibiotics, and recombinant
human transforming growth factor-.beta.1 (TGF-.beta.1).
[0200] Stem cells cultured in the chondrogenic media on nanotube,
nanowire and/or nanopillar-comprising products of manufacture of
this invention proliferate and differentiate into the chondrogenic
lineage as the stem cell differentiation; which in some embodiments
is influenced by both the cell-substrate interactions from the
topographical cues of the surface in addition to the chemical cues
of the inducing media. In some embodiments, nanotopography of
nanotube, nanowire and/or nanopillar surfaces of nanotube, nanowire
and/or nanopillar-comprising products of manufacture of this
invention thus play an essential role in mimicking the cell and
extracellular matrix (ECM) organization that is found, for example,
in the natural cartilage zone, that would play a role in directing
MSC differentiation into chondrocytes. By combining synthetic
TiO.sub.2 nanostructures having topographical cues combined with
the biochemical cues (e.g., TGF-.beta.1 and/or BMPs), the products
and methods of the invention further enhance chondrocyte growth
and/or differentiation to chondrocytes from progenitor cells (e.g.,
stem cells), and in alternative embodiments the products and
methods of the invention enhance the upregulation of chondrogenic
maker expressions (genes, proteins, ECM, etc.) in colony-forming
unit-fibroblast (CFU-F), marrow stromal cell or mesenchymal stem
cell (MSC), stem cell, totipotent cell, multipotent progenitor cell
and/or a pluripotent cell cultures.
[0201] In alternative embodiments, the nanotube, nanopillar and/or
nanoribbon scaffolds (comprising e.g., a metal or a metal oxide
such as Ti or TiO.sub.2), and/or microcavities or macrocavities,
are configured so as to store and release chemicals, drugs and/or
biological agents, e.g., growth factors, e.g., chondrogenic growth
factors such as FGF, EGF, BMPs and/or TGF-.beta.1 and the like, in
a well-controlled fashion. The chemicals, drugs or biological
agents, e.g., growth factors, can be stored either in nanodepot
cavities of nanotubes or nanopillars, or between nanotube,
nanopillar and/or nanoribbon scaffolds, as illustrated for example
in FIGS. 6, 13, 14, 17, 18, 20, 21, 26, or microcavities or macro
cavities such as shown in FIGS. 23 and 29.
[0202] While the naturally occurring stem cells in human or animal
body contribute somewhat to the growth of bones and cartilages, in
some embodiments this invention comprises use of stem cells and/or
chondrogenic growth factors with products of manufacture of the
invention to further accelerate the cartilage growth. In
alternative embodiments, the stem cells themselves can be supplied
either as a part of the cartilage growth media or can be stored and
supplied from macro/micro cavities of products of manufacture of
the invention, e.g., in the exemplary structures illustrated in
FIGS. 23 and 29. In alternative embodiments, the nanodepot entrance
to the nanopores or nanotubes on Ti implants or on scaffold wires
is geometrically modified to have controlled diameter bottleneck
structure first. In alternative embodiments, the nanodepot storage
space is then loaded with drugs and/or growth factors such as FGF,
EGF, BMPs and/or TGF-.beta.1 and the like, and allowed to follow
precisely planned release rate of the growth factor over desired
period of time, e.g., from 1 day to more than 60 days, or more,
according to the pre-designed pore entrance size of the
nano-depots.
[0203] FIG. 17 illustrates exemplary TiO.sub.2 nanotube or nanopore
arrays on the surfaces of exemplary hairy-shaped, or
mesh-screen-shaped Ti (or other biocompatible wire like stainless
steel) bonded onto the Ti implant. Optionally, biological agents
can be stored in the nanotubes or nanopores for growth factor, drug
delivery, etc. FIG. 17(a) illustrates exemplary protruding (from
the surface) TiO.sub.2 nanotubes (left) and exemplary recessed
(from the surface) TiO.sub.2 nanotubes (right); wherein the
illustrated example protruding structures comprising hairy or mesh
screens Ti or other alloys are bent or angled to minimize tissue
irritation (tissue or cell "poking"); the substrate upon which the
nanotubes are fixed can also be made of Ti alloy or TiO.sub.2. FIG.
