U.S. patent application number 11/194270 was filed with the patent office on 2007-02-01 for use of adipose tissue-derived stromal cells in spinal fusion.
Invention is credited to Jeffrey M. Gimble, Mandi Lopez.
Application Number | 20070027543 11/194270 |
Document ID | / |
Family ID | 37695366 |
Filed Date | 2007-02-01 |
United States Patent
Application |
20070027543 |
Kind Code |
A1 |
Gimble; Jeffrey M. ; et
al. |
February 1, 2007 |
Use of adipose tissue-derived stromal cells in spinal fusion
Abstract
The present invention encompasses methods and compositions for
treating a bone condition. The isolated adipose tissue-derived
stromal cell of the invention and products related thereto have a
plethora of uses, including but not limited to research,
diagnostic, and therapeutic applications such as in spinal fusion
procedures.
Inventors: |
Gimble; Jeffrey M.; (Baton
Rouge, LA) ; Lopez; Mandi; (Saint Gabriel,
LA) |
Correspondence
Address: |
DRINKER BIDDLE & REATH;ATTN: INTELLECTUAL PROPERTY GROUP
ONE LOGAN SQUARE
18TH AND CHERRY STREETS
PHILADELPHIA
PA
19103-6996
US
|
Family ID: |
37695366 |
Appl. No.: |
11/194270 |
Filed: |
August 1, 2005 |
Current U.S.
Class: |
623/17.11 ;
623/23.63 |
Current CPC
Class: |
A61L 27/3608 20130101;
A61L 2430/38 20130101; A61P 19/00 20180101; A61K 35/28 20130101;
A61L 27/3856 20130101; A61L 27/3658 20130101; A61P 25/00 20180101;
A61F 2/4455 20130101; A61P 19/02 20180101; A61L 27/3852 20130101;
A61P 19/08 20180101; A61P 19/10 20180101; A61F 2002/445 20130101;
A61P 43/00 20180101; A61L 27/3804 20130101; A61L 27/3654 20130101;
A61F 2002/30677 20130101 |
Class at
Publication: |
623/017.11 ;
623/023.63 |
International
Class: |
A61F 2/44 20060101
A61F002/44 |
Claims
1. A method of enhancing the fusion of bone following a spinal
fusion procedure in a mammal, the method comprising administering
an isolated adipose tissue-derived adult stromal (ADAS) cell to the
spine of said mammal, wherein said ADAS cell differentiates in vivo
into a cell that expresses at least one characteristic of a bone
cell.
2. The method of claim 1, wherein said ADAS cell is cultured in
vitro for a period of time without being induced to differentiate
prior to the administration of said cell to the mammal.
3. The method of claim 1, wherein said ADAS cell is allogeneic with
respect to said mammal.
4. The method of claim 1, wherein said ADAS cell induces bone
formation for intervertebral body spinal fusion.
5. The method of claim 1, wherein said ADAS cell induces bone
formation for intertransverse process spinal fusion.
6. The method of claim 1, wherein said ADAS cell further comprises
a biocompatible matrix.
7. The method of claim 1, wherein said biocompatible matrix is
selected from the group consisting of calcium alginate, agarose,
fibrin, collagen, laminin, fibronectin, glycosaminoglycan,
hyaluronic acid, heparin sulfate, chondroitin sulfate A, dermatan
sulfate, and bone matrix gelatin.
8. The method of claim 1, wherein said ADAS cell is genetically
modified.
9. The method of claim 1, wherein said ADAS cell is administered
into one or more interbody spaces in the spine of the mammal.
10. The method of claim 1, wherein the spinal fusion is in a
segment of the spine selected from the group consisting of
cervical, thoracic, lumbar, lumbosacral and sacro-iliac (SI)
joint.
11. The method of claim 1, wherein said ADAS cell is administered
into one or more interbody spaces by an approach selected from the
group consisting of a posterior approach, a posterolateral
approach, an anterior approach, an anterolateral approach, and a
lateral approach.
12. The method of claim 1, wherein said mammal is a human.
13. A method of performing one or more spinal fusions in a mammal,
the method comprising administering an isolated adipose
tissue-derived adult stromal (ADAS) cell to the spine of said
mammal to facilitate a single or multi level spinal fusion.
14. The method of claim 13, wherein said ADAS cell differentiates
in vivo into a cell that expresses at least one characteristic of a
bone cell.
15. The method of claim 13, wherein said ADAS cell is cultured in
vitro for a period of time without being induced to differentiate
prior to the administration of said cell to said mammal.
16. The method of claim 13, wherein said ADAS cell is allogeneic
with respect to the mammal.
17. The method of claim 13, wherein the ADAS cell induces bone
formation for intervertebral body spinal fusion.
18. The method of claim 13, wherein the ADAS cell induces bone
formation for intertransverse process spinal fusion.
19. The method of claim 13, wherein said ADAS cell further
comprises a biocompatible matrix.
20. The method of claim 13, wherein said biocompatible matrix is
selected from the group consisting of calcium alginate, agarose,
fibrin, collagen, laminin, fibronectin, glycosaminoglycan,
hyaluronic acid, heparin sulfate, chondroitin sulfate A, dermatan
sulfate, and bone matrix gelatin.
21. The method of claim 13, wherein said ADAS cell is genetically
modified.
22. The method of claim 13, wherein said ADAS cell is administered
into one or more interbody spaces in the spine of said mammal.
23. The method of claim 13, wherein the spinal fusion is in a
segment of the spine selected from the group consisting of
cervical, thoracic, lumbar, lumbosacral and SI joint.
24. The method of claim 13, wherein said ADAS cell is administered
into one or more interbody spaces by an approach selected from the
group consisting of a posterior approach, a posterolateral
approach, an anterior approach, an anterolateral approach, and a
lateral approach.
25. The method of claim 13, wherein said mammal is a human.
Description
BACKGROUND OF THE INVENTION
[0001] There generally are two types of bone conditions: 1)
non-metabolic bone conditions, such as bone fractures, bone/spinal
deformation, osteosarcoma, myeloma, bone dysplasia and scoliosis,
and 2) metabolic bone conditions, such as osteoporosis,
osteomalacia, rickets, fibrous osteitis, renal bone dystrophy and
Paget's disease of bone. Osteoporosis, a metabolic bone condition,
is a systemic disease characterized by increased bone fragility and
fracturability due to decreased bone mass and change in fine bone
tissue structure, its major clinical symptoms including spinal
kyphosis, and fractures of dorsolumbar bones, vertebral centra,
femoral necks, lower end of radius, ribs, upper end of humerus, and
others. In bone tissue, bone formation and destruction due to bone
resorption occur constantly. Upon deterioration of the balance
between bone formation and bone destruction due to bone resorption,
a quantitative reduction in bone occurs. Traditionally, bone
resorption suppressors such as estrogens, calcitonin and
bisphosphonates have been mainly used to treat osteoporosis.
[0002] With respect to bone/spinal conditions, over 75% of the
American population suffers from back pain sometime during their
life. Underlying medical illnesses can contribute to back pain.
These include scoliosis, spinal stenosis, degenerative disc
disease, infectious processes, tumors, and trauma. The repair of
large segmental defects in diaphyseal bone is a significant problem
faced by orthopaedic surgeons today. Although such bone loss may
occur as the result of acute injury, these massive defects commonly
present secondary to congenital malformations, benign and malignant
tumors, osseous infection, and fracture non-union. The use of fresh
autologous bone graft material has been viewed as the historical
standard of treatment but is associated with substantial morbidity
including infection, malformation, pain, and loss of function (Kahn
et al., 1995, Clin. Orthop. Rel. Res. 313:69-75). The complications
resulting from graft harvest, combined with its limited supply,
have inspired the development of alternative strategies for the
repair of clinically significant bone defects. The primary approach
to this problem has focused on the development of effective bone
implant materials.
[0003] Three general classes of bone implants have emerged from
these investigational efforts, and these classes may be categorized
as osteoconductive, osteoinductive, or directly osteogenic.
Allograft bone is probably the best known type of osteoconductive
implant. Although widely used for many years, the risk of disease
transmission, host rejection, and lack of osteoinduction compromise
its desirability (Leads, 1988, JAMA 260:2487-2488). Synthetic
osteoconductive implants include titanium fibermetals and ceramics
composed of hydroxyapatite and/or tricalcium phosphate. The
favorably porous nature of these implants facilitate bony ingrowth,
but their lack of osteoinductive potential limits their utility. A
variety of osteoinductive compounds have also been studied,
including demineralized bone matrix, which is known to contain bone
morphogenic proteins (BMP). Since the original discovery of BMPs,
others have characterized, cloned, expressed, and implanted
purified or recombinant BMPs in orthotopic sites for the repair of
large bone defects (Gerhart et al., 1993, Clin. Orthop. Rel. Res.
293:317-326; Stevenson et al., 1994, J. Bone Joint Surg.
76:1676-1687; Wozney et al., 1988 Science 242:1528-1534). The
success of this approach has hinged on the presence of mesenchymal
cells capable of responding to the inductive signal provided by the
BMP (Lane et al., 1994, In First International Conference on Bone
Morphogenic Proteins). It is these mesenchymal progenitors which
undergo osteogenic differentiation and are ultimately responsible
for synthesizing new bone at the surgical site.
[0004] One alternative to the osteoinductive approach is the
implantation of living cells which are directly osteogenic. Since
bone marrow has been shown to contain a population of cells which
possess osteogenic potential, some have devised experimental
therapies based on the implantation of fresh autologous or
syngeneic marrow at sites in need of skeletal repair (Grundel et
al., 1991, Clin. Orthop. Rel. Res. 266:244-258; Werntz et al.,
1996, J. Orthop. Res. 14:85-93; Wolff et al., 1994, J. Orthop. Res.
12:439-446). Though sound in principle, the practicality of
obtaining enough bone marrow with the requisite number of
osteoprogenitor cells is limiting.
[0005] The emerging field of regenerative medicine seeks to combine
biomaterials, growth factors, and cells as novel therapeutics to
repair damaged tissues and organs. As this specialty grows, there
is a demand for a reliable, safe, and effective source of human
adult stem cells to serve in tissue engineering applications. For
regulatory purposes, these cells must be defined by quantifiable
measures of purity. For practical purposes at the clinical level,
these cells should be available as an "off the shelf" product
immediately available upon demand at the point of care. From a
commercial standpoint, the ability to use allogeneic, as opposed to
autologous, adult stem cells for transplantation would have a
significant positive impact on product development. Under these
circumstances, a single lot of cells derived from one donor could
be transplanted to multiple mammals, reducing the costs of both
quality control and quality assurance.
[0006] Studies have demonstrated the existence of adult stem cells
in multiple tissue sites. Cells derived from bone marrow, known as
mesenchymal stem cells (MSC) or bone marrow stromal cells (BMSC),
have been extensively characterized (Castro-Malaspina et al., 1980,
Blood 56:289-30125; Piersma et al., 1985, Exp. Hematol 13:237-243;
Simmons et al., 1991, Blood 78:55-62; Beresford et al., 1992, J.
Cell. Sci. 102:341-3 51; Liesveld et al., 1989, Blood 73:1794-1800;
Liesveld et al., Exp. Hematol 19:63-70; Bennett et al., 1991, J.
Cell. Sci. 99:131-139). Recent studies have demonstrated that
allogeneic bone marrow-derived MSCs can be transplanted
(Bartholomew et al., 2002, Exp. Hematol. 30:42-8), and used to
repair a critical sized orthopedic defect in a canine model
(Arinzeh et al., 2003, J. Bone Joint Surg. Am. 85-A:1927-35).
However, MSCs represent approximately 1 out of every 10,000 to
100,000 nucleated bone marrow cells or about 200 cells per ml of
bone marrow aspirate (Bruder et al., 2000, Principles of Tissue
Engineering, 2.sup.nd Edition, Academic Press). In order to obtain
MSC numbers sufficient for tissue engineering applications, it is
necessary to expand the bone marrow-derived MSCs through multiple
passages in vitro.
[0007] In contrast to bone marrow, adipose tissue is easily
accessible for surgical harvest and abundant in the average adult
American. Recently, it has been demonstrated that adipose tissue
can serve as a source of stem cells (known as adipose derived adult
stem cells or ADAS cells). These cells are capable of
differentiating along multiple lineage pathways. In response to
specific chemicals, hormones, and/or cytokines, human and rodent
ADAS cells express biochemical and histological characteristics
consistent with adipose, bone, cartilage, muscle, and neuronal
cells. In a recent study, murine ADAS cells accelerated the repair
a critical sized calvarial defect (Cowan et al., 2004, Nat.
Biotechnol. 22:560-7).
[0008] Bone grafting is often used for the treatment of bone
conditions. Indeed, more than 1.4 million bone grafting procedures
are performed in the world annually. The success or failure of bone
grafting is dependent upon a number of factors including the
vitality of the site of the graft, the graft processing, and the
immunological compatibility of the engrafted tissue. In view of the
prevalence of bone conditions, there is a need for novel sources of
bone for therapeutic, diagnostic, and research uses. The present
invention satisfies this need.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention includes a method of enhancing the fusion of
bone following a spinal fusion procedure in a mammal comprising
administering an isolated adipose tissue-derived adult stromal
(ADAS) cell to the spine of the mammal, wherein the ADAS cell
differentiates in vivo into a cell that expresses at least one
characteristic of a bone cell.
[0010] The invention also includes a method of performing one or
more spinal fusions in a mammal comprising administering an ADAS
cell to the spine of the mammal to facilitate a single or multi
level spinal fusion. Preferably, following administration of the
ADAS into the spine of the mammal, the ADAS cell differentiates in
vivo into a cell that expresses at least one characteristic of a
bone cell.
[0011] In one aspect the ADAS cell is cultured in vitro for a
period of time without being induced to differentiate prior to the
administration of the cell to the mammal.
[0012] In another aspect, the ADAS cell is allogeneic with respect
to the mammal.
[0013] In yet another aspect, the ADAS cell induces bone formation
for intervertebral body spinal fusion.
[0014] In another aspect, the ADAS cell induces bone formation for
intertransverse process spinal fusion.
[0015] In one aspect, the ADAS cell further comprises a
biocompatible matrix. Preferably, the biocompatible matrix is
selected from the group consisting of calcium alginate, agarose,
fibrin, collagen, laminin, fibronectin, glycosaminoglycan,
hyaluronic acid, heparin sulfate, chondroitin sulfate A, dermatan
sulfate, and bone matrix gelatin.
[0016] In another aspect, the ADAS cell is genetically
modified.
[0017] In yet another aspect, the ADAS cell is administered into
one or more interbody spaces in the spine of the mammal.
[0018] In a further aspect, the spinal fusion is in a segment of
the spine selected from the group consisting of cervical, thoracic,
lumbar, lumbosacral and sacro-iliac (SI) joint.
[0019] In yet a further aspect, the ADAS cell is administered into
one or more interbody spaces by an approach selected from the group
consisting of a posterior approach, a posterolateral approach, an
anterior approach, an anterolateral approach, and a lateral
approach.
[0020] In yet another aspect, the mammal is a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0022] FIG. 1 is an image depicting a spinal fusion procedure. FIG.
1A depicts the intervertebral space in the lumbar spine. FIGS. 1B
and 1C demonstrates introduction of a mechanical device and bone
grafts to stabilize the space, respectively. FIG. 1D is an image
depicting the spine.
[0023] FIG. 2 is an image depicting the potential of ADAS cells to
differentiate along multiple lineage pathways. In response to
specific cocktails of chemicals and growth factors, human ADAS
cells can differentiate into chondrocytes, osteoblasts, adipocytes,
and neuronal- and glial-like cells in vitro.
[0024] FIG. 3 is an image depicting osteogenesis of human ADAS
cells.
[0025] FIG. 4 is a chart depicting the aldehyde phosphatase
expression in ADAS cells during adipogenic and osteogenic
differentiation.
[0026] FIG. 5 is an image demonstrating that ADAS cells form bone
in vivo.
DETAILED DESCRIPTION
[0027] The present invention encompasses methods and compositions
for treating a bone disease. In a preferred embodiment, an isolated
adipose tissue-derived adult stromal (ADAS) cell of the invention
is used to enhance the fusion of bone following a spinal fusion
procedure in a mammal.
Definitions
[0028] As used herein, each of the following terms has the meaning
associated with it in this section.
[0029] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0030] The term "about" will be understood by persons of ordinary
skill in the art and will vary to some extent on the context in
which it is used.
[0031] The term "adipose tissue-derived cell" refers to a cell that
originates from adipose tissue. The initial cell population
isolated from adipose tissue is a heterogeneous cell population
including, but not limited to stromal vascular fraction (SVF)
cells.
[0032] As used herein, the term "adipose derived stromal cells,"
"adipose tissue-derived stromal cells," "adipose tissue-derived
adult stromal (ADAS) cells," or "adipose-derived stem cells (ASCs)"
are used interchangeably and refer to stromal cells that originate
from adipose tissue which can serve as stem cell-like precursors to
a variety of different cell types such as but not limited to
adipocytes, osteocytes, chondrocytes, muscle and neuronal/glial
cell lineages. "Adipose" refers to any fat tissue. The adipose
tissue may be brown or white adipose tissue. Preferably, the
adipose tissue is subcutaneous white adipose tissue. Such cells may
comprise a primary cell culture or an immortalized cell line. The
adipose tissue may be from any organism having fat tissue.
Preferably the adipose tissue is mammalian, most preferably the
adipose tissue is human. A convenient source of human adipose
tissue is that derived from liposuction surgery. However, the
source of adipose tissue or the method of isolation of adipose
tissue is not critical to the invention.
[0033] "Allogeneic" refers to a graft derived from a different
animal of the same species.
[0034] As used herein, the term "autologous" is meant to refer to
any material derived from the same individual to which it is later
to be re-introduced into the individual.
[0035] "Xenogeneic" refers to a graft derived from a mammal of a
different species.
[0036] As used herein, the term "biocompatible lattice," is meant
to refer to a substrate that can facilitate formation into
three-dimensional structures conducive for tissue development.
Thus, for example, cells can be cultured or seeded onto such a
biocompatible lattice, such as one that includes extracellular
matrix material, synthetic polymers, cytokines, growth factors,
etc. The lattice can be molded into desired shapes for facilitating
the development of tissue types. Also, at least at an early stage
during culturing of the cells, the medium and/or substrate is
supplemented with factors (e.g., growth factors, cytokines,
extracellular matrix material, etc.) that facilitate the
development of appropriate tissue types and structures.
[0037] As used herein, the term "bone condition (or injury or
disease)" refers to disorders or diseases of the bone including,
but is not limited to, acute, chronic, metabolic and non-metabolic
conditions of the bone. The term encompasses conditions caused by
disease, trauma or failure of the tissue to develop normally.
Examples of bone conditions include, but are not limited, a bone
fracture, a bone/spinal deformation, osteosarcoma, myeloma, bone
dysplasia, scoliosis, osteoporosis, osteomalacia, rickets, fibrous
osteitis, renal bone dystrophy, and Paget's disease of bone.
[0038] "Differentiation medium" is used herein to refer to a cell
growth medium comprising an additive or a lack of an additive such
that a stem cell, adipose derived adult stromal cell or other such
progenitor cell, that is not fully differentiated when incubated in
the medium, develops into a cell with some or all of the
characteristics of a differentiated cell.
[0039] "Expandability" is used herein to refer to the capacity of a
cell to proliferate, for example, to expand in number or in the
case of a cell population to undergo population doublings.
[0040] "Graft" refers to a cell, tissue, organ or otherwise any
biological compatible lattice for transplantation.
[0041] By "growth factors" is intended the following specific
factors including, but not limited to, growth hormone,
erythropoietin, thrombopoietin, interleukin 3, interleukin 6,
interleukin 7, macrophage colony stimulating factor, c-kit
ligand/stem cell factor, osteoprotegerin ligand, insulin, insulin
like growth factors, epidermal growth factor (EGF), fibroblast
growth factor (FGF), nerve growth factor, ciliary neurotrophic
factor, platelet derived growth factor (PDGF), and bone
morphogenetic protein at concentrations of between picogram/ml to
milligram/ml levels.
[0042] As used herein, the term "growth medium" is meant to refer
to a culture medium that promotes growth of cells. A growth medium
will generally contain animal serum. In some instances, the growth
medium may not contain animal serum.
[0043] An "isolated cell" refers to a cell which has been separated
from other components and/or cells which naturally accompany the
isolated cell in a tissue or mammal.
[0044] As used herein, the term "multipotential" or
"multipotentiality" is meant to refer to the capability of a stem
cell of the central nervous system to differentiate into more than
one type of cell.
[0045] As used herein, the term "modulate" is meant to refer to any
change in biological state, i.e. increasing, decreasing, and the
like.
[0046] As used herein, the term "non-immunogenic" is meant to refer
to the discovery that ADAS cells do not induce proliferation of T
cells in an MLR. However, non-immunogenic should not be limited to
T cell proliferation in an MLR, but rather should also apply to
ADAS cells not inducing T cell proliferation in vivo.
[0047] "Proliferation" is used herein to refer to the reproduction
or multiplication of similar forms, especially of cells. That is,
proliferation encompasses production of a greater number of cells,
and can be measured by, among other things, simply counting the
numbers of cells, measuring incorporation of .sup.3H-thymidine into
the cell, and the like.
[0048] "Progression of or through the cell cycle" is used herein to
refer to the process by which a cell prepares for and/or enters
mitosis and/or meiosis. Progression through the cell cycle includes
progression through the G1 phase, the S phase, the G2 phase, and
the M-phase.
[0049] The terms "precursor cell," "progenitor cell," and "stem
cell" are used interchangeably in the art and herein and refer
either to a pluripotent, or lineage-uncommitted, progenitor cell,
which is potentially capable of an unlimited number of mitotic
divisions to either renew itself or to produce progeny cells which
will differentiate into the desired cell type. Unlike pluripotent
stem cells, lineage-committed progenitor cells are generally
considered to be incapable of giving rise to numerous cell types
that phenotypically differ from each other. Instead, progenitor
cells give rise to one or possibly two lineage-committed cell
types.
[0050] The term "stromal cell medium" as used herein, refers to a
medium useful for culturing ADAS cells. A non-limiting example of a
stromal cell medium is a medium comprising DMEM/F 12 Ham's, 10%
fetal bovine serum, 100 U penicillin/100 .mu.g streptomycin/0.25
.mu.g Fungizone. Typically, the stromal cell medium comprises a
base medium, serum and an antibiotic/antimycotic. However, ADAS
cells can be cultured with stromal cell medium without an
antibiotic/antimycotic and supplemented with at least one growth
factor. Preferably the growth factor is human epidermal growth
factor (hEGF). The preferred concentration of hEGF is about 1-50
ng/ml, more preferably the concentration is about 5 ng/ml. The
preferred base medium is DMEM/F12 (1:1). The preferred serum is
fetal bovine serum (FBS) but other sera may be used including horse
serum or human serum. Preferably up to 20% FBS will be added to the
above media in order to support the growth of stromal cells.
However, a defined medium could be used if the necessary growth
factors, cytokines, and hormones in FBS for stromal cell growth are
identified and provided at appropriate concentrations in the growth
medium. It is further recognized that additional components may be
added to the culture medium. Such components include but are not
limited to antibiotics, antimycotics, albumin, growth factors,
amino acids, and other components known to the art for the culture
of cells. Antibiotics which can be added into the medium include,
but are not limited to, penicillin and streptomycin. The
concentration of penicillin in the culture medium is about 10 to
about 200 units per ml. The concentration of streptomycin in the
culture medium is about 10 to about 200 .mu.g/ml. However, the
invention should in no way be construed to be limited to any one
medium for culturing stromal cells. Rather, any media capable of
supporting stromal cells in tissue culture may be used.
[0051] The term "pharmaceutically acceptable carrier (or medium)"
which may be used interchangeably with the term biologically
compatible carrier or medium, refers to reagents, cells, compounds,
materials, compositions, and/or dosage forms which are, within the
scope of being suitable for use in contact with the tissues of
human beings and animals without excessive toxicity, irritation,
allergic response, or other complication commensurate with a
reasonable benefit/risk ratio.
[0052] A "suitable interbody space" as the term is used herein
means the space between adjacent vertebrae where a disc resides in
a healthy spine but which is either at least partially devoid of
disc material due to wear and tear on the vertebral column or has
been prepared using techniques known in the art to surgically
create a void in the disc space.
[0053] As used herein, a "therapeutically effective amount" is the
amount of ADAS cells sufficient to provide a beneficial effect to
the subject to which the cells are administered.
[0054] "Treating (or treatment of)" refers to ameliorating the
effects of, or delaying, halting or reversing the progress of, or
delaying or preventing the onset of, a bone condition.
[0055] As used herein "endogenous" refers to any material from or
produced inside an organism, cell or system.
[0056] "Exogenous" refers to any material introduced from or
produced outside an organism, cell, or system.
[0057] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0058] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. Nucleotide sequences that encode proteins and RNA
may include introns.
[0059] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, i.e., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, i.e., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, i.e., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (i.e., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0060] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0061] The phrase "under transcriptional control" or "operatively
linked" as used herein means that the promoter is in the correct
location and orientation in relation to the polynucleotides to
control RNA polymerase initiation and expression of the
polynucleotides.
[0062] As used herein, the term "promoter/regulatory sequence"
means a nucleic acid sequence which is required for expression of a
gene product operably linked to the promoter/regulatory sequence.
In some instances, this sequence may be the core promoter sequence
and in other instances, this sequence may also include an enhancer
sequence and other regulatory elements which are required for
expression of the gene product. The promoter/regulatory sequence
may, for example, be one which expresses the gene product in a
tissue specific manner.
[0063] A "constitutive" promoter is a nucleotide sequence which,
when operably linked with a polynucleotide which encodes or
specifies a gene product, causes the gene product to be produced in
a cell under most or all physiological conditions of the cell.
[0064] An "inducible" promoter is a nucleotide sequence which, when
operably linked with a polynucleotide which encodes or specifies a
gene product, causes the gene product to be produced in a cell
substantially only when an inducer which corresponds to the
promoter is present in the cell.
[0065] A "tissue-specific" promoter is a nucleotide sequence which,
when operably linked with a polynucleotide which encodes or
specifies a gene product, causes the gene product to be produced in
a cell substantially only if the cell is a cell of the tissue type
corresponding to the promoter.
[0066] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses. Thus, the term "vector" includes an
autonomously replicating plasmid or a virus. The term should also
be construed to include non-plasmid and non-viral compounds which
facilitate transfer of nucleic acid into cells, such as, for
example, polylysine compounds, liposomes, and the like. Examples of
viral vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, and the
like.
[0067] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, such as cosmids, plasmids
(i.e., naked or contained in liposomes) and viruses that
incorporate the recombinant polynucleotide.
[0068] Description
[0069] The present invention relates to the discovery that adipose
tissue-derived adult stromal (ADAS) cells can differentiate into a
variety of different cell types including, but not limited to,
adipocytes, osteocytes, chondrocytes, muscle and neuronal/glial
cell lineages. Particularly, the invention relates to the
observation that ADAS cells can differentiate along the osteogenic
lineage in vivo.
[0070] Based on the present disclosure, an ADAS cell can be
successfully used in cell and/or gene therapy for
experimental/therapeutic purposes. For example, the cells can be
used in the treatment of bone diseases. Preferably, the cells are
used to enhance the fusion of bone following a spinal fusion
procedure. Spinal fusion is a common orthopedic and neurosurgical
procedure used to treat back pain in mammals suffering from
degenerative disc disease, spinal stenosis, scoliosis, spinal
fracture, tumor, and the like.
Isolation and Culturing of ADAS
[0071] The ADAS cells useful in the methods of the present
invention may be isolated by a variety of methods known to those
skilled in the art. For example, such methods are described in U.S.
Pat. No. 6,153,432, which is incorporated herein in its entirety.
In a preferred method, ADAS cells are isolated from a mammalian
subject, preferably a human subject. In humans, the ADAS cells are
typically isolated from liposuction material. If the cells of the
invention are to be transplanted into a human subject, it is
preferable that the ADAS cells be isolated from that same subject
so as to provide for an autologous transplant.
[0072] In another aspect of the invention, the administered ADAS
cells may be allogeneic with respect to the recipient. The
allogeneic ADAS cells are isolated from a donor that is a different
individual of the same species as the recipient. Following
isolation, the cells are cultured using the methods disclosed
herein to produce an allogeneic product. The invention also
encompasses ADAS cells that are xenogeneic with respect to the
recipient.
[0073] Without limiting the invention in anyway, stromal cells from
adipose tissue can be isolated using the methods disclosed herein.
Briefly, human adipose tissue from subcutaneous depots are removed
by liposuction surgery. The adipose tissue is then transferred from
the liposuction cup into a 500 ml sterile beaker and allowed to
settle for about 10 minutes. Precipitated blood is removed by
suction. About a 125 ml volume (or less) of the tissue is
transferred to a 250 ml centrifuge tube, and the tube is then
filled with Krebs-Ringer Buffer. The tissue and buffer are allowed
to settle for about three minutes or until a clear separation is
achieved, and then the buffer is removed by aspiration. The tissue
can be washed with Krebs-Ringer Buffer for an additional four to
five times or until the tissue becomes orange-yellow in color and
until the buffer becomes light tan in color.
[0074] The stromal cell of the adipose tissue can be dissociated
using collagenase treatment. Briefly, the buffer is removed from
the tissue and replaced with about 2 mg collagenase/ml Krebs Buffer
(Worthington, Me.) solution at a ratio of 1 ml collagenase
solution/ml tissue. The tubes are incubated in a 37.degree. C.
water bath with intermittent shaking for about 30 to 35
minutes.
[0075] Stromal cells are isolated from other components of the
adipose tissue by centrifugation for 5 minutes at 500.times.g at
room temperature. The oil and adipocyte layer are removed by
aspiration. The remaining fraction can be resuspended in
approximately 100 ml of phosphate buffered saline (PBS) by vigorous
swirling, divided into 50 ml tubes and centrifuged for five minutes
at 500.times.g. The buffer is removed by aspiration, leaving the
stromal cells. The stromal cells are then resuspended in stromal
cell medium, and plated at an appropriate cell density and
incubated at 37.degree. C. in 5% CO.sub.2 overnight. Once attached
to the tissue culture dish or flask, the cultured stromal cells can
be used immediately or maintained in culture for a period of time
or a number of passages before using the cells according to the
methods disclosed herein. However, the invention should in no way
be construed to be limited to any one method of isolating stromal
cells. Rather, any method of isolating ADAS cells should be
encompassed in the present invention.
[0076] Any medium capable of supporting fibroblasts in cell culture
may be used to culture ADAS. Media formulations that support the
growth of fibroblasts include, but are not limited to, Minimum
Essential Medium Eagle, ADC-1, LPM (bovine serum albumin-free), F10
(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and
without Fitton-Jackson Modification), Basal Medium Eagle (BME-with
the addition of Earle's salt base), Dulbecco's Modified Eagle
Medium (DMEM-without serum), Yamane, IMEM-20, Glasgow Modification
Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium,
Medium M199 (M199E-with Earle's salt base), Medium M199 (M199H-with
Hank's salt base), Minimum Essential Medium Eagle (MEM-E-with
Earle's salt base), Minimum Essential Medium Eagle (MEM-H-with
Hank's salt base) and Minimum Essential Medium Eagle (MEM-NAA with
non-essential amino acids), and the like. A preferred medium for
culturing ADAS is DMEM, more preferably DMEM/F12 (1:1).
[0077] Additional non-limiting examples of media useful in the
methods of the invention can contain fetal serum of bovine or other
species at a concentration at least 1% to about 30%, preferably at
least about 5% to 15%, most preferably about 10%. Embryonic extract
of chicken or other species can be present at a concentration of
about 1% to 30%, preferably at least about 5% to 15%, most
preferably about 10%.
[0078] Following isolation, ADAS cells are incubated in stromal
cell medium in a culture apparatus for a period of time or until
the cells reach confluency before passing the cells to another
culture apparatus. The culturing apparatus can be of any culture
apparatus commonly used in culturing cells in vitro. Preferably,
the level of confluence is greater than 70% before passing the
cells to another culture apparatus. More preferably, the level of
confluence is greater than 90%. A period of time can be any time
suitable for the culture of cells in vitro. Stromal cell medium may
be replaced during the culture of the ADAS cells at any time.
Preferably, the stromal cell medium is replaced every 3 to 4 days.
ADAS cells are then harvested from the culture apparatus whereupon
the ADAS cells can be used immediately or cryopreserved to be
stored for use at a later time. ADAS cells may be harvested by
trypsinization, EDTA treatment, or any other procedure used to
harvest cells from a culture apparatus.
[0079] Various terms are used to describe cells in culture. Cell
culture refers generally to cells taken from a living organism and
grown under controlled condition. A primary cell culture is a
culture of cells, tissues or organs taken directly from an organism
and before the first subculture. Cells are expanded in culture when
they are placed in a growth medium under conditions that facilitate
cell growth and/or division, resulting in a larger population of
the cells. When cells are expanded in culture, the rate of cell
proliferation is typically measured by the amount of time required
for the cells to double in number, otherwise known as the doubling
time.
[0080] Each round of subculturing is referred to as a passage. When
cells are subcultured, they are referred to as having been
passaged. A specific population of cells, or a cell line, is
sometimes referred to or characterized by the number of times it
has been passaged. For example, a cultured cell population that has
been passaged ten times may be referred to as a P10 culture. The
primary culture, i.e., the first culture following the isolation of
cells from tissue, is designated P0. Following the first
subculture, the cells are described as a secondary culture (P1 or
passage 1). After the second subculture, the cells become a
tertiary culture (P2 or passage 2), and so on. It will be
understood by those of skill in the art that there may be many
population doublings during the period of passaging; therefore the
number of population doublings of a culture is greater than the
passage number. The expansion of cells (i.e., the number of
population doublings) during the period between passaging depends
on many factors, including but is not limited to the seeding
density, substrate, medium, and time between passaging.
Genetic Modification
[0081] The cells of the present invention can also be used to
express a foreign protein or molecule for a therapeutic purpose or
in a method of tracking the assimilation of the cell and/or its
differentiation in the recipient. Thus, the invention encompasses
expression vectors and methods for the introduction of exogenous
DNA into ADAS cells with concomitant expression of the exogenous
DNA in the ADAS cells. Methods for introducing and expressing DNA
in a cell are well known to the skilled artisan and include those
described, for example, in Sambrook et al. (2001, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York), and in Ausubel et al. (1997, Current Protocols in Molecular
Biology, John Wiley & Sons, New York).
[0082] The isolated nucleic acid can encode a molecule used to
track the migration, assimilation, and survival of ADAS cells once
they are introduced in the recipient. Proteins useful for tracking
a cell include, but are not limited to, green fluorescent protein
(GFP), any of the other fluorescent proteins (e.g., enhanced green,
cyan, yellow, blue and red fluorescent proteins; Clontech, Palo
Alto, Calif.), or other tag proteins (e.g., LacZ, FLAG-tag, Myc,
His.sub.6, and the like).
[0083] Tracking the migration, assimilation and/or differentiation
of an ADAS cell of the present invention is not limited to the use
of detectable molecules expressed by a vector or virus. The
migration, assimilation, and/or differentiation of a cell can also
be assessed using a series of probes that facilitate localization
of transplanted ADAS cells within a mammal. Tracking an ADAS cell
transplant may further be accomplished using antibodies or nucleic
acid probes for cell-specific markers detailed elsewhere
herein.
[0084] The term "genetic modification" as used herein refers to the
stable or transient alteration of the genotype of an ADAS cell by
intentional introduction of exogenous DNA. DNA may be synthetic, or
naturally derived, and may contain genes, portions of genes, or
other useful DNA sequences. The term "genetic modification" as used
herein is not meant to include naturally occurring alterations such
as that which occurs through natural viral activity, natural
genetic recombination, or the like.
[0085] Exogenous DNA may be introduced to an ADAS cell using viral
vectors (retrovirus, modified herpes viral, herpes-viral,
adenovirus, adeno-associated virus, lentiviral, and the like) or by
direct DNA transfection (lipofection, calcium phosphate
transfection, DEAE-dextran, electroporation, and the like).
[0086] When the purpose of genetic modification of the cell is for
the production of a biologically active substance, the substance
will generally be one that is useful for the treatment of a given
disorder. For example, it may be desired to genetically modify
cells so that they secrete a certain growth factor product
associated with bone formation.
[0087] The cells of the present invention can be genetically
modified by having exogenous genetic material introduced into the
cells, to produce a molecule such as a trophic factor, a growth
factor, a cytokine, and the like, which is beneficial to culturing
the cells. In addition, by having the cells genetically modified to
produce such a molecule, the cell can provide an additional
therapeutic effect to the mammal when transplanted into a mammal in
need thereof. For example, the genetically modified cell can
secrete a molecule that is beneficial neighboring cells in the
mammal.
[0088] As used herein, the term "growth factor product" refers to a
protein, peptide, mitogen, or other molecule having a growth,
proliferative, differentiative, or trophic effect on a cell. For
example, growth factor products useful in the treatment of bone
disorders include, but are not limited to, FGF, TGF-.beta.,
insulin-like growth factor, and bone morphogenetic protein.
[0089] According to the present invention, gene constructs which
comprise nucleotide sequences that encode heterologous proteins are
introduced into the ADAS cells. That is, the cells are genetically
altered to introduce a gene whose expression has therapeutic effect
in the mammal. According to some aspects of the invention, ADAS
cells from the mammal to be treated or from another mammal, may be
genetically altered to replace a defective gene and/or to introduce
a gene whose expression has therapeutic effect in the mammal being
treated.
[0090] In all cases in which a gene construct is transfected into a
cell, the heterologous gene is operably linked to regulatory
sequences required to achieve expression of the gene in the cell.
Such regulatory sequences typically include a promoter and a
polyadenylation signal.
[0091] The gene construct is preferably provided as an expression
vector that includes the coding sequence for a heterologous protein
operably linked to essential regulatory sequences such that when
the vector is transfected into the cell, the coding sequence will
be expressed by the cell. The coding sequence is operably linked to
the regulatory elements necessary for expression of that sequence
in the cells. The nucleotide sequence that encodes the protein may
be cDNA, genomic DNA, synthesized DNA or a hybrid thereof or an RNA
molecule such as mRNA.
[0092] The gene construct includes the nucleotide sequence encoding
the beneficial protein operably linked to the regulatory elements
and may remain present in the cell as a functioning cytoplasmic
molecule, a functioning episomal molecule or it may integrate into
the cell's chromosomal DNA. Exogenous genetic material may be
introduced into cells where it remains as separate genetic material
in the form of a plasmid. Alternatively, linear DNA which can
integrate into the chromosome may be introduced into the cell. When
introducing DNA into the cell, reagents which promote DNA
integration into chromosomes may be added. DNA sequences which are
useful to promote integration may also be included in the DNA
molecule. Alternatively, RNA may be introduced into the cell.
[0093] The regulatory elements for gene expression include: a
promoter, an initiation codon, a stop codon, and a polyadenylation
signal. It is preferred that these elements be operable in the
cells of the present invention. Moreover, it is preferred that
these elements be operably linked to the nucleotide sequence that
encodes the protein such that the nucleotide sequence can be
expressed in the cells and thus the protein can be produced.
Initiation codons and stop codons are generally considered to be
part of a nucleotide sequence that encodes the protein. However, it
is preferred that these elements are functional in the cells.
Similarly, promoters and polyadenylation signals used must be
functional within the cells of the present invention. Examples of
promoters useful to practice the present invention include but are
not limited to promoters that are active in many cells such as the
cytomegalovirus promoter, SV40 promoters and retroviral promoters.
Other examples of promoters useful to practice the present
invention include but are not limited to tissue-specific promoters,
i.e. promoters that function in some tissues but not in others;
also, promoters of genes normally expressed in the cells with or
without specific or general enhancer sequences. In some
embodiments, promoters are used which constitutively express genes
in the cells with or without enhancer sequences. Enhancer sequences
are provided in such embodiments when appropriate or desirable.
[0094] The cells of the present invention can be transfected using
well known techniques readily available to those having ordinary
skill in the art. Exogenous genes may be introduced into the cells
using standard methods where the cell expresses the protein encoded
by the gene. In some embodiments, cells are transfected by calcium
phosphate precipitation transfection, DEAE dextran transfection,
electroporation, microinjection, liposome-mediated transfer,
chemical-mediated transfer, ligand mediated transfer or recombinant
viral vector transfer.
[0095] In some embodiments, recombinant adenovirus vectors are used
to introduce DNA with desired sequences into the cell. In some
embodiments, recombinant retrovirus vectors are used to introduce
DNA with desired sequences into the cells. In some embodiments,
standard CaPO.sub.4, DEAE dextran or lipid carrier mediated
transfection techniques are employed to incorporate desired DNA
into dividing cells. Standard antibiotic resistance selection
techniques can be used to identify and select transfected cells. In
some embodiments, DNA is introduced directly into cells by
microinjection. Similarly, well-known electroporation or particle
bombardment techniques can be used to introduce foreign DNA into
the cells. A second gene is usually co-transfected or linked to the
therapeutic gene. The second gene is frequently a selectable
antibiotic-resistance gene. Transfected cells can be selected by
growing the cells in an antibiotic that will kill cells that do not
take up the selectable gene. In most cases where the two genes are
unlinked and co-transfected, the cells that survive the antibiotic
treatment have both genes in them and express both of them.
[0096] It should be understood that the methods described herein
may be carried out in a number of ways and with various
modifications and permutations thereof that are well known in the
art. It may also be appreciated that any theories set forth as to
modes of action or interactions between cell types should not be
construed as limiting this invention in any manner, but are
presented such that the methods of the invention can be more fully
understood.
Therapeutic Use of ADAS Cells
[0097] In addition to the fact that ADAS cells can differentiate
along different cell lineages, the invention also relates to the
discovery that ADAS cells lack immunogenic characteristics with
respect to inducing proliferation of T cells. This characteristic
is an indication that there is a reduced likelihood of an immune
rejection by the recipient's immune cells. In addition, ADAS cells
have been shown not to stimulate allogeneic PBMCs in a mixed
lymphocyte reaction.
[0098] In some embodiments of the invention, it may not be
necessary or desirable to immunosuppress a mammal prior to
initiation of cell/gene therapy with ADAS cells. Accordingly,
transplantation with allogeneic, or even xenogeneic, ADAS cells is
included in the invention.
[0099] The use of ADAS cells for the treatment of a disease,
disorder, or a condition of the bone provides an additional
advantage in that the ADAS cells can be introduced into a recipient
without the requirement of an immunosuppressive agent. Successful
transplantation of a cell is believed to require the permanent
engraftment of the donor cell without inducing a graft rejection
immune response generated by the recipient. Typically, in order to
prevent a graft rejection response, nonspecific immunosuppressive
agents such as cyclosporine, methotrexate, steroids and FK506 are
used. These agents are administered on a daily basis and if
administration is stopped, graft rejection usually results.
However, an undesirable consequence in using nonspecific
immunosuppressive agents is that they function by suppressing all
aspects of the immune response (general immune suppression),
thereby greatly increasing a recipient's susceptibility to
infection and other diseases.
[0100] The present invention provides a method of treating a
disease, disorder, or a condition of the bone by introducing
undifferentiated or differentiated ADAS cells into the recipient
without the requirement of immunosuppressive agents. There is
therefore a reduced susceptibility for the recipient of the
transplanted ADAS cell to incur infection and other diseases,
including cancer relating conditions that is associated with
immunosuppression therapy.
[0101] The present invention includes the administration of an
allogeneic or a xenogeneic ADAS cell, or otherwise an ADAS cell
that is genetically disparate from the recipient, into a recipient
to provide a benefit to the recipient. The present invention
provides a method of using ADAS cells to treat a disease, disorder
or condition of the bone without the requirement of using
immunosuppressive agents when administering the cells to a
recipient.
[0102] In a further embodiment, the ADAS cell used in the present
invention can be isolated, from adipose tissue of any species of
mammal, including but is not limited to, human, mouse, rat, ape,
gibbon, bovine. Preferably, the ADAS cell is isolated from a human,
a mouse, or a rat. More preferably, the ADAS cell is isolated from
a human.
[0103] The ADAS cell may be administered to a mammal following a
period of in vitro culturing. The ADAS cell may be cultured in a
manner that induces the ADAS cell to differentiate in vitro.
However, it is preferred that the ADAS cell is implanted into the
recipient in an undifferentiated state and that the implanted ADAS
cell differentiates to express at least one characteristic of a
bone cell in vivo.
[0104] The ADAS cells of this invention can be transplanted into a
mammal using techniques known in the art such as i.e., those
described in U.S. Pat. Nos. 5,082,670 and 5,618,531, each
incorporated herein by reference, or into any other suitable site
in the body. Transplantation of the cells of the present invention
can be accomplished using techniques well known in the art as well
as those described herein or as developed in the future. The
present invention comprises a method for transplanting, grafting,
infusing, or otherwise introducing the cells into a mammal,
preferably, a human.
[0105] The number of ADAS cells administered to a mammal may be
related to, for example, the cell yield after adipose tissue
processing. A portion of the total number of cells may be retained
for later use or cyropreserved. In addition, the dose delivered
depends on the route of delivery of the cells to the mammal.
[0106] The dosage of the ADAS cells varies within wide limits and
may be adjusted to the individual requirements in each particular
case. The number of cells used depends on the weight and condition
of the recipient, the number and/or frequency of administrations,
and other variables known to those of skill in the art. This number
can be adjusted by orders of magnitude to achieve the desired
therapeutic effect.
[0107] Between about 10.sup.5 and about 10.sup.13 ADAS cells per
100 kg body weight can be administered to the individual. In some
embodiments, between about 1.5.times.10.sup.6 and about
1.5.times.10.sup.12 cells are administered per 100 kg body weight.
In some embodiments, between about 1.times.10.sup.9 and about
5.times.10.sup.11 cells are administered per 100 kg body weight. In
other embodiments, between about 4.times.10.sup.9 and about
2.times.10.sup.11 cells are administered per 100 kg body weight. In
yet other embodiments, between about 5.times.10.sup.8 cells and
about 1.times.10.sup.10 cells are administered per 100 kg body
weight.
[0108] ADAS cells can be suspended in an appropriate diluent, at a
concentration of from about 0.01 to about 5.times.10.sup.6
cells/ml. Suitable excipients for injection solutions are those
that are biologically and physiologically compatible with the ADAS
cells and with the recipient, such as buffered saline solution or
other suitable excipients. The composition for administration can
be formulated, produced and stored according to standard methods
complying with proper sterility and stability.
[0109] The cells may also be encapsulated and used to deliver
biologically active molecules, according to known encapsulation
technologies, including microencapsulation (see, e.g., U.S. Pat.
Nos. 4,352,883; 4,353,888; and 5,084,350, herein incorporated by
reference), or macroencapsulation (see, e.g., U.S. Pat. Nos.
5,284,761; 5,158,881; 4,976,859; and 4,968,733; and International
Publication Nos. WO 92/19195; WO 95/05452, all of which are
incorporated herein by reference). For macroencapsulation, cell
number in the devices can be varied; preferably, each device
contains between 10.sup.3-10.sup.9 cells, most preferably, about
10.sup.5 to 10.sup.7 cells. Several macroencapsulation devices may
be implanted in the mammal. Methods for the macroencapsulation and
implantation of cells are well known in the art and are described
in, for example, U.S. Pat. No. 6,498,018.
[0110] The mode of administration of the cells of the invention to
the mammal may vary depending on several factors including the type
of disease being treated, the age of the mammal, whether the cells
are differentiated or not, whether the cells have heterologous DNA
introduced therein, and the like. The cells may be introduced to
the desired site by direct injection, or by any other means used in
the art for the introduction of compounds administered to a mammal
suffering from a particular disease or disorder of the bone.
[0111] The ADAS cells can be administered into a host in a wide
variety of ways. Modes of administration include, but are not
limited to, intravascular, intracerebral, parenteral,
intraperitoneal, intravenous, epidural, intraspinal, intrastemal,
intra-articular, intra-synovial, intrathecal, intra-arterial,
intracardiac, or intramuscular. Preferably, the cells are used in
spinal fusion procedures.
Composition
[0112] The invention also provides a matrix for implantation into a
mammal, wherein the matrix comprises an ADAS cell of the invention.
The matrix can also include, but is not limited to, an ADAS cell,
an ADAS cell lysate, an ADAS cell conditioned medium, and an
extracellular matrix produced by an ADAS cell.
[0113] The matrix may also contain or be treated with one or more
bioactive factor including, but not limited to an anti-apoptotic
agent (i.e., erythropoietin, thrombopoietin, insulin-like growth
factor I and insulin-like growth factor II, hepatocyte growth
factor, caspase inhibitors); an anti-inflammatory agent (i.e., p38
MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1
inhibitors, and non-steroidal anti-inflammatory drugs); an
immunosupressive/immunomodulatory agent; an mTOR inhibitor; an
anti-proliferative agent; a corticosteroid (i.e., prednisolone,
hydrocortisone); an anti-thrombogenic agent; and an anti-oxidant.
The presence of a bioactive factor can contribute to the
proliferation and/or differentiation of the ADAS cells.
[0114] The invention further provides in some aspects methods of
regenerating bone tissue in a mammal in need thereof by
administering a composition comprising an ADAS cell, a matrix, an
ADAS cell lysate, an ADAS-product of the invention (i.e. molecules
secreted by the ADAS cell), or any combination thereof in a mammal.
As such, the invention encompasses a pharmaceutical composition,
wherein the composition may be used in the treatment of a bone
condition. For example, the bone condition includes, but is not
limited to, a bone fracture, a bone/spinal deformation,
osteosarcoma, myeloma, bone dysplasia, scoliosis, osteoporosis,
osteomalacia, rickets, fibrous osteitis, renal bone dystrophy, and
Paget's disease of bone. Preferably, the invention provides
compositions and methods for enhancing fusion of bone following a
spinal fusion procedure.
[0115] In a non-limiting embodiment, a formulation comprising the
cells of the invention is prepared for administration directly to
the site where the production of new bone tissue is desired. For
example, the cells of the invention may be suspended in a hydrogel
solution for injection. Alternatively, the hydrogel solution
containing the cells may be allowed to harden, for instance in a
mold, to form a matrix having cells dispersed therein prior to
implantation, or once the matrix has hardened, the cell formations
may be cultured so that the cells are mitotically expanded prior to
implantation. The hydrogel is an organic polymer (natural or
synthetic) which is cross-linked via covalent, ionic, or hydrogen
bonds to create a three-dimensional open-lattice structure which
entraps water molecules to form a gel. Examples of materials which
can be used to form a hydrogel include polysaccharides such as
alginate and salts thereof, peptides, polyphosphazines, and
polyacrylates, which are crosslinked ionically, or block polymers
such as polyethylene oxide-polypropylene glycol block copolymers
which are crosslinked by temperature or pH, respectively.
[0116] In some embodiments, the polymers are at least partially
soluble in aqueous solutions, such as water, buffered salt
solutions, or aqueous alcohol solutions, that have charged side
groups, or a monovalent ionic salt thereof. Examples of polymers
with acidic side groups that can be reacted with cations are
poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids),
copolymers of acrylic acid and methacrylic acid, poly(vinyl
acetate), and sulfonated polymers, such as sulfonated polystyrene.
Copolymers having acidic side groups formed by reaction of acrylic
or methacrylic acid and vinyl ether monomers or polymers can also
be used. Examples of acidic groups are carboxylic acid groups,
sulfonic acid groups, halogenated (preferably fluorinated) alcohol
groups, phenolic OH groups, and acidic OH groups.
[0117] Examples of polymers with basic side groups that can be
reacted with anions are poly(vinyl amines), poly(vinyl pyridine),
poly(vinyl imidazole), and some imino substituted polyphosphazenes.
The ammonium or quaternary salt of the polymers can also be formed
from the backbone nitrogens or pendant imino groups. Examples of
basic side groups are amino and imino groups.
[0118] Other examples of polymers include, but are not limited to
poly-alpha-hydroxy esters, polydioxanone, propylene fumarate,
poly-ethylene glycol, poly-erthoesters, polyanhydrides and
polyurethanes, poly-L-lactic acid, poly-glycolic acid, and
poly-lactic-co-glycolic acid.
[0119] Transplantation of ADAS Cells Using Scaffolds
[0120] The cells of the invention can be seeded onto or into a
three-dimensional scaffold and implanted in vivo, where the seeded
cells proliferate on the framework and form a replacement tissue in
vivo in cooperation with the cells of the mammal.
[0121] In some aspects of the invention, the scaffold comprises
extracellular matrix, cell lysate (e.g., soluble cell fractions),
or combinations thereof, of the ADAS cells. In some embodiments,
the scaffold comprises an extracellular matrix protein secreted by
the cells of the invention. Alternatively, the extracellular matrix
is an exogenous material selected from the group consisting of
calcium alginate, agarose, fibrin, collagen, laminin, fibronectin,
glycosaminoglycan, hyaluronic acid, heparin sulfate, chondroitin
sulfate A, dermatan sulfate, and bone matrix gelatin. In some
aspects, the matrix comprises natural or synthetic polymers.
[0122] The invention includes biocompatible scaffolds, lattices,
self-assembling structures and the like, whether biodegradable or
not, liquid or solid. Such Scaffolds are known in the arts of
cell-based therapy, surgical repair, tissue engineering, and wound
healing. Preferably the scaffolds are pretreated (e.g., seeded,
inoculated, contacted with) with the cells, extracellular matrix,
conditioned medium, cell lysate, or combination thereof. In some
aspects of the invention, the cells adhere to the scaffold. The
seeded scaffold can be introduced into a mammal's body in anyway
known in the art, including but not limited to implantation,
injection, surgical attachment, transplantation with other tissue,
injection, and the like. The scaffold of the invention may be
configured to the shape and/or size of a tissue or organ in vivo.
For example, but not by way of limitation, the scaffold may be
designed such that the scaffold structure supports the seeded cells
without subsequent degradation; supports the cells from the time of
seeding until the tissue transplant is remodeled by the host
tissue; and allows the seeded cells to attach, proliferate, and
develop into a tissue structure having sufficient mechanical
integrity to support itself.
[0123] Scaffolds of the invention can be administered in
combination with any one or more growth factors, cells, drugs or
other components described elsewhere herein that stimulate tissue
formation or otherwise enhance or improve the practice of the
invention. The ADAS cells to be seeded onto the scaffolds may be
genetically engineered to express growth factors or drugs.
[0124] In another preferred embodiment, the cells of the invention
are seeded onto a scaffold where the material exhibits specified
physical properties of porosity and biomechanical strength to mimic
the features of true bone, thereby promoting stability of the final
structure and access and egress of metabolites and cellular
nutrients. That is, the material should provide structural support
and can form a scaffolding into which host vascularization and cell
migration can occur. In the preferred embodiment, ADAS cells are
first mixed with a carrier material before application to a
scaffold. Suitable carriers include, but are not limited to,
calcium alginate, agarose, types I, II, IV or other collagen
isoform, fibrin, poly-lactic/poly-glycolic acid, hyaluronate
derivatives, gelatin, laminin, fibronectin, starch,
polysaccharides, saccharides, proteoglycans, synthetic polymers,
calcium phosphate, and ceramics (i.e. hydroxyapatite, tricalcium
phosphate).
[0125] The external surfaces of the three-dimensional framework may
be modified to improve the attachment or growth of cells and
differentiation of tissue, such as by plasma coating the framework
or addition of one or more proteins (e.g., collagens, elastic
fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g.,
heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate,
dermatan sulfate, keratin sulfate), a cellular matrix, and/or other
materials such as, but not limited to, gelatin, alginates, agar,
and agarose.
[0126] In some embodiments, it is important to re-create in culture
the cellular microenvironment found in vivo, such that the extent
to which the cells of the invention are grown prior to implantation
in vivo. In addition, growth factors, osteogenic inducing agents,
and angiogenic factors may be added to the culture medium prior to,
during, or subsequent to inoculation of the cells to trigger
differentiation and tissue formation by the ADAS cells following
implantation into the mammal.
Therapeutic Applications of ADAS Cells
[0127] The present invention encompasses methods for administering
an ADAS cell to a mammal, including a human, in order to treat a
disease where the introduction of the ADAS cells provide a
therapeutic relief. The cells of the invention may be administered
alone or as admixtures with other cells and/or a bioactive factor
as discussed elsewhere herein. A cell that may be administered in
conjunction with ADAS cells of the invention include, but is not
limited to, other multipotent or pluripotent cells, an osteocyte,
an osteoblast, an osteoclast, a bone lining cell, a stem cell, and
a bone marrow cell. The different types of cells may be admixed
with the ADAS cells immediately or shortly prior to administration
to a mammal, or they may be co-cultured together for a period of
time prior to administration to a mammal.
[0128] The skilled artisan will readily understand that ADAS cells
can be transplanted into a mammal whereby upon receiving signals
and cues from the surrounding milieu, the cells differentiate into
mature cells in vivo dictated by the neighboring cellular milieu.
Preferably, the ADAS cells differentiate into a cell that exhibits
at least one characteristic of a bone cell. Alternatively, the ADAS
cells can be differentiated in vitro into a desired cell type and
the differentiated cell can be administered to a mammal in need
thereof.
[0129] The invention also encompasses grafting ADAS cells in
combination with other therapeutic procedures to treat diseases of
the bone. Preferably, the cells are useful in enhancing fusion of
bone following a spinal fusion procedure. ADAS cells can be
co-grafted with other cells, both genetically modified and
non-genetically modified cells which exert beneficial effects on
the mammal. Therefore the methods disclosed herein can be combined
with other therapeutic procedures as would be understood by one
skilled in the art once armed with the teachings provided
herein.
[0130] The ADAS cells may be administered with other beneficial
drugs or biological molecules (growth factors, trophic factors).
When the ADAS cells are administered with other agents, they may be
administered together in a single pharmaceutical composition, or in
separate pharmaceutical compositions, simultaneously or
sequentially with the other agents (either before or after
administration of the other agents). Bioactive factors which may be
co-administered include, but are not limited to, an anti-apoptotic
agent (i.e., erythropoietin, thrombopoietin, insulin-like growth
factor I and insulin-like growth factor II, hepatocyte growth
factor, caspase inhibitors); an anti-inflammatory agent (i.e., p38
MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1
inhibitors, and non-steroidal anti-inflammatory drugs); an
immunosupressive/immunomodulatory agent; an mTOR inhibitor; an
anti-proliferative (i.e., azathioprine, mycophenolate mofetil); a
corticosteroid (i.e., prednisolone, hydrocortisone); an
anti-thrombogenic agent; and an anti-oxidant.
[0131] The invention encompasses administering ADAS cells to a
mammal as undifferentiated cells, i.e., as cultured in growth
medium. Alternatively, ADAS cells may be administered following
exposure in culture to conditions that stimulate differentiation
toward a desired phenotype, for example, an osteogenic
phenotype.
[0132] The cells of the invention may be surgically implanted,
injected, delivered (e.g., by way of a catheter or syringe), or
otherwise administered directly or indirectly to the site in need
of repair or augmentation. The cells may be administered by way of
a matrix (e.g., a three-dimensional scaffold). The cells may be
administered with conventional pharmaceutically acceptable
carriers. Routes of administration of the cells of the invention or
components (e.g., extracellular matrix, cell lysate, conditioned
medium) thereof include intramuscular, ophthalmic, parenteral
(including intravenous), intraarterial, subcutaneous, oral, and
nasal administration. Particular routes of parenteral
administration include, but are not limited to, intramuscular,
subcutaneous, intraperitoneal, intracerebral, intraventricular,
intracerebroventricular, intrathecal, intracisternal, intraspinal
and/or peri-spinal routes of administration. Preferably, the cells
are used in spinal fusion procedures.
[0133] The cells of the invention can be introduced alone or in
admixture with a composition useful in the repair of bone wounds
and defects. Such compositions include, but are not limited to bone
morphogenetic proteins, hydroxyapatite/tricalcium phosphate
particles (HA/TCP), gelatin, poly-L-lysine, and collagen. For
example, the cells of the invention may be combined with
demineralized bone matrix (DBM) or other matrices to make the
composite osteogenic (bone forming in it own right) as well as
osteoinductive.
[0134] To enhance the differentiation, survival or activity of
implanted cells, additional bioactive factors as discussed
elsewhere herein may be added. For example, a bioactive factor can
include, but is not limited to bone morphogenetic protein, vascular
endothelial growth factor, fibroblast growth factors, and other
cytokines that have either osteoconductive and/or osteoinductive
capacity. To enhance vascularization and survival of transplanted
bone tissue, angiogenic factors such as VEGF, PDGF or bFGF can be
added either alone or in combination with endothelial cells or
their precursors.
[0135] Alternatively, ADAS cells to be transplanted may be
genetically engineered to express such growth factors,
antioxidants, antiapoptotic agents, anti-inflammatory agents, or
angiogenic factors.
Pharmaceutical Compositions
[0136] Also encompassed within the scope of the invention are ADAS
cell-products, including but not limited to extracellular matrices
secreted by the ADAS cells themselves, cell lysates (e.g., soluble
cell fractions) of ADAS cells, and ADAS cell-conditioned medium. As
such, in terms of administering a composition comprising an ADAS
cell, the invention includes a pharmaceutical composition
comprising at least one of the following: an ADAS cell, an
extracellular matrix produced thereby, a cellular lysate thereof,
or an ADAS-conditioned medium. The pharmaceutical composition of
the invention preferably includes a pharmaceutically acceptable
carrier or excipient. The pharmaceutical composition is preferably
used for treating bone conditions as defined herein.
[0137] Pharmaceutical compositions of the invention may comprise
homogeneous or heterogeneous populations of ADAS cells,
extracellular matrix or cell lysate thereof, or conditioned medium
thereof in a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers for the cells of the invention include organic
or inorganic carrier substances suitable which do not deleteriously
react with the cells of the invention or compositions or components
thereof. To the extent they are biocompatible, suitable
pharmaceutically acceptable carriers include water, salt solution
(such as Ringer's solution), alcohols, oils, gelatins, and
carbohydrates, such as lactose, amylose, or starch, fatty acid
esters, hydroxymethylcellulose, and polyvinyl pyrolidine. Such
preparations can be sterilized, and if desired, mixed with
auxiliary agents such as lubricants, preservatives, stabilizers,
wetting agents, emulsifiers, salts for influencing osmotic
pressure, buffers, and coloring. Pharmaceutical carriers suitable
for use in the present invention are known in the art and are
described, for example, in Pharmaceutical Sciences (17.sup.th Ed.,
Mack Pub. Co., Easton, Pa.) and WO 96/05309, each of which are
incorporated by reference herein.
[0138] As another example but not by way of limitation, the cells
of the invention may be administered alone, in a pharmaceutically
acceptable carrier, or seeded on or in a matrix as described
elsewhere herein, can be used to repair or replace damaged or
destroyed bone tissue, to augment existing bone tissue, to
introduce new or altered tissue, or to modify artificial
prostheses.
[0139] When cells are administered in semi-solid or solid devices,
surgical implantation into a precise location in the body is
typically a suitable means of administration. In the case where the
cells are administered in the form of a liquid or fluid
pharmaceutical composition, the cells may be administered to a more
general location (i.e. throughout a diffusely affected area), from
which they migrate to a particular location (i.e. by responding to
chemical signals).
[0140] Other embodiments encompass methods of treatment by
administering pharmaceutical compositions comprising ADAS cellular
components (e.g., cell lysates or components thereof) or products
(e.g., extracellular matrix, trophic and other biological factors
produced naturally by ADAS cells or through genetic modification,
conditioned medium from ADAS culture). Again, these methods may
further comprise administering other active agents as disclosed
elsewhere herein.
[0141] The ADAS cells may also be applied with additives to
enhance, control, or otherwise direct the intended therapeutic
effect. Similarly, the cells may be applied with a biocompatible
matrix which facilitates in vivo tissue engineering by supporting
and/or directing the fate of the implanted cells.
[0142] Prior to the administration of the ADAS cells into a mammal,
the cells may be stably or transiently transfected or transduced
with a nucleic acid of interest using a plasmid, viral or
alternative vector strategy. The cells may be administered
following genetic manipulation such that they express gene products
that intended to promote the therapeutic response(s) provided by
the cells.
[0143] ADAS cells of the invention may be used to treat mammals
requiring the repair or replacement of bone tissue resulting from
disease or trauma or failure of the tissue to develop normally.
Treatment may entail the use of the cells of the invention to
produce new bone tissue. For example, the undifferentiated or
osteogenic differentiation-induced cells of the invention may be
used to treat bone conditions, including metabolic and
non-metabolic bone diseases. Examples of a bone condition includes,
but is not limited, a bone fracture, a bone/spinal deformation,
osteosarcoma, myeloma, bone dysplasia, scoliosis, osteoporosis,
osteomalacia, rickets, fibrous osteitis, renal bone dystrophy, and
Paget's disease of bone.
Spinal Fusion
[0144] As set forth herein, back pain remains a major public health
problem, especially among aged people. Persistent and severe back
pain often causes debility and disability. This pain is closely
associated with intervertebral disc abnormalities of the spine.
Based on the present disclosure, degenerated discs may be treated
by restoring the damaged tissues within the disc. The ADAS cells of
the invention may be used to stimulate bone development and thereby
restore the intervertebral discs at various stages of
degeneration.
[0145] However, it is often necessary to remove at least a portion
of the damaged and/or malfunctioning back component. For example,
when a disc becomes ruptured, a discectomy surgical procedure can
be performed to remove the ruptured disc and to fuse the two
vertebrae between the removed disc together. Spinal fusion is a
process by which two or more of the vertebrae that make up the
spinal column are fused together with bone grafts and internal
devices (such as rods) that heal into a single solid bone. Spinal
fusion can eliminate unnatural motion between the vertebrae and, in
turn, reduce pressure on nerve endings. In addition, spinal fusion
can be used to treat, for example, injuries to spinal vertebrae
caused by trauma; protrusion and degeneration of the cushioning
disc between vertebrae (sometimes called slipped disc or herniated
disc); abnormal curvatures (such as scoliosis or kyphosis); and
weak or unstable spine caused by infections or tumors. The present
invention encompasses compositions and methods for improving the
success rates of spinal fusion procedures.
[0146] Since the ADAS cells of the present invention have been
shown to form bone in vivo, the ADAS cells may be used in the place
of bone grafts conventionally used in spinal fusion surgeries.
Specifically, the ADAS cells may be used to stimulate bone
formation between two adjacent vertebrae (within the vertebral
body), as well as between adjacent transverse processes (within the
intertransverse process spaces on either side of the spine).
[0147] ADAS cells of the present invention have numerous
applications in the treatment of spine disorders, including
promoting the production of proteoglycan rich matrix in
intervertebral disc repair, producing bone for the intervertebral
body in intertransverse process spinal fusion, and producing bone
for long bone fracture healing. The present invention is based on
the discovery that ADAS cells can be used to facilitate bone fusion
in a spinal fusion procedure.
[0148] When a disc becomes ruptured, a discectomy surgical
procedure can be performed to remove the ruptured disc and to fuse
the two vertebrae between the removed disc together. Details
regarding typical implementations of methods for fusing vertebrae
are disclosed in U.S. Pat. Nos. 6,033,438 and 5,015,247, the
contents of which are incorporated in their entireties herein by
reference.
[0149] Disc degeneration is commonly treated with a segment fusion,
whereby both the anterior and posterior spine elements of the
interbody space are fused. It is important to consider the
mechanical stresses place on the anterior and posterior elements
when considering a fusion technique. The anterior motion segment
elements (vertebral bodies and disc) bear approximately 80% of the
compressive force at that given level in the spine. The posterior
1/3 of the vertebral body and disc represent the center point for
axial compression in the spine. These mechanics are critical for
assessing what type of fusion will have the best clinical outcome
for a given pathology. The invention provides a spinal fusion
procedure in which ADAS cells are used as a source of a bone
substitute.
[0150] In one embodiment, the invention includes methods for
performing one or more spinal fusions in a mammal comprising
introducing an effective amount of ADAS cells into one or more
suitable interbody spaces in the mammal by injection the cells
through a syringe, catheter, or cannula to facilitate single, or
multi level spinal fusion. The ADAS cells are to set under
physiological conditions, i.e., in vivo, over time where the cells
differentiate and form bone. The presence of the ADAS cells enhance
the fusion of bone in a spinal fusion procedure.
[0151] In another embodiment, the invention includes methods for
performing one or more spinal fusion on a mammal comprising placing
in the posterior portion of at least one suitable interbody space a
metallic implant selected from rods and pedicle screws or plates
and pedicle screws by attachment thereof to adjacent vertebrae;
injecting into the anterior portion of the interbody space an
effective amount of ADAS cells; and allowing the ADAS cells to
differentiate into a cell that exhibits at least one characteristic
of a bone cell and thereby form bone in vivo.
[0152] The invention includes methods for performing spinal fusions
by using either an anterior, posterior, or posterolateral approach
to the interbody space. The posterolateral approach (unilateral or
bilateral) reduces surgical morbidity over an anterior approach,
but caution is required while working around the cauda equina and
exiting nerve roots in the spinal canal. Posterior access and
visualization of the interbody space is more limited than with the
anterior approach, but many spinal surgeons are trained in how to
deal with those circumstances.
[0153] As discussed elsewhere herein, the ADAS cells can also
comprise an amount of one or more active bioactive agents suitable
to promote bone growth, such as a growth factor, a bone morphology
protein, or a pharmaceutical carrier therefor.
[0154] One mechanism by which the ADAS cells may provide a
therapeutic or structural benefit is by incorporating themselves or
their progeny into newly generated, existing or repaired tissues or
tissue components. For example, ADAS cells and/or their progeny may
incorporate into newly generated bone other structural or
functional tissue and thereby cause or contribute to a therapeutic
or structural improvement. Another mechanism by which the ADAS
cells may provide a therapeutic or structural benefit is by
expressing and/or secreting molecules, e.g., growth factors, that
promote creation, retention, restoration, and/or regeneration of
structure or function of a given tissue or tissue component.
[0155] The ADAS cells may also be used in combination with other
cells or devices such as synthetic or biologic scaffolds, materials
or devices that deliver factors, drugs, chemicals or other agents
that modify or enhance the relevant characteristics of the cells as
further described herein.
[0156] In accordance with the invention disclosed herein, the ADAS
cells can be delivered to the mammal soon after harvesting the
adipose tissue from the mammal. For example, the cells may be
administered immediately after processing of the adipose tissue and
obtaining a composition of ADAS cells. Ultimately, the timing of
delivery will depend upon mammal availability and the processing
time required to process the adipose tissue. In another embodiment,
the timing for delivery may be relatively longer if the cells to be
re-infused to the mammal are subject to additional modification,
purification, stimulation, or other manipulation, as discussed
herein. The number of cells administered to a mammal may be related
to, for example, the cell yield after adipose tissue processing. A
portion of the total number of cells may be retained for later use
or cyropreserved.
[0157] The following examples further illustrate aspects of the
present invention. However, they are in no way a limitation of the
teachings or disclosure of the present invention as set forth
herein.
EXAMPLES
[0158] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only, and the invention is not limited to these
Examples, but rather encompasses all variations which are evident
as a result of the teachings provided herein.
[0159] The following experiments were performed to determine the
role of ADAS cells on the outcome of a spinal fusion procedure. For
example the effect of syngeneic and allogeneic ADAS cells on spinal
fusion procedures. The results herein demonstrate that ADAS cells
are osteogenic and contribute to improvement in spinal fusion.
Based on the present disclosure, ADAS cells can be used to treat
mammals including, but are not limited to, trauma victims,
osteoporotic mammals lacking suitable numbers of osteogenic cells,
and mammals with non-union fractures.
Example 1
Alternatives to Autograft Bone in Spinal Fusion Surgery
[0160] Over 75% of the American population suffers from back pain.
In some instances, underlying medical illnesses can contribute to
back pain. These include scoliosis, spinal stenosis, degenerative
disc disease, infectious processes, tumors, and trauma. For 1% of
the population, the back pain is so severe that they are forced to
go onto lifetime disability; an additional 1% of the population is
incapacitated by back pain for a limited time period. The majority
of the mammals with back pain are treated with conservative
therapies; however, when modalities such as bed rest and medication
fail, physicians often recommend spinal fusion surgery. The goal of
this operation is to form ectopic bone between two or more adjacent
vertebra, "fusing" them together into a solid structure.
Immobilization of the vertebral joint reduces the pressure on nerve
roots leaving the spinal cord and the resulting painful sensation.
Surgeons use a poster lateral approach to the lumbar spine,
introducing a bone graft or osteoinductive material with a
mechanical support between two vertebral bodies to form ectopic
bone (FIG. 1).
[0161] Without wishing to be bound by any particular theory, it is
believed that the "ideal" graft material for a spinal fusion would
provide the following properties: mechanical support (material
stabilizes the spine/surgical site during the recovery period);
osteoconductive (material facilitates the ingrowth and integration
of adjacent bone upon itself); osteoinductive (material recruits
and stimulates the formation and growth of bone from cells that may
not naturally do so); and osteogenic (material contains cells that
themselves are capable of forming new bone).
[0162] At present, the "gold standard" for spinal fusion repair is
autologous bone, usually harvested from the iliac crest of the
individual. Surgeons transplant the mammal's own bone to the site
of need. Nevertheless, this is far from a perfect solution. In 30%
of mammals, the donor site becomes infected, bruised, fractured, or
painful following the surgery. Indeed, when a mammal requires
autograft bone for multiple spinal fusions, the iliac crest may not
provide sufficient material. The underlying health of the mammal
further influences the outcome of spinal fusion surgery. Mammals
with osteoporosis or vascular insufficiency due to diabetes,
smoking, or age display reduced new bone formation and non-union at
the site of the spinal fusion (Whang et al., 2003, Spine J.
3:155-65). Thus, there is a need for alternatives to autograft bone
in spinal fusion surgery.
[0163] While there are alternative materials available, all face a
common limitation; none display osteogenic capability. Allograft
bone from cadavers can be sterilized, stored, and used in the
operating room as needed. These materials can be pre-shaped for
specific use or powdered, allowing them to be applied as a paste at
the surgical site; however, allografts can cause inflammation,
elicit an immune response, and have been an infectious source in a
limited number of cases (Whang et al., 2003, Spine J. 3:155-65).
Because allograft bone is sterilized, it no longer contains viable
native bone forming cells (osteoblasts, osteocytes) and lacks
osteogenic properties. In clinical trials, allograft bone is
inferior to autograft bone in multilevel spinal fusions (Whang et
al., 2003, Spine J. 3:155-65). Ceramic materials, such as
hydroxyapatite and tricalcium phosphate (HA/TCP), are
osteoconductive and promote new bone formation. However, they lack
osteogenic and osteoinductive properties, limiting their utility.
While osteoinductive growth factors such as bone morphogenetic
proteins (BMP) are available commercially (Infuse.TM. from
Sofamor/Medtronics), they require the presence of osteogenic cells
within the spinal fusion site to promote new bone formation (Whang
et al., 2003, Spine J. 3:155-65). The development of an osteogenic
cell has the potential to improve the outcome with any of these
alternative spinal fusion materials.
[0164] A number of animal species have served as models in
pre-clinical spinal fusion trials. These include the rat, rabbit,
dog, sheep, goat, and non-human primate (Khan et al., 2004,
Biomaterials 25:1475-85; Liebschner et al., 2004, Biomaterials
25:1697-714; Sandhu et al., 2001, Eur. Spine J. 10:S122-31). Of
these, the rat (Boden et al., 1998, Spine 23:2486-92; Cui et al.,
2001, Spine 26:2305-10; Wang et al., 2003, J. Bone Joint Surg. Am.
85-A:905-11) and rabbit (Khan et al., 2004, Biomaterials
25:1475-85; Kruyt et al., 2004, Biomaterials 25:1463-73) have been
used for "proof of concept" studies due to the animal's size and
cost. In each species, surgeons can use a postero lateral approach
to the lumbar spine, similar to that used to treat other mammals.
The rabbit is more commonly used for spinal fusion feasibility
studies (Khan et al., 2004, Biomaterials 25:1475-85), in part due
to the animal's size and the confirmed observation that the rate of
spinal fusion in the rabbit is similar to that observed in a human.
Nevertheless, the rabbit presents certain disadvantages compared to
the rat model. Unlike rats, where well-characterized inbred strains
are available, laboratory rabbits do not display syngeneic or
congenic haplotypes. Thus, it may not be possible to routinely
transplant cells from one rabbit to the other without the risk of
rejection. The rat poster lateral spinal fusion model has been
employed successfully to demonstrate the osteoinductive effect of
bone morphogenetic protein 7 when presented in a collagen scaffold
(Salamon et al., 2003, J. Spinal Disord. Tech. 16:90-5). Several
groups have used the rat model successfully to evaluate the
osteogenic effect of bone marrow stromal cells on spinal fusion
(Boden et al., 1998, Spine 23:2486-92; Cui et al., 2001, Spine
26:2305-10; Wang et al., 2003, J. Bone Joint Surg. Am.
85-A:905-11). They achieved statistically significant improvements
in spinal fusion within 4 to 9 weeks following implantation of bone
marrow stromal cells compared to scaffold alone. Each study used
cohorts of n=4 to 8 animals.
[0165] The following experiments are designed to assess the role of
ADAS cells in spinal fusion procedures.
Isolation of ADAS Cells
[0166] Subcutaneous adipose tissue is harvested from male Fischer
rats (8 to 10 weeks of age, n=25, yielding approximately 3 grams
tissue per rat). ADAS cells are prepared according to published
methodologies (Aust et al., 2004, Cytotherapy 6:7-14; Halvorsen et
al., 2001, Metabolism 50:407-413; Sen et al., 2001, Journal of
Cellular Biochemistry 81:312-319). Breifly, adipose tissue is
minced, washed, and suspended in an equal volume of phosphate
buffered saline containing 1% bovine serum albumin and 0.1%
collagenase type I (Worthington Biochemical, Lakewood N.J.).
Following a 60-minute digestion at 37.degree. C. with agitation (50
rpm), the suspension is centrifuged at 1200 rpm for 5 minutes at
room temperature and the stromal vascular fraction cells pelleted.
The stromal vascular cells are plated at a density of 0.1 grams of
tissue digest per cm.sup.2 in "Stromal Media" (DMEM/F-12 Ham's
Media supplemented with 10% fetal bovine serum (Hyclone, Logan
Utah) and 1% antibiotic/antimycotic. The cells are incubated for 3
to 6 days in a humidified 5% CO.sub.2 incubator until they reach
75% confluency. This yields approximately 25-30.times.10.sup.4
cells/cm.sup.2. At that time, ADAS cells are harvested by digestion
with trypsin/EDTA and passaged at a plating density of
5.times.10.sup.3 cells/cm.sup.2. Cells are expanded for up to 2
passages to obtain >60 million cells (Table 1). Cells are
evaluated in vitro for their osteogenic and adipogenic capacity
using standard assays over a 1 to 3 week inductive period as
described in (Halvorsen et al., 2001, Tissue Eng. 7:729-41; Hicok
et al., 2004, Tissue Engineering 10:371-380). Cells can be
cryopreserved in liquid nitrogen prior to use.
[0167] To track the cells histologically, the cells are labeled
during the initial passage with a retroviral vector carrying the
LacZ gene expressing .beta.-galactosidase to provide a trackable
marker. Stable integration of retroviral vectors reduces the risk
that the marker will be lost during the time of implantation. This
method has been used routinely to track implanted cells.
TABLE-US-00001 TABLE 1 Estimated ADAS Cell Yield and Expansion
Passage Initial Passage Second Passage ADAS Cells/gm 2.5 .times.
10.sup.5 1.25 .times. 10.sup.6 ADAS Cells/Adipose 18.75 .times.
10.sup.6 93.75 .times. 10.sup.6 tissue from 25 rats (.about.75
gm)
Example 2
ADAS Cell Osteogenesis In Vitro
[0168] It has been demonstrated that human ADAS cells display an
osteogenic phenotype in vitro when cultured in the presence of
ascorbate, .beta.-glycerophosphate, dexamethasone, and 1,25
dihydroxyvitamin D.sub.3 (Halvorsen et al., 2001, Tissue Eng.
7:729-41). Under these conditions, the ADAS cells mineralize their
extracellular matrix as demonstrated by positive staining with
either Alizarin Red or von Kossa for calcium phosphate deposition
(FIG. 3).
[0169] It was observed that human ADAS cell osteogenesis over a
10-day period was accompanied by an increase in alkaline
phosphatase activity. At the end of the culture period, osteogenic
cells (mineralized) displayed a 3-fold higher level of alkaline
phosphatase relative to cells maintained under control conditions
(FIG. 4). Likewise, there was a time dependent increase in secreted
levels of osteocalcin protein under osteogenic conditions. The ADAS
cells expressed a number of gene markers consistent with an
osteoblast phenotype, including osteocalcin, osteopontin, bone
morphogenetic proteins (BMP) 2 and 4, and the BMP receptors IA, IB,
and II.
[0170] It has also been demonstrated that ADAS cells have the
potential to differentiate along multiple lineage pathways. In
response to specific cocktails of chemicals and growth factors,
ADAS cells can differentiate into chondrocytes, osteoblasts,
adipocytes, and neuronal- and glial-like cells in vitro (FIG.
2).
Example 3
ADAS Cells are Osteogenic In Vivo
[0171] To extend the in vitro findings, human ADAS cells were
transplanted into immunodeficient SCID mice. The ADAS cells were
loaded onto three cm.sup.3 cubes of hydroxyapatite/tricalcium
phosphate (HA/TCP) scaffold and implanted subcutaneously. After a
6-week period, the implants were harvested, fixed, decalcified, and
stained with Hematoxylin/Eosin or with human nuclear antigen
specific antibodies (FIG. 5). Based on H&E staining, it was
observed that new bone formed adjacent to the
hydroxyapatite/tricalcium phosphate scaffold in the presence of the
human ADAS cells. The human cells were identified within the bone
based on immunofluorescent analysis with the human antigen specific
antibody. In the presence of the scaffold alone (no cells), new
bone did not form and no human cells were detected. These studies
demonstrate that ADAS cells are capable of osteogenesis in
vivo.
Example 4
ADAS Cells can be Transplanted Allogeneically
[0172] The following experiments serve to provide proof of concept
regarding the allogeneic transplantation of ADAS cells in the
spinal fusion model. It has been demonstrated that ADAS cells fail
to elicit a proliferative response from allogeneic lymphocytes in a
mixed lymphocyte reaction. Without wishing to be bound by any
particular theory, it is believed that ADAS cells release a factor
that inhibits the lymphocyte's immune response to allogeneic
antigens. The presence of ADAS cells prolonged skin graft survival
in the baboon model, and therefore indicates that adult stem cells
can be transplanted allogeneically for tissue engineering
applications.
[0173] Using a canine model, a critical sized segmental defect in
the femoral diaphysis of dogs can be created. The defects can be
repaired with hydroxyapatite/tricalcium phosphate scaffolds alone
or in combination with either autologous or allogeneic ADAS cells;
the allogeneic cells are mismatched for both the HLA-1 and HLA-2
antigens (Table 2). The transplant recipients do not receive any
immunosuppressive therapy. The animals are sacrificed 16 week
later, and the degree of bone repair observed in the presence of
ADAS cells can be compared with transplant of scaffold alone (no
ADAS cells). Without wishing to be bound by any particular theory,
it is believed that there will be no observable significant
difference between the repair obtained with autologous versus
allogeneic ADAS cells, nor will there be evidence of any immune
response to the allogeneic cells. These experiments serve to
demonstrate the fact that allogeneic transplantation of adult stem
cells in a tissue engineered construct is feasible, and in some
instances does not require immunosuppressive therapy.
TABLE-US-00002 TABLE 2 Histomorphometric Analysis of Bone and
Ceramic in Canine Segmental Defects** Percent Implant Type Ceramic
Percent Bone Allogeneic MSC-ceramic implants (n = 4) 35 .+-. 3% 49
.+-. 12%.sup.# Autologous MSC-ceramic implants (n = 6) 33 .+-. 5%
42 .+-. 5%.sup.# Cell free ceramic implants 30 .+-. 6% 25 .+-. 12%
**Percent Ceramic was the percentage of the implant total area
occupied by the ceramic, and the Percent Bone was the percentage of
the porous space occupied by bone. Values are given as the mean
.+-. standard deviation. .sup.#Compared to the cell free implants,
the difference was significant (p < 0.05) (Arinzeh et al., 2003,
J. Bone Joint Surg. Am. 85-A: 1927-35).
Example 5
Syngeneic ADAS Cells on Spinal Fusion
[0174] The following experiments serve to address the hypothesis
that ADAS cells are osteogenic in vivo and, in combination with a
suitable biomaterial carrier, can improve and accelerate spinal
fusion in animal models. Table 3 summarizes the experimental
design. The initial studies are conducted with syngeneic ADAS cells
(cells from the same strain of rat), to mimic the conditions
existing in a human autologous cell transplant. By removing issues
relating to immune response and rejection, these experiments focus
on the osteogenic capacity of ADAS cells for spinal fusion. These
experiments using rat as a spinal fusion model are patterned to
methods known in the art (Boden et al., 1995, Spine 20:412-20; Wang
et al., 2003, J. Bone Joint Surg. Am. 85-A:905-11; Cui et al.,
2001, Spine 26:2305-10; Sandhu et al., 2001, Eur. Spine J. 10
Suppl. 2:S122-31; Wang et al., 2003, Spine J. 3:155-65).
Surgical Procedure and Euthanasia
[0175] A single level intertransverse spinal arthrodesis (L4-L5) on
96 female Fischer rats are performed as described by Cui (Cui et
al., 2001, Spine 26:2305-10). Animals are anesthetized with
ketamine (80 mg/kg) and xylazine (7 mg/kg), shaved, draped, and
their skin disinfected with Betadine and 70% ethanol. A midline
posterior longitudinal incision is made from L3 to L5. The
periosteum is raised along the spinous processes and lamina to the
lateral aspect of the facets. The facets are removed using a
rongeur and the wound is irrigated with saline solution. Animals
are randomized into cohorts of n=32. Cohort A receives no
treatment. Cohort B receives the implantation of
hydroxyapatite/tricalcium phosphate (40 mg) alone into the fusion
bed. Cohort C receives implantation of hydroxyapatite/tricalcium
phosphate (40 mg) in combination with 2.times.10.sup.6 ADAS cells
derived from the subcutaneous adipose tissue of Fischer rats
(syngeneic cells) into the fusion bed. Following the placement of
the implant, the deep fascia and skin incisions are closed. Animals
receiving buprenorphine hydrochloride (0.1 mg/kg) for
post-operative analgesia are monitored for recovery of mobility and
function for up to 24 hours following the procedure. Groups of 16
animals from each Cohort are sacrificed by CO.sub.2 asphyxiation 6
and 12 weeks after the surgical procedure. At that time, serum
specimens and the lumbar spine are collected for analysis.
Radiographic Follow-Up
[0176] Animals are subjected to posteroanterior and lateral
radiographs of the lumbosacral spine following surgery and at 6
week intervals following surgery. The radiographic analysis serve
to detect ectopic bone formation and callus formation in the lumbar
spine at the surgical site. Micro computerized tomography
(micro-CT) are performed on the dissected specimens following
sacrifice. The structure and volume of new bone formation can be
determined using methods known in the art (Mankani et al., 2004,
Radiology 230:369-76).
Manual Palpation of Spinal Fusion
[0177] At the time of sacrificing the animals, the L3-L5 lumbar
spine are dissected from the animals. The specimens are palpated
for extension and flexion at L3-4 and L4-5. The specimens are
graded for the presence or absence of any motion. Those specimens
with motion in any direction receive a score of "0" while those
without motion in any dimension are considered "fused" with a score
of "1" (Cui et al., 2001, Spine 26:2305-10; Grauer et al., 2004,
Spine J. 4:281-6).
Biomechanical Testing of Spinal Fusion
[0178] Before testing, all muscle are cleared and the
intervertebral disc at L4-5 are divided so that only the fusion
mass is connecting the two vertebrae. Steel k-wire (3.2 mm) pins
are placed in an antero-posterior direction into the vertebral
bodies. Uniaxial tensile testing are performed at a displacement
rate of 0.5 cm/minute with the load applied through the k-wire.
Displacements are measured by extensometers and the loads measured
by a load cell. The peak load to failure is measured from computer
generated load displacement plot. Stiffness is determined as the
slope of the line between two points (at 50% and 75% load to
failure) on the load displacement curve. The adjacent segment at
L3-4 is tested in a similar manner.
Histological Analysis
[0179] The lumbar spine specimens (n=8 at each time point for each
Cohort) is fixed in formalin for 48 hours, decalcified in 0.25 M
ethylenediaminetetraacetic acid in phosphate buffered saline for 2
weeks at 4.degree. C., and incubated for 16 hours in a solution of
X-gal (1 mg/ml) at 37.degree. C. The specimen is paraffin embedded,
sectioned transversely (5 .mu.m), and stained with hematoxylin and
eosin. Ten sections from each specimen are analyzed using the
Medivue (Nikon) software to quantify the mean percentage
(.+-.standard deviation) of each implant occupied by ectopic
bone.
[0180] Without wishing to be bound by any particular theory, it is
believed that ADAS cells are successful for spinal fusion if the
following outcomes are achieved: 1) minimal evidence of fusion
(manual manipulation fusion score of 0 in 90% of animals, no
radiographic evidence of ectopic bone, and less than 5% of the area
of the surgical site occupied by bone matrix in 10 sections per
specimen based on histology and CT analysis) in Cohort A (no
treatment) at the 6 and 12 week time points is observed; 2)
detection of the HA/TCP scaffold in histological analysis of
animals in Cohorts B and C at the 6 and 12 week time points; 3)
minimal evidence of fusion (manual manipulation fusion score of 0
in 90% of animals, no radiographic evidence of ectopic bone, and
less than 5% of the area of the surgical site occupied by bone
matrix in 10 sections per specimen based on histology and CT
analysis) in Cohort B (HA/TCP alone) at the 6 and 12 week time
points; 4) detection of transplanted ADAS cells in Cohorts C for 6
weeks following surgery based on .beta.-galactosidase enzyme
activity or immunodetection on histological analysis; and 5)
superior spinal fusion in the presence of ADAS cells (Cohorts C)
(manual manipulation fusion score of "1" in 90% of animals,
radiographic evidence of ectopic bone at the surgical site, and
greater than 30% of the area of the HA/TCP implant occupied by bone
matrix in 10 sections per specimen based on histology and by CT
analysis) relative to scaffold alone (Cohort B) or empty lesion
(Cohort A) controls at the 6 and 12 week time points.
[0181] The experiments set forth in this Example serve to address
the utility of ADAS cells to accelerate and improve lumbar spinal
fusion in a rat model. TABLE-US-00003 TABLE 3 Outline of
Experimental Design Cohort A B C No Rx Scaffold Only Scaffold +
Syngeneic Cells Intertransverse N = 96 Fischer rats Spinal L4-L5
Arthrodesis Implants N = 32 N = 32 N = 32 HA-TCP - + + scaffold
Fischer ADAS - - + cells (2 .times. 10.sup.6) Euthanize at 6 N = 16
N = 16 N = 16 weeks Euthanize at 12 N = 16 N = 16 N = 16 weeks In
vivo Micro CT analysis, X-ray analysis, manual determination
analyses of spinal fusion (blinded analysis, 2 independent
observers) In vitro Histology (H&E) on decalcified tissue,
biomechanical analyses testing.
Example 6
Allogeneic ADAS Cells on Spinal Fusion
[0182] The following experiments serve to address the hypothesis
that ADAS cells can be transplanted allogeneically with a
biomaterial scaffold to achieve a superior spinal fusion as
compared to a biomaterial scaffold alone. Table 4 summarizes the
experimental design. It has been shown that it is possible to
transplant bone marrow derived MSCs to repair bone defects without
evidence of significant immune rejection (Arinzeh et al., 2003, J.
Bone Joint Surg. Am. 85-A:1927-35). The experiments disclosed
herein demonstrate the utility of allogeneic ADAS cells in a lumbar
spinal fusion model.
[0183] Fischer and ACI inbred rat strains are selected for the
following experiments based on previous studies in the literature
(Akahane et al., 1999, J. Bone Miner Res. 14:561-8; Yoshikawa et
al., 2000, J. Bone Miner Res. 15:1147-57). These animals display a
histocompatibility antigen mismatch and reject osteogenic tissue
transplants unless given immunosuppressive therapy (Akahane et al.,
1999, J. Bone Miner Res. 14:561-8; Yoshikawa et al., 2000, J. Bone
Miner Res. 15:1147-57).
[0184] Isolated allogeneic ADAS cells from ACI rats are used for
implantation into Fischer rat lumbar spinal fusion. The experiments
herein can be conducted in parallel with the experiments relating
to syngeneic autologous ADAS cells, thereby allowing for
comparative analysis. The absence or presence of an immune response
to the allogeneic ADAS cells can be assessed based on one-way mixed
lymphocyte reactions and flow cytometric analysis of serum samples
obtained from the Cohorts. TABLE-US-00004 TABLE 4 Outline of
Experimental Design Cohort A B D No Rx Scaffold Only Scaffold +
Allogeneic Cells Intertransverse N = 96 Fischer rats Spinal L4-
L5Arthrodesis Implants N = 32 N = 32 N = 32 HA/TCP - + + ACI ADAS -
- - cells (2 .times. 10.sup.6) Euthanize at 6 N = 16 N = 16 N = 16
weeks Euthanize at 12 N = 16 N = 16 N = 16 weeks In vivo Micro CT
analysis, X-ray analysis, manual determination analyses of spinal
fusion (blinded analysis, 2 independent observers) In vitro
Histology (H&E) on decalcified tissue, biomechanical analyses
testing, one-way mixed lymphocyte reaction, flow cytometric
analysis of serum samples (to include Cohort C).
[0185] Subcutaneous adipose tissue are harvested from male ACI rats
(8 to 10 weeks of age, n=25, yielding approximately 3 grams tissue
per rat) as discussed elsewhere herein. The number of cells
obtained with each passage follows the estimates outlined in Table
1. The ADAS cells from the ACI rats are subjected to the same in
vitro analyses as those employed for the Fischer rat ADAS cells.
The surgical and follow up procedures (i.e. radiographic follow-up,
manual palpation of spinal fusion) are performed as described
elsewhere herein. The HA/TCP implants can contain about
2.times.10.sup.6 cells in a 100 .mu.l volume.
[0186] For histological analysis, the lumbar spine specimens are
fixed in formalin for 48 hours, decalcified in 0.25 M
ethylenediaminetetraacetic acid in phosphate buffered saline for 2
weeks at 4.degree. C., and incubated for 16 hours in a solution of
X-gal (1 mg/ml) at 37.degree. C. The specimen are paraffin
embedded, sectioned transversely (5 .mu.m), and stained with
hematoxylin and eosin. Ten sections from each specimen are analyzed
using the Medivue (Nikon) software to quantify the mean percentage
(.+-.standard deviation) of each implant occupied by ectopic bone.
Sections are evaluated for the presence or absence of infiltrating
lymphocytes. Without wishing to be bound by any particular theory,
an antibody against the pan-hematopoietic antibody (anti-CD45) can
be used to immunohistochemical staining the cells to identify any
immune cells within or around the implants. The number of
infiltrating lymphocytes can be determined in 10 sections per
specimen and quantified using the Medivue software program.
Serum Immune Response
[0187] Serum antibody binding to the ACI strain ADAS cells is
evaluated by flow cytometry. ADAS cells from ACI rats are quickly
thawed from liquid nitrogen storage and placed in culture for 5
days to facilitate maximum viability and surface antigen
expression. The cells are harvested by trypsinization, washed in
staining buffer (1.times.DPBS, 5% FBS, 0.5% BSA, 0.1% Sodium
Azide), and resuspended at 5.times.10.sup.6 cells/ml. 90 .mu.l of
cells (5.times.10.sup.5 cells) are aliquotted into 2 ml Eppendorf
tubes. 10 .mu.l of undiluted rat serum, or serum diluted 1:10 in
staining buffer, is added to each tube to give effective dilutions
of serum that are 1:10 and 1:100. All tubes are incubated on ice
for 30 minutes, washed with wash buffer (1.times.DPBS, 0.5% BSA and
0.1% sodium azide), and then resuspended in 100 .mu.l of staining
buffer. Goat anti-rat (IgG/IgM) FITC secondary antibody is added to
all tubes at a final dilution of 1:100. Control tubes receive ACI
ADAS cells with secondary antibody only (negative control), or with
a positive control Fischer anti-ACI rat serum that is produce by
repeated immunization of Fischer rats with ACI ADAS cells. The
suspensions are incubated in the dark on ice for 15 minutes and
washed twice with wash buffer as discussed elsewhere herein. The
cells are then fixed in 200 .mu.l of 1% paraformaldehyde and
allowed to incubate on ice, in fixative for at least 15 minutes
prior to acquisition. 20,000 events are acquired for flow cytometry
analysis. Results are expressed as the percentage of ACI cells
stained with the secondary antibody based on increased mean
fluorescent intensity relative to the secondary antibody alone
negative control.
One-Way Mixed Lymphocyte Reaction (MLR)
[0188] This assay is based on the following rationale: if T cells
are primed in vivo to ACI alloantigens, they will respond to
restimulation in vitro at a faster kinetic rate. Recipient rat T
cell activation to allogeneic ACI strain ADAS cells can be
evaluated by the MLR assay. MLR assays are performed on individual
rats using pooled mesenteric plus cervical LN cells as responder
cells. Eight animals per group from 3 groups: No Treatment (Cohort
A); scaffold only (Cohort B); scaffold+allogeneic cells (Cohort D)
are assessed (Table 5). The assay is set up by culturing the
responder cells in medium, with irradiated (5000R) syngeneic
Fischer spleen stimulator cells, or with irradiated allogeneic ACI
spleen stimulator cells. The T cell proliferation in response to
medium or to syngeneic spleen cells represents background
responses; the syngeneic response is typically subtracted from the
response to allogeneic cells to assess true proliferation. As
positive and negative controls, assays are set up with irradiated
allogeneic ACI (positive) and syngeneic Fischer (negative)
lymphocytes; their expression of both allogeneic versus syngeneic
HLA 1 and 2 antigens insure either a robust proliferative response
by the responder, Fischer derived lymphocytes, or no response. The
MLR assay are performed in 96 well plates using triplicate wells
per treatment. Responder cells are plated at 4.times.10.sup.5
cells/well and spleen cell stimulators are plated at
1.times.10.sup.5 cells/well. The culture medium used is Iscove's
Modified Dulbecco's Medium plus 10% FBS (Hyclone) supplemented with
non-essential amino acids, sodium pyruvate, 2-mercaptoethanol, and
antibiotics/antimycotics. Replicate culture plates are prepared for
harvesting on days 3 and 7 of culture. Cultures are pulsed on days
2 or 6 with .sup.3H-thymidine (1 .mu.Ci/well) and the cells are
harvested approximately 16 hours later for scintillation counting.
Results are reported as counts per minute (cpm) which reflect the
degree of T cell proliferation in the culture wells. TABLE-US-00005
TABLE 5 One-Way Mixed Lymphocyte Reaction Responder Cells
Stimulator Cells Cohort A - Medium Alone Syngeneic Allogeneic Lymph
Node Fischer Splenic ACI Splenic Cell Cells Cells Cohort B - Lymph
Node Cell Cohort C - Lymph Node Cell
[0189] Without wishing to be bound by any particular theory, it is
believed that allogeneