U.S. patent application number 11/045620 was filed with the patent office on 2005-12-08 for bone tissue engineering by ex vivo stem cells ongrowth into three-dimensional trabecular metal.
Invention is credited to Bunger, Cody, Li, Haisheng, Xuenong, Zou.
Application Number | 20050272153 11/045620 |
Document ID | / |
Family ID | 35449477 |
Filed Date | 2005-12-08 |
United States Patent
Application |
20050272153 |
Kind Code |
A1 |
Xuenong, Zou ; et
al. |
December 8, 2005 |
Bone tissue engineering by ex vivo stem cells ongrowth into
three-dimensional trabecular metal
Abstract
Adult autologous stem cells cultured on a porous,
three-dimensional tissue scaffold-implant for bone regeneration by
the use of a hyaluronan and/or dexamethasone to accelerate bone
healing alone or in combination with recombinant growth factors or
transfected osteogenic genes. The scaffold-implant may be machined
into a custom-shaped three-dimensional cell culture system for
support of cell growth, reservoir for peptides, recombinant growth
factors, cytokines and antineoplastic drugs in the presence of a
hyaluronan and/or dexamethasone alone or in combination with growth
factors or transfected osteogenic genes, to be assembled ex vivo in
a tissue incubator for implantation into bone tissue.
Inventors: |
Xuenong, Zou; (Aarhus,
DK) ; Li, Haisheng; (Viby, DK) ; Bunger,
Cody; (Auning, DK) |
Correspondence
Address: |
DUANE MORRIS LLP
PO BOX 5203
PRINCETON
NJ
08543-5203
US
|
Family ID: |
35449477 |
Appl. No.: |
11/045620 |
Filed: |
January 27, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60539661 |
Jan 27, 2004 |
|
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|
Current U.S.
Class: |
435/395 ;
435/384; 623/23.63; 623/23.76 |
Current CPC
Class: |
A61F 2002/30225
20130101; A61F 2230/0063 20130101; A61L 2300/64 20130101; A61F
2002/0086 20130101; A61F 2310/00982 20130101; A61L 27/38 20130101;
A61F 2/34 20130101; A61F 2310/00161 20130101; A61F 2/32 20130101;
A61F 2/4644 20130101; A61L 27/54 20130101; A61F 2002/2817 20130101;
A61L 27/00 20130101; A61L 27/365 20130101; A61F 2/44 20130101; A61F
2002/4648 20130101; A61L 2400/18 20130101; A61F 2/30767 20130101;
A61L 2430/00 20130101; A61F 2310/00293 20130101; A61F 2310/00976
20130101; A61F 2002/30677 20130101; A61F 2/36 20130101; A61F
2310/00544 20130101; A61L 27/20 20130101; A61L 27/22 20130101; C12N
5/0068 20130101; A61F 2/28 20130101; A61F 2310/00491 20130101; A61L
2300/606 20130101; A61F 2002/3093 20130101; A61F 2310/00341
20130101; A61L 2300/222 20130101; A61L 27/3608 20130101; A61L
2300/252 20130101; A61F 2/38 20130101; A61F 2002/2835 20130101;
A61F 2002/30199 20130101; C12N 5/0662 20130101; A61F 2230/0069
20130101; A61F 2/3094 20130101; A61L 27/56 20130101 |
Class at
Publication: |
435/395 ;
435/384; 623/023.63; 623/023.76 |
International
Class: |
C12N 005/08; A61F
002/02; A61F 002/28 |
Claims
What is claimed is:
1. A three-dimensional tissue scaffold-implant for supporting
tissue on-growth, the scaffold-implant comprising: a lattice having
a matrix of interconnected pores which form surfaces in three
dimensions; an inert, bio-compatible material covering the
surfaces; and at least one of a hyaluronan, dexamethasone, protein,
peptide, transcript factor, cytokine, therapeutic agent, chitosan,
polymer, osteogenic gene and growth factor, covering the
material.
2. The tissue scaffold-implant of claim 1, wherein the inert,
biocompatible material comprises a metal.
3. The tissue scaffold-implant of claim 2, wherein the metal
comprises tantalum.
4. The tissue scaffold-implant of claim 1, wherein the inert,
biocompatible material comprises a metal alloy.
5. The tissue scaffold-implant of claim 1, further comprising
living cells covering the biocompatible material, the cells
selected from the group consisting of bone marrow cells,
osteoblasts, mesenchymal stem cells, embryonic stem cells, gene
transfected cells, endothelial cells and combinations thereof.
6. The tissue scaffold-implant of claim 1, further comprising
tissue grown over the bio-compatible material.
7. The tissue scaffold-implant of claim 6, wherein the tissue
comprises bone.
8. A method of forming tissue, the method comprising: providing a
three-dimensional tissue scaffold comprising a lattice having a
matrix of interconnected pores which form surfaces in three
dimensions, and an inert, biocompatible material covering the
surfaces; covering the material covered surfaces of the scaffold
with living cells; and culturing the scaffold to grow tissue on and
in the scaffold.
9. The method of claim 8, wherein prior to the culturing step,
further comprising the step of applying at least one of a
hyaluronan, dexamethasone, protein, peptide, transcript factor,
cytokine, therapeutic agent, chitosan, polymer, osteogenic gene and
growth factor, to the material covered surfaces.
10. The method of claim 9, wherein the applying step includes
encapsulating the at least one of the hyaluronan, dexamethasone,
protein, peptide, transcript factor, cytokines therapeutic agent,
chitosan, polymer, osteogenic gene and growth factor.
11. The method of claim 10, wherein the encapsulation is performed
by complex sandwich conjugation with a polymeric material.
12. The method of claim 11, wherein the culturing step is performed
by placing the scaffold in a medium and incubating the medium and
scaffold.
13. The method of claim 12, further comprising the step of
implanting the scaffold in a body of one of an animal and a human
being.
14. The method of claim 9, wherein the culturing step is performed
by implanting the scaffold in the body of one of an animal and a
human being.
15. The method of claim 10, wherein the culturing step is performed
by implanting the scaffold in the body of one of an animal and a
human being.
16. The method of claim 8, wherein the culturing step is performed
by placing the scaffold in a medium and incubating the medium and
scaffold.
17. The method of claim 16, further comprising the step of
implanting the scaffold in a body of one of an animal and a human
being.
18. The method of claim 8, wherein the culturing step is performed
by implanting the scaffold in the body of one of an animal and a
human being.
19. The method of claim 8, wherein the covering step is performed
by cell transplantation.
20. The method of claim 8, wherein the inert, biocompatible metal
comprises tantalum.
21. The method of claim 8, wherein the living cells are selected
from the group consisting of bone marrow cells, osteoblasts,
mesenchymal stem cells, embryonic stem cells, gene transfected
cells, endothelial cells and combinations thereof.
22. The method of claim 8, wherein the tissue comprises bone.
23. A method of making an implant for supporting tissue on-growth,
the method comprising: providing a three-dimensional tissue
scaffold comprising a lattice having a matrix of interconnected
pores which form surfaces in three dimensions, and an inert,
biocompatible metal covering the surfaces; and covering the metal
covered surfaces of the scaffold with living cells.
24. The method of claim 23, further comprising the step of applying
at least one of a hyaluronan, dexamethasone, protein, peptide,
transcript factor, cytokine, therapeutic agent, chitosan, polymer,
osteogenic gene and growth factor, to the metal covered
surfaces.
25. The method of claim 24, wherein the applying step includes
encapsulating at least one of the hyaluronan, dexamethasone,
protein, peptide, transcript factor, cytokines, therapeutic agent,
chitosan, polymer, osteogenic gene and growth factor.
26. The method of claim 25, wherein the encapsulation is performed
by complex sandwich conjugation with a polymeric material.
27. The method of claim 26, further comprising the step of
culturing the scaffold to grow tissue on and in the scaffold.
28. The method of claim 27, wherein the culturing step is performed
by placing the scaffold in a medium and incubating the medium and
scaffold.
29. The method of claim 23, further comprising the step of
culturing the scaffold to grow tissue on and in the scaffold.
30. The method of claim 29, wherein the culturing step is performed
by placing the scaffold in a medium and incubating the medium and
scaffold.
31. The method of claim 25, wherein the culturing step is performed
by implanting the scaffold in the body of one of an animal and a
human being.
32. The method of claim 23, wherein the covering step is performed
by cell transplantation.
33. The method of claim 23, wherein the inert, biocompatible
material comprises tantalum.
34. The method of claim 23, wherein the living cells are selected
from the group consisting of bone marrow cells, osteoblasts,
mesenchymal stem cells, embryonic stem cells, gene transfected
cells, endothelial cells and combinations thereof.
35. The method of claim 34, wherein cells are encapsulated in at
least one of a hyaluronan and collagen, or chitosan with a
polymeric material by complex sandwich conjugation.
36. The method of claim 28, wherein the culturing method is
performed in one of or any combination of a static, dynamic medium
flow, pulsatile flow, microgravity and multidirectional gravity
culturing environment.
37. The method of claim 23, wherein the tissue comprises bone.
Description
RELATED APPLICATIONS
[0001] The application claims the benefit of U.S. Provisional
Application No. 60/539,661, filed on Jan. 27, 2004, which is
incorporated herein in its entirety.
FIELD
[0002] The invention relates to tissue generation. More
particularly, the invention relates to three-dimensional tissue
generation by ex vivo three-dimensional cell culture methods using
porous, three-dimensional tissue scaffold-implants.
BACKGROUND
[0003] The current trend of tissue engineering technology is toward
the development of biomaterials for repairing tissue defects or to
enhance fixation of implants to the host tissue. Basic requirements
include a scaffold-implant conductive to cell attachment and
maintenance of cell function, together with a rich source of
progenitor cells. Biomaterials in combination with cells from
ex-vivo cultures will not only accelerate the tissue healing, but
also increase the biocompatibility of scaffold-implants to shorten
the hospitalization, and improved long-term function of the
devices. In particular, patients with large defects, impaired bone
healing and cancer disease in the region of repair shall benefit
from this new technology. One regenerative tissue engineering
approach involves a process known as "tissue induction", whereby a
two or three-dimensional polymer or mineral scaffold-implant
without cells is implanted into a patient. With tissue induction,
tissue generation occurs through ingrowth of surrounding tissue
into the scaffold-implant.
[0004] Another approach to tissue generation, known as "cell
transplantation", involves seeding a scaffold-implant with cells,
cytokines, and other growth-related molecules, then culturing and
implanting the scaffold-implant into the subject to induce the
growth of new tissue. Cultured cells are infused in a biodegradable
or non-biodegradable scaffold-implant, which may be placed in a
bioreactor in-vitro to allow the cells to proliferate before the
cells containing scaffold-implant is implanted in the patient.
Alternatively, the cell-seeded scaffold-implant may be directly
implanted, in which case the patient's body acts as an in-vivo
bioreactor. Once implanted, in-vivo cellular proliferation and, in
the case of absorbable scaffold-implants, concomitant
bio-absorption of the scaffold-implant, proceeds.
[0005] In both types of tissue engineering, i.e., tissue induction
and cell transplantation, the scaffold-implant, whether or not
bio-absorbable, must be biocompatible, such that it does not invoke
an adverse immune response from, or result in toxicity to, the
patient.
[0006] Several types of materials have been investigated for use as
seeding scaffold-implants, including metals, ceramics, polymers,
and polymer-coated metals and ceramics. Existing scaffold-implants
may be manufactured by solvent casting, shaping sections with
machining, 3D printing, or molded collagen/cell constructs. While
the aforementioned scaffold-implant materials are primarily for
industrial applications, the fabrication of hydroxyapatite
scaffold-implants using selective laser sintering and
polymer-coated calcium phosphate powder, have been investigated.
Additional post-processing, such as high temperature heating which
burns out the binder, and then higher temperature sintering which
fuses the powder together, is required to strengthen the
scaffold-implant.
[0007] Whichever type of scaffold-implant is selected, a purpose of
the scaffold-implant is to support cells, which, after being seeded
into the device, cling to the interstices of the scaffold-implant,
replicate, produce their own extracellular matrices, and organize
into the target tissue. For example, in the case of bone
regeneration, the optimal pore size for maximum tissue growth
ranges from 200-400 mircons (.mu.m). Therefore, the material or
materials used for fabricating the scaffold-implant should have
this pore size (200-400 .mu.m) and have sufficient rigidity and
biomechanical properties to support loads that are used for
generating bone tissue.
[0008] Scaffold-implants fabricated from a material such as
hydroxyapatite, which is useful for supporting bone cells, are too
brittle and non-pliable to act as scaffolding for muscle or
tendons. Many of the polymers, and the polymer-coated metals and
ceramics present a challenge to seeding cells in three-dimensional
scaffolds. None of the known scaffold-implant materials allow
growth of cells to a depth of greater than about 250 .mu.m, which
is a generally accepted practical limit on the depth to which cells
and nutrients can diffuse into scaffold-implants having the desired
porosities. Even if cells could be made to diffuse to greater
depths, it is generally believed that to support cell growth and
avoid or at least curtail apoptosis at these depths, the
scaffold-implant must also support some form of vasculature to
promote angiogenesis; none of the earlier mentioned
scaffold-implant fabrication methods, however, allow for
incorporation of blood vessels.
[0009] Porous, three-dimensional metallic structures have recently
been developed for potential application in reconstructive
orthopaedics and other surgical disciplines. Such structures are
described in U.S. Pat. No. 5,282,861 entitled "Open Cell Tantalum
Structures For Cancellous Bone Implants And Cell And Tissue
Receptors" issued to Kaplan; U.S. Pat. No. 5,443,515 entitled
"Vertebral Body Prosthetic Implant With Slidably Positionable
Stabilizing Member" issued to Cohen et al.; U.S. Pat. No. 5,755,809
entitled "Femoral Head Core Channel Filling Prothesis" issued to
Cohen et al.; U.S. Pat. No. 6,063,442 entitled "Bonding Of Porous
Materials To Other Materials Utilizing Chemical Vapor Deposition"
issued to Cohen et al.; and U.S. Pat. No. 6,087,553 entitled
"Implantable Metallic Open-Celled Lattice/Polyethylene Composite
Material And Devices" issued to Cohen et al., the disclosures of
which are incorporated herein by reference. The porous,
three-dimensional metallic structure is a bio-compatible material
having a three-dimensional network of continuously interconnected
channels or pores which define a three-dimensional porosity, i.e.,
volume porosity, ranging from 50 to 90% (higher than all other
known implant materials). This high bulk volume porosity readily
facilitates nutrient diffusion and media circulation.
[0010] The porous, three-dimensional metallic structures may be
fabricated using a vapor deposition/infiltration process wherein
tantalum, which has a long history of medical uses, or other
bio-compatible metal or material is vaporized at high temperature
and precipitated as a thin layer onto a carbon lattice. The coating
of tantalum or other metal enhances or improves the strength or
mechanical characteristics of the carbon lattice.
[0011] As a scaffold for "bone induction," preliminary animal
studies with transcortical (bone conduction) porous,
three-dimensional metallic structures have been shown to support
rapid and extensive bone ingrowth. For example, tissue response to
porous tantalum acetabular cups indicates that the porous tantalum
material is effective for biologic fixation. The biomechanical
property of the porous tantalum biomaterial is sufficient to
withstand physiological load for specific applications, such as an
acetabular cup, a spinal fusion, and a vertebral body replacement
in fractures or in metastatic cancer disease. As a scaffold for
"cell transplantation," porous, three-dimensional metallic
structures can extend culturing of multipotent hematopoietic
progenitors without cytokine augmentation and enhance maintenance
and retroviral transduction of primitive hematopoietic progenitor
cells.
[0012] Hyaluronan possesses biochemical and physical properties
suitable to perform an important role in the early events of
osteogenesis as well as in many other tissues. A low-molecular
weight hyaluronan fully expresses the in-vitro steogenic potential
of mesenchymal cells through the subsequent proliferation and
differentiation of osteoprogenitor cells using proper conditions.
Locally applied high-molecular hyaluronan of MW 1900 kDa also has
been shown to be capable of accelerating new bone formation through
mesenchymal cell differentiation in femur wounds. Hyaluronan at a
low concentration (0.5 mg/mL) has been shown to increase the
development of porcine embryos in culture.
SUMMARY
[0013] One aspect is a three-dimensional tissue scaffold-implant
for supporting tissue (e.g., bone) on-growth. The tissue
scaffold-implant comprises a lattice having a matrix of
interconnected pores which form surfaces in three dimensions; and
an inert, biocompatible material covering the surfaces. The
surfaces of the scaffold-implant define a high surface area
relative to a volume of the scaffold-implant.
[0014] Another aspect is a method of forming tissue (e.g., bone).
The method comprises providing a three-dimensional tissue scaffold
comprising a lattice having a matrix of interconnected pores which
form surfaces in three dimensions, and an inert, biocompatible
material covering the surfaces; covering the biocompatible material
covered surfaces of the scaffold with cells; and culturing the
scaffold to grow tissue on and in the scaffold.
[0015] Yet another aspect is a method of making an implant for
supporting tissue (e.g., bone) on-growth. This method comprises
providing a three-dimensional tissue scaffold comprising a lattice
having a matrix of interconnected pores which form surfaces in
three dimensions, and an inert, biocompatible material covering the
surfaces; and covering the biocompatible material covered surfaces
of the scaffold with cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a flow chart depicting an embodiment of the method
of the present invention.
[0017] FIG. 2 is a bar graph quantifying stem cells binding to a
coralline hydroxyapatite disc and uncoated and coated porous
tantalum discs, after a 24-hour incubation at 37.degree. C. and
normalized to the uncoated TA disc.
[0018] FIGS. 3A and 3B are Hoechst stained fluorescent micrographs
at 50.times. original magnification and 20.times. original
magnification respectively, showing the growing stem cells in the
pores of porous tantalum after 7 days of incubation.
[0019] FIGS. 4A and 4B are histological micrographs at 20.times.
original magnification and 6.25.times. original magnification
respectively, after 8 weeks of implantation in pigs.
[0020] FIG. 5 is a scanning electronic micrograph of the
three-dimensional tissue scaffold-implant.
[0021] FIG. 6 is a histological micrograph of the three-dimensional
tissue scaffold-implant after 12 weeks of implantation in a
pig.
DETAILED DESCRIPTION
[0022] The tissue formation method of the present invention
utilizes an ex vivo cell culture system and a porous,
three-dimensional metallic structure or tissue scaffold of a
desired shape and size, which will be implanted into the body of an
animal or human being (hereinafter scaffold-implant). The cell
culture system induces early stage cell proliferation and
differentiation on and in the tissue scaffold-implant, resulting in
tissue generation. The tissue formation method of the present
invention is especially useful for generating bone tissue. The
method of the invention may also be used to generate connective
tissue and hematopoietic tissue.
[0023] The flow chart of FIG. 1 depicts an embodiment of the tissue
formation method of the present invention. In step 10 of the
method, a porous, three-dimensional tissue scaffold-implant is
fabricated in a desired shape and size, e.g., hip implant, spinal
implant, knee implant, etc. For example, the scaffold-implant may
be shaped and sized as a prosethetic acetabular cup such as the one
disclosed in U.S. Pat. No. 5,443,519 entitled "Prosthetic
Ellipsoidal Acetabular Cup," issued to Averill et al. In another
example, the scaffold-implant may be shaped and sized as a
prosethetic femoral component such as the one disclosed in U.S.
Pat. No. 5,702,487 entitled "Prosethetic Device" issued to Averill
et al.
[0024] In one embodiment, the scaffold-implant may be fabricated as
a single unitary member. In an alternative embodiment, the
scaffold-implant may be fabricated as a single, integral member
formed by two or more separately fabricated sections which are
mechanically assembled together in a conventional manner. In still
another embodiment, the scaffold-implant may be fabricated as an
assembly of two or more cooperating, unitary and/or integral
members (e.g., acetabular cup and femoral stem/ball assembly).
[0025] The porous, three-dimensional tissue scaffold-implant may
comprise a carbon lattice having a strut or ligament skeleton which
forms a three-dimensional network of continuously interconnected
channels or pores each roughly approximating a dodecahedron, which
create a series of continuous microniches and form surfaces of the
lattice in three dimensions; and a thin film of an inert,
bio-compatible metal or other bio-compatible material, which covers
the surfaces.
[0026] The carbon lattice may be formed as a single, unitary member
of a desired shape and size, or in sections of desired shapes and
sizes to be mechanically assembled. The carbon lattice is
substantially rigid, therefore, it may be machined into a bone
regeneration tool of a desired shape and size using conventional
machining methods.
[0027] The inert, bio-compatible metal or other bio-compatible
material may be applied to the surfaces of the carbon lattice using
conventional vapor depositing and infiltrating methods. In a
preferred embodiment, the inert, biocompatible metal comprises
tantalum. In other embodiments, the inert, biocompatible metal may
comprise niobium or alloys of tantalum and niobium.
[0028] The completed porous, three-dimensional tissue
scaffold-implant forms a three-dimensional network of continuously
interconnected, channels or pores which define a three-dimensional
porosity (volume porosity). In one embodiment, the tissue scaffold
implant may comprise channels or pores having an average diameter
of 400 to 500 .mu.m and a volume porosity ranging from about 50 to
about 90%. The geometry of the interconnected pores and surface
texturing arising from the metal vapor deposition process produce
high surface area-to-volume ratio. The large pores and surfaces
allow attachment of proteins, peptides and differentiated and
undifferentiated cells. After fabrication, the scaffold-implant may
be coated with substrate molecules such as fibronectin and
collagens which aid in the attachment of the proteins, peptides and
differentiated and undifferentiated cells.
[0029] In step 20, a hyaluronan (also referred to as hyaluronic
acid or sodium hyaluronate) or a hyaluronan, dexamethasone, one or
more growth factors and/or osteogenic genes is (are) applied to the
surfaces of the tissue scaffold-implant to stimulate early cell
proliferation and differentiation, therefore accelerating tissue
generation. Sodium hyaluronate is a natural high-viscosity anionic
mucopolysaccharide with alternating beta (1-3) glucuronide and beta
(1-4) glucosaminidic bonds. It is commonly found in the umbilical
cord, in vitreous humor, in synovial fluid, in pathologic joints,
in group A and C hemolytic streptococci, and in Wharton's jelly.
Dexamethasone is a synthetic steroid compound. In one embodiment,
the tissue scaffold-implant may be treated with a low concentration
(4 mg/mL) of sodium hyaluronate to induce in-vitro, early stage
stem cell proliferation and differentiation on and in the tissue
scaffold-implant (after performing steps 30 and 40 to be described
further on). In another embodiment, the tissue scaffold-implant may
be treated with a high concentration (10-20 mg/mL) of sodium
hyaluronate which forms a hydro gel with the stem cells in the
tissue scaffold-implant intraoperatively.
[0030] In step 30 of the method, a cell transplantation process is
performed on the porous, three-dimensional tissue scaffold-implant.
In an embodiment of the cell transplantation process, the tissue
scaffold-implant is seeded with living cells, which may comprise
differentiated, undifferentiated or gene transfected cells.
Examples of differentiated or undifferentiated cells include
without limitation bone marrow cells, osteoblasts, mesenchymal stem
cells, embryonic stem cells, endothelial cells. In another
embodiment of the cell transplantation process, the tissue
scaffold-implant is seeded with living cells and proteins,
peptides, transcript factors, osteogenic genes, cytokines,
therapeutic agents, and growth factors.
[0031] The living cells and other factors can be entrapped and
delivered in the tissue scaffold-implant by means of a versatile
self-assembly method. In this self-assembly method cellular matrix
fibrils are formed with methylated collagen (type I) and hyaluronic
acid, or chitosan, which entrap and deliver living cells and other
factors. The cellular matrix fibrils are then combined with an
outer-layer membrane comprising a polymer such as alginate,
hydroxylethyl methacrylate (HEMA), or a terpolymer of hydroxylethyl
methacrylate (HEMA), methy methacrylate (MMA) and methylacric acid
(MAA) by complex sandwich conjugation achieved, for example, using
a complex coacervation process, to protect transplanted allogeneic
cells from immune attacks and to sustain release of the stimulating
factors and therapeutic agents. In one embodiment, the membrane may
be several micrometers to about 100 micrometers thick. The
thickness of the membrane may be adjusted by controlling the
concentrations and contact time of polyelectrolytes in the complex
sandwich conjugation process.
[0032] The surface features (the texture on the surface of the
metal resulting from the CVD of the metal) and the open, highly
interconnected pores of the tissue scaffold-implant readily
facilitate nutrient diffusion and media circulation and thus will
operate as conduits for cell infusion, adhesion, mass transfer, or
to stimulate angiogenesis for blood flow.
[0033] In an alternate embodiment, the application of the
hyaluronan or the hyaluronan, dexamethasone, one or more growth
factors and/or osteogenic genes, to the surfaces of the tissue
scaffold-implant (step 20) may be performed during the cell
transplantation process of step 30.
[0034] In step 40, after seeding, the scaffold-implant is cultured
in a bioreactor to generate the desired tissue. In one embodiment,
the culturing step is an ex-vivo process. Ex-vivo culturing may be
performed in a broth medium, e.g., Dulbecco's modified Eagle's
medium (DMEM) available from HyClone, plus 10% fetal calf serum,
which is placed in an incubator e.g., perfusion or spiner flask
bioreactor or a rotating bioreactor. The broth medium and incubator
operate as an in-vitro bioreactor. In one embodiment, the incubator
may provide a humidified atmosphere of 95% air and 5% CO.sub.2 at
37.degree. C. In addition, the incubator may be of the type which
provides static, dynamic medium flow, pulsatile air flow,
microgravity and multidirectional gravity culturing conditions. The
scaffold-implant may then be implanted (in-vivo) into an animal or
patient's body.
[0035] In another embodiment, the culturing step is an in-vivo
process. In-vivo culturing may be performed in an animal or a
patient by directly implanting the scaffold-implant in the animal
or the patient. In this embodiment, the animal or the patient' body
operates as an in-vivo bioreactor.
[0036] In still an alternate embodiment, the culturing step can be
performed intraoperatively. In this embodiment, cells are taken
from the animal or patient and applied to the scaffold-implant. The
scaffold-implant is then implanted in the animal or patient.
[0037] FIG. 2 is a bar graph quantifying stem cells binding to 1) a
coralline hydroxyapatite (HA) disc; 2) an uncoated porous,
tantalum-based, three-dimensional tissue scaffold-implant (TA)
configured as a disc; 3) a TA disc coated with gelatin; 4) a TA
disc coated with type I collagen; and 5) a TA disc coated with
fibronectin, n=9 (repeated test), after a 24-hour incubation at
37.degree. C. and normalized to the uncoated TA disc. In the graph,
the stem cells are .sup.3H-thymidine labeling cells.
[0038] FIGS. 3A and 3B are fluorescent micrographs showing the
growing cells in the pores of a porous, tantalum-based,
three-dimensional tissue scaffold-implant configured as a disc
after 7 days of incubation (Hoechst staining). As can be seen in
FIG. 3A, at day 7, porcine bone marrow stem cells depicted
funicular proliferations of spindle cells on the pore surface and
within the pores. As shown in FIG. 3B, growing stem cells in the
pores mainly distributed on the surface areas of disc (superior)
where the cells were loaded on. Only a few stem cells had grown
into the central pores and down to other surface areas of the disc
(inferior) where the disc was seated on a well.
[0039] FIGS. 4A and 4B are histological micrographs which show,
after 8 weeks of implantation in pigs, ectopic bone formation after
autologous bone marrow stem cells cultured with a tantalum-based,
three-dimensional tissue scaffold-implant for 7 days of incubation.
Basic fuchsin and light green staining revealed the bone is green G
and fibrous tissue is red R. The black structure B is porous
tantalum strut. Specifically, FIG. 4A shows bone forming in the
pore surface and the pores and FIG. 4B shows a layer of de novo
bone formation at the surface area of the scaffold-implant.
[0040] FIG. 5 is a scanning electronic micrograph of the
three-dimensional tissue scaffold-implant. As can be seen, the
scaffold-implant has a volume porosity of about 50% to about 90%
with interconnecting pores, allowing approximately 2-3 times
greater bone ingrowth compared to conventional porous coatings.
[0041] FIG. 6 is a histological micrograph of the three-dimensional
tissue scaffold-implant after 12 weeks of implantation in a pig. As
can be observed, there is bone formation from intraoperative
conjugation of autologous bone marrow stem cells and hyaluronic
acid gel in the tissue scaffold-implant. Basic fuchsin and light
green staining revealed the bone is green G and fibrous tissue is
red R. The black structure B is porous tantalum strut.
[0042] While the foregoing invention has been described with
reference to the above, various modifications and changes can be
made without departing from the spirit of the invention.
Accordingly, all such modifications and changes are considered to
be within the scope of the appended claims.
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