17(b) illustrates an in vivo or in vitro environment with cell
growth nutritional media (e.g., human or tissue culture medium),
where cells, e.g., stem cells, chondrocytes, hMSCs or a mixture of
chondrocytes and hMSCs, or any combination of cells; and in
alternative embodiments the media comprises drugs, growth factors
and/or antibiotics; FIG. 17(b) also illustrates how the invention
can promote three-dimensional and geometrically secured cartilage
growth around exemplary embodiments comprising e.g., compliant,
hairy or spring-shaped Ti having nanotubes, e.g., TiO.sub.2
nanotubes, or other nanostructures.
[0204] FIG. 18(a) illustrates an embodiment of the invention
comprising exemplary TiO.sub.2 nanotube or nanopore arrays;
hairy-shaped or mesh-screen shaped surface structures; this
exemplary structure comprises springy and compliant Ti microwires,
e.g., 10 to 100 um in diameter; comprising on the wire surface (as
the illustration highlight notes) TiO.sub.2 nanotubes or nanopores,
for e.g., large surface cell (e.g., stem cell or chondrocyte)
growth and cartilage and/or ECM formation; in one embodiment this
allows for "pseudo-vertical" growth of cartilage where spring wires
are vertically enlongated. FIG. 18(b) schematically illustrates an
embodiment of three-dimensional and geometrically secured cartilage
growth around compliant spring-shaped Ti wires having TiO.sub.2
nanotubes or other nanostructures.
[0205] FIG. 19(a) to (d) illustrate an embodiment of the invention
comprising diffusional bonding of Ti hairy-shaped or Ti mesh-screen
shaped surface structures onto an exemplary composition of the
invention, e.g., a Ti implant; FIG. 19(a) illustrates an exemplary
protruding structure comprising e.g., hairy or mesh screen wires,
e.g., Ti wires on a Ti surface; FIG. 19(b) illustrates an
embodiment comprising anchoring thick film Ti deposits; e.g.,
between about 100 to 2000 .mu.m thick, optionally having an oblique
incidence plus rotating substrate; FIG. 19(c) illustrates an
embodiment comprising diffusion annealed and bonded Ti layer at
e.g. between about 500 to 1000.degree. C. for between about 0.1 to
100 hours; FIG. 19(d) illustrates an embodiment comprising both Ti
wire surfaces and flat Ti surfaces anodized to have a (e.g.,
TiO.sub.2) nanopore or nanotube structure.
[0206] FIG. 20(a) to (d) illustrates an embodiment of the invention
comprising melt-bonding of Ti or stainless steel hairy-shaped or
mesh-screen shaped surface structures onto an exemplary composition
of the invention, e.g., a Ti implant. FIG. 20(a) illustrates an
embodiment comprising heating of nanotubes or nanowires (which here
comprise the protruding "hairy" or mesh screen shaped embodiments
on a Ti base) by e.g. induction heating using radio frequency (RF)
waves, electron-beam ("e-beam") heating, laser heating, torch
heating and/or furnace heating; FIG. 20(b) illustrates the
"melt-bonding" subsequent to the heating described in FIG. 20(a).
FIG. 20(c) illustrates an embodiment comprising protruding
TiO.sub.2 nanotube structures on an exemplary wire surface made
e.g., by anodization; and FIG. 20(d) illustrates an embodiment
comprising recessed TiO.sub.2 nanotube structures on an exemplary
wire surface.
[0207] FIG. 21(a) to (d) illustrate an embodiment of the invention
comprising spot-welding of Ti or stainless steel hairy-shaped or
mesh-screen shaped surface structures onto an exemplary composition
of the invention, e.g., a Ti implant. FIG. 21(a) illustrates an
embodiment comprising spot welding of nanotubes or nanowires onto a
base, e.g., a TiO.sub.2 base, the circled "i" representing current
running between an upper electrode and a lower electrode for
compression spot welding. In alternative embodiments the spot
welding upper electrode is in the shape of a disk, plate, grid,
frame and the like. In alternative embodiments the upper electrode
contact region is e.g., Au, Pt, Pd or an alloy of one or more of
these Au, Pt, Pd or other metals. This illustration also includes
protruding "hairy" or mesh screen Ti wires. FIG. 21(b) illustrates
the spot welded Ti region subsequent to the welding described in
FIG. 21(a). FIG. 21(c) illustrates an embodiment comprising
protruding TiO.sub.2 nanotube structures on an exemplary wire
surface made e.g., by anodization; and FIG. 21(d) illustrates an
embodiment comprising recessed TiO.sub.2 nanotube structures on an
exemplary wire surface.
[0208] FIG. 22(a) and FIG. 22(b) illustrate an embodiment of the
invention comprising exemplary TiO.sub.2 nanotube or nanopore
arrays; a side view of exemplary hairy-shaped or mesh-screen shaped
surface structures are shown. FIG. 22(a) illustrates an embodiment
comprising spot welding, or induction melting-bonding, or
electron-beam ("e-beam") bonding, or laser bonding, or a
braze-bonded Ti wire mesh, e.g., as a single or a multi-layer)
comprising a surface nanopore or nanotube array. In alternative
embodiments the construction (e.g., as an implant) can be as a
flat, round and/or curved surface, e.g., a Ti surface. FIG. 22(b)
illustrates an embodiment comprising three-dimensionally secured
cartilage growth around woven or compliant, gauze Ti wire mesh; in
this embodiment the cartilage is "mechanically locked" onto the
mesh and thus has enhanced toughness and strength.
[0209] In alternative embodiments, for the three-dimensional,
wire-containing scaffold structures of the invention, e.g., as in
the exemplary structures illustrated in FIG. 17 to FIG. 22, the
springy and compliant metallic wires or microwires have a diameter
in the range of e.g. between about 10 to 100 um in diameter. In
alternative embodiments, these wires have a surface structure of
nanostructures, e.g., nanotubes, nanowires, nanoribbons and/or
nanopores for e.g., enhanced chondrocyte adhesion and cartilage
growth, or for enhanced bone adhesion and growth.
[0210] In alternative embodiments, the material used for the
three-dimensional springy, coil, wire, or mesh screen scaffold of
FIGS. 17 to 22 comprises a metal or alloy selected from Ti, Zr, Hf,
Nb, Ta, Mo or W, or alloys containing at least one of these
elements, or stainless steel, or Co--Cr--Ni--Mo alloy (commonly
known as MP35N alloy), or oxides comprising one of these elements
or alloys, or any mixture thereof.
[0211] FIG. 23(a) FIG. 23(b) and FIG. 23(c) illustrate embodiments
of the invention comprising exemplary flat or dual-structured Ti
implants having compliant, three-dimensional wire assembly
structure, as described in detail, below. FIG. 23(a) illustrates an
embodiment comprising bonded protruding hairy or springy Ti wire or
mesh with surface nanopores or nanowires (made e.g., of TiO.sub.2)
on a Ti flat surfaced base. FIG. 23(a) illustrates an embodiment
comprising hairy or springy Ti wire or mesh with surface nanopores
or nanowires (made e.g., of TiO.sub.2) and a dual-structured
macroscopically cell-locking or cartilage-locking (e.g., adhesion)
surface (e.g., of an implant). FIG. 23(c) illustrates an embodiment
comprising "bottle-necked" shapes, e.g., having constricted
entrances, to slow and control release of compositions and/or
material within the microscopic/macroscopic cavities/chambers on
the implant surface; the compositions and/or material within the
cavities/chambers can comprise proteins, growth factors, or
antibiotics and the like. The "bottle-necked" shapes of the
cavities/chambers also can facilitate having cells (e.g., stem
cells, hMSCs, chondrocytes) adhere to the surface.
[0212] FIG. 24(a), FIG. 24(b) and FIG. 24(c) illustrate embodiments
of the invention comprising exemplary TiO.sub.2 compositions of the
invention comprising Ti or Ti alloy particles or fibers. FIG. 24(a)
illustrates an embodiment comprising protruding micro or macro
particles or fibers (which can be Ti, Ti alloys or other material)
as a cross-sectional view of the particles or fibers, attached on a
Ti implant surface; which can be attached by e.g., induction
melting-bonding, or electron-beam ("e-beam") melt bonding, or laser
bonding, or spot welding, or braze bonding, etc. In alternative
embodiments the construction (e.g., as an implant) can be as a
flat, round and/or curved surface, e.g., a Ti surface. FIG. 24(b)
illustrates an embodiment comprising surface-modified particles,
e.g., Ti particles, of FIG. 24(a); which can be modified e.g. by
anodization-induced modification of a TiO.sub.2 nanotube or
nanopore surface. FIG. 24(c) illustrates an embodiment comprising
"locked in" bone and/or cartilage growth around and/or in Ti
particles or fibers (which in different embodiments can be on a
micro- and/or nano-scale size) using the exemplary embodiments
described in FIG. 24(a) and FIG. 24(b).
[0213] FIG. 25 illustrates an embodiment of the invention
comprising sintering exemplary Ti or Ti alloy particles or fibers
onto an exemplary composition of the invention, e.g., a Ti implant;
where the arrow points to exemplary "protruding" surface-modified
Ti particle aggregates, or exemplary "protruding" surface-modified
Ti fiber aggregates; made e.g., using an anodization-induced
TiO.sub.2 nanotube or nanopore on the particle and/or fiber
surface. This embodiment has the aggregates on a flat surface; but
alternatively as with any surface of a composition of the
invention, the surface also can be shaped in any way, e.g., round
or curved.
[0214] FIG. 26(a), FIG. 26(b) and FIG. 26(c) illustrate embodiments
of the invention comprising Ti or Ti alloy wire or ribbon arrays
unidirectionally or vertically aligned, with the surface of wires
or ribbons anodized to form TiO.sub.2 nanotubes. FIG. 26(a)
illustrates Ti wire or ribbons on a Ti base formed e.g., by bonding
onto the Ti base surface, or etching onto the Ti base surface. FIG.
26(b) illustrates TiO.sub.2 nanotube and/or nanopore arrays on the
surfaces of the wires or ribbons. FIG. 26(c) illustrates that in
this exemplary embodiment the chondrocyte or cartilage growth is
guided somewhat vertically along the Ti wire or ribbon direction.
In one embodiment the wires or ribbons are "compliant" on an
implant surface for increased surface area and enhanced cell (e.g.,
stem cell, chondrocyte), cartilage and/or bone growth.
[0215] FIG. 27(a) and FIG. 27(b) illustrate embodiments comprising
exemplary compositions of the invention comprising compliant,
springy or bent Ti or Ti alloy wires, ribbons, columns, mesh
screens or ribbon arrays on a Ti or Ti alloy base or surface. This
embodiment has the aggregates on a flat surface; but alternatively
as with any surface of a composition of the invention, the surface
also can be shaped in any way, e.g., round or curved. In
alternative embodiments, the FIG. 27(a) wires, mesh screens or
ribbon arrays can be compliant, springy and/or bent, and can be Ti
or Ti alloy, or TiO.sub.2 or TiO.sub.2 alloy, or a pure metal
(e.g., Au, Pt, Pd) or a metal (e.g., Au, Pt, Pd) alloy. FIG. 27(b)
illustrates an embodiment where a Ti mesh screen is bonded onto a
Ti surface, e.g., an implant. FIG. 27(a) and FIG. 27(b) also
illustrates an optional embodiment having spacers and/or
"protectors" between sections of wires, ribbons, columns, mesh
screens or ribbon arrays.
[0216] FIG. 28(a), FIG. 28(b) and FIG. 28(c) illustrate embodiments
of the invention comprising exemplary compositions of this
invention comprising nanotubes and/or nanopores on non-Ti surfaces
such as ceramics, polymers, plastics and other non-Ti metals (e.g.,
Si, Au, Pt, Al) deposited or made by e.g., anodization. FIG. 28(a)
illustrates an embodiment comprising "pre-patterned" a surface
comprising a Ti or Ti alloy, or a pure metal (e.g., Au, Pt, Pd) or
a metal (e.g., Au, Pt, Pd) alloy, or any mixture thereof; where in
alternative embodiments the surface can have a "pre-patterned"
regular or irregular shape. In alternative embodiments, a
"pre-patterned" regular shape is made by machining or mask
patterning. In alternative embodiments, a "pre-patterned" irregular
shape is made by sandblasting or chemical etching. FIG. 28(b)
illustrates an embodiment comprising a nanotube or nanopore layer
on or within the surface of the product of manufacture of FIG.
28(a); in one embodiment this is made by depositing a Ti and/or Ti
alloy on the surface and anodizing to make a nanotube and/or
nanopore on or in the surface if the surface already does not
comprise Ti or a Ti alloy. FIG. 28(c) illustrates the enhanced
growth of cartilage and/or bone on the exemplary surfaces
illustrated in FIG. 28(b) and FIG. 28(b).
[0217] FIG. 29(a) and FIG. 29(b) illustrate embodiments of the
invention comprising exemplary compositions comprising nanotubes
and/or nanopores on non-Ti surfaces such as ceramics, polymers,
plastics and other non-Ti metals (e.g., Si, Au, Pt, Al) deposited
or made by e.g., anodization, as described above for FIG. 28. FIG.
29(a) illustrates nanotubes and/or nanopores on the surface of
"lock-in" structures, which can be made as "pre-patterned"
substrates, as described above for FIG. 28. FIG. 28(c) illustrates
the enhanced growth of cartilage and/or bone on the exemplary
surfaces.
[0218] It is understood that the above-described embodiments are
illustrative of only a few of the many possible specific
embodiments which can represent applications of the invention.
Numerous and varied other arrangements can be made by those skilled
in the art without departing from the spirit and scope of the
invention. For example, in alternative embodiments of the invention
the materials used to make the products of manufacture do not have
to be Ti oxide nanotubes on Ti-based metals, as in alternative
embodiments the nanotubes and nanopillars of this invention are
adhered to other biocompatible materials, or non-biocompatible
materials coated with biocompatible and bioactive surface layer,
e.g., in an alternative embodiments a biocompatible surface layer
comprises Ti, a portion of which can be converted into a TiO.sub.2
nanotube, nanowire and/or nanopillar array configuration.
Kits
[0219] The invention provides kits comprising compositions of the
invention (e.g., the products of manufacture of the invention, such
as implants); and optionally also comprising materials for
practicing methods of the invention, and optionally also comprises
instructions for practicing the methods of this invention.
[0220] The invention will be further described with reference to
the following examples; however, it is to be understood that the
invention is not limited to such examples.
EXAMPLES
Example 1
[0221] The following examples describe TiO.sub.2 nanotubes of the
invention having various dimensions, and their fabrication; and
demonstrate how they enhance chondrocyte growth and accelerate
extracellular matrix formation.
[0222] Fabrication of Nanotube Array Structure for Chondrocyte
Culture Experiments.
[0223] Shown in FIG. 2 are exemplary TiO.sub.2 nanotube structures
of the invention prepared for chondrocyte culture. Primary bovine
cartilage chondrocyte (BCC) was utilized for the experiments. The
vertically aligned TiO.sub.2 nanotubule array structures with
different nanotube diameters, as shown in the scanning electron
microscopy photographs illustrated in FIG. 2, were fabricated by
anodization technique using a Ti sheet (0.25 mm thick, 99.5%
purity) which is electrochemically processed in a 0.5% HF solution
at 20, 15, 10, or 5 V for 30 min at room temperature. A platinum
electrode (thickness: 0.1 mm, purity: 99.99%) was used as the
cathode. To crystallize the as-deposited, amorphous-structured
TiO.sub.2 nanotubes into the desired anatase phase, the specimens
were heat-treated at 500.degree. C. for 2 hrs. In this application,
it is preferred that the amorphous TiO.sub.2 nanotubes is
crystallized to anatase phase by heat treatment, because an
amorphous TiO.sub.2 phase tends to be more susceptible to breakage
by external stresses as compared to a crystalline phase.
[0224] The SEM images illustrated in FIG. 2 show highly ordered
nanotubes with four different pore sizes between 30-100 nm created
by controlling potentials ranging from 5 to 20V. The geometrical
features used for the chondrocyte culture were 30, 50, 70, 100 nm
diameter titania (TiO.sub.2) nanotubular surfaces prepared by
anodization. The height of the nanotubes was in the range of
.about.250-300 nm. The dimensions of the nanotubes were varied in
order to determine how the size of the nanotubes influences the
chondrocyte behavior as previous studies have shown for other types
of cells, e.g., see Park (2007) Nano Lett. 7(6):1686-1691.
[0225] Chondrocyte Culture and SEM Analysis of Cell Morphology
[0226] FIG. 7 shows comparative SEM images of bovine cartilage
chondrocytes (BCCs) cultured 2 hours, 24 hours, and 5 days on flat
Ti vs. different diameter (30, 50, 70, 100 nm) on exemplary
TiO.sub.2 nanotube surfaces. At 2 hours (FIG. 7 top row),
chondrocytes initially appear like they are beginning to spread out
on all surfaces (indicated by the ring of dark conceivable matrix
like material surrounding the cells) except for the 100 nm
TiO.sub.2 nanotube surface. Uniquely, the cells on the nanotubes
with the large 100 nm pores remain spherical on the surface having
no dark surrounding material deposition. Unlike any other surface,
cells on the 100 nm diameter TiO.sub.2 nanotubes elicit
extracellular matrix (ECM) fibrils (arrow) forming inter-cellular
bridges between adjacent chondrocytes at the very early time point
of 2 hours of culture incubation.
[0227] At 24 hours of culture (FIG. 7 middle row), the chondrocytes
on the exemplary Ti substrate continue to show signs of possible
flattening and spreading on the surface indicated by the dark
matrix areas on the periphery of the cells. On the exemplary
nanotube surfaces however, flattening has been reduced and much
increased ECM fibrils are present (arrows), especially on the 70 nm
and 100 nm pore size nanotubes. The nanotubes substrates appear
that they are inducing a positive response from the chondrocytes
because it is observed that the cells begin initiating ECM
deposition and fibril organization within the initial 24 hours of
culture.
[0228] Higher magnification SEM observations of BCCs (24 hours of
culture) in FIG. 8 reveal a striking difference in the formation of
ECM between the flat Ti (as illustrated in FIG. 8(a)) vs. nanotube
(30 nm diameter TiO.sub.2 nanotubes shown in this image) (as
illustrated in FIG. 8(b)) surfaces. There is a deposition of dense,
fibril material on the nanotubular surfaces of this invention as
illustrated in FIG. 8(b), most likely collagen Type II, a primary
ECM molecule produced by chondrocytes and a ground substance in
cartilage tissue. It appears that the nanotubes are actually
regulating the cells by facilitating a more intricate, nanoscale
order of ECM deposition. The nanotopography supports ECM molecule
distribution atop the walls of the nanotubes of this invention,
allowing for a type of guidance of fibril formation.
[0229] By contrast, the ECM deposited upon the flat Ti surface (as
illustrated in FIG. 8(a)) is irregular, sparse, and thus lacks
surface structuring cues for signaling ECM fibril organization.
Moreover, it could be that fibers of bio active material such as
collagen in this case are "nano-inspired" to form on the nanotube
structure of this invention because of the fine scale cues and top
surface (tip of the vertical wall) of TiO.sub.2 nanotubes having a
physically confined geometry which could aid in fibril formation.
It was demonstrated previously that the nanotubes produced
bio-active nanostructured formations of sodium titanate nanofibers
on the top of TiO.sub.2 nanotubes when nanotubes were exposed to
the NaOH solution, see e.g., Oh (2005) Biomaterials
23:2945-2954.
[0230] With increased ECM production as seen in the SEM micrographs
at early time points (2 and 24 hours), nanotubes most likely
promote the proper ECM structuring of molecules much faster and
more efficiently than the flat substrate of Ti. In addition, the
naturally present pore configuration within the nanotubes can
possibly be utilized as nano-depots to store and entrap extra
biomolecules and nutrients while the fluid spaces in-between the
nanotube walls allow for the exchange of gas, nutrients, and cell
signaling molecules for an overall enhanced cell environment. The
increased surface area with 100 nm diameter TiO.sub.2 nanotubes
having approximately 20.times. the amount of surface area compared
to flat Ti quite possibly increases the ECM storage capacity.
[0231] At even longer culture times of 5 days, the chondrocytes on
the nanotube structures appear to be encased in think beds of
extracellular matrix FIG. 7 (bottom row). The arrows indicate a
type of cell cluster formation within ECM beds that seem to link
the cells together, connecting long strings of chondrocytes. Flat
Ti had less cell clusters and more fibroblastic or elongated type
cells spread over a large area where more cells on the nanotube
substrates seemed to be spherical, retaining the phenotypical
chondrocyte shape. In one aspect, the nanotube geometry seen in
FIG. 2 can aids in preserving this morphology because of the
distinct structure of the surface, where cells may be localized
atop the pores, anchored possibly and confined by the tube contour.
The chondrocytes on the flat Ti seem to spread along the surface
probably because the necessary structuring cues and nanopores
needed for shape confinement are absent.
[0232] Cell Viability
[0233] In order to show the viability of chondrocytes on the
substrates, chondrocytes on control polystyrene (Nunc 12-well
plate), flat Ti, and 30, 50, 70, 100 nm TiO.sub.2 nanotube
substrates were incubated with fluorescein diactate (FDA)
cytoplasmic fluorescent dye after 5 days of culture. The FDA (green
fluorescence) images are illustrated in the panels of FIG. 9. More
notably round shaped cells were observed on the nanotube substrates
while more cell flattening and fibroblastic shaped cells were
apparent on the polystyrene and Ti surfaces comparatively. This
flattening may indicate loss of the chondrogenic phenotype on
polystyrene and Ti.
[0234] FIG. 9 schematically illustrates six panels of
immunofluorescent images of cells grown on exemplary compositions
of this invention (having nanotube surfaces) to show the viability
of bovine cartilage chondrocytes (BCCs) using fluorescein diacetate
(FDA) cytoplasmic staining (a viability-staining technique); the
cells were cultured for 5 days on control polystyrene culture
dishes, flat Ti and 30, 50, 70, 100 nm diameter TiO.sub.2 nanotube
surfaces of this invention; viability-staining demonstrates that
practically all the cells were alive on all surfaces, as described
in detail, below.
[0235] Cell Shape Analysis
[0236] The morphological analysis based on the FDA observations, as
graphically illustrated in FIG. 10, may further imply that the
nanotubes induce a more spherical chondrocyte shape. The percentage
of round cells was significantly lower for bovine cartilage
chondrocytes (BCCs) on the polystyrene, Ti, and the smallest
diameter (30 nm) nanotube substrates compared to the 50 nm, 70 nm,
and 100 nm TiO.sub.2 nanotube surfaces. The highest percentage of
round cells on the 70 nm pore size sample reached approximately
80%.
[0237] Functional Inspection: Extracellular Matrix (ECM)
Formation
[0238] Cartilage consists of two main components, chondrocyte cells
and their matrix. The structure of the matrix is composed of two
basic macromolecules that are essential for the structural and
functional integrity of cartilage, namely type II collagen and
aggrecan, see e.g., Muir, H., The chondrocyte, architect of
cartilage. Biomechanics, structure, function and molecular biology
of cartilage matrix macromolecules. Bioessays, 1995. 17(12): p.
1039-48. Aggrecan consists of both a core protein and keratin
sulfate glycosamionoglycan (GAG) chains which fill the narrow
spaces within the collagen, see e.g., Muir (1995) supra.
[0239] In FIG. 11, the glycosaminoglycan (GAG) amount secreted in
the media in contact with the different substrates was evaluated
relative to control polystyrene culture dishes. Clearly, the
nanotubular surfaces of this invention up-regulate GAG secretion.
There is a general trend that shows increasing amounts of GAG
secretion with increasing size of nanotube diameter, reaching its
highest at approximately 70 nm diameter, with twice as much GAG
secreted in the media compared to flat Ti (the Ti surface is
covered with thin native TiO.sub.2 oxide layer). While the exact
mechanism is yet to be thoroughly understood, and the invention is
not limited by any particular mechanism of action, the nanotube
functionality trend is likely due to the slight change in
cytoskeletal tension and focal adhesion distances due to pore size
of the nanotubes.
[0240] Naturally, aggrecan draws water into the tissue and swells
against the collagen network, thereby resisting compression and
allowing for proper joint movement, see e.g., Muir (1995) supra.
While the invention is not limited by any particular mechanism of
action, in some aspects the up-regulation of GAG chains is
indicative of the increased aggrecan production observed on the
larger sized nanotube pores in exemplary structures of this
invention; and this could imply that because there are increased
storage volume capabilities as pore size increases it triggers a
higher rate of production because the molecule retention ability of
the cellular environment has been inflated.
[0241] To further evaluate the response of BCCs for this
comparative surface morphology, the functional modification of
collagen type II expression by the different surface physiological
conditions was also measured. Comparative immunofluorescent images
of collagen type II ECM fibrils produced by BCCs on flat Ti vs 100
nm diameter TiO.sub.2 nanotube surfaces are illustrated in FIG.
12(a). We observed an up-regulated collagen type II expression on
the nanotube surface where fibrils have formed intricate networks
connecting cells over a long range order which is basically absent
on the Ti surface. The images in FIG. 12(b) represent higher
magnification immunofluorescent images of collagen type II (red)
and DAPI (blue) nuclear staining of BCCs on polystyrene, flat Ti,
and 30, 50, 70, 100 nm TiO.sub.2 nanotube surfaces after 5 days of
culture. Large aggregates and cell assemblies expressing collagen
type II are seen on the nanotube surfaces. In a similar trend as
the GAG secretion, as nanotube size generally increased, the
collagen production and network activation between cells increased
(data not shown). The collagen type II expression on the nanotube
surface also reveal dense beds and clusters of ECM structures and
lacunae type structures possibly mimicking the natural matrix
cavities in an actual cartilage environment. While the invention is
not limited by any particular mechanism of action, the nanotubes of
the products of manufacture of the invention may be facilitating a
more natural and active response of BCCs.
[0242] The results obtained demonstrate that the presence of the
nanotube structures of this invention significantly up-regulate
glycosaminoglycan (GAG) secretion and collagen Type II production
by chondrocytes, which is beneficial for cartilage repair. It was
found that increasing the diameter of the nanotubes to the
approximate regime of 70 nm to 100 nm in this invention increased
the cartilage related productivity. Nanotube diameter sizes larger
than 100 nm can also be used, and these larger sizes, to some
extent, may also increase the productivity threshold as compared
with an implant surface with no nanotube structure.
[0243] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
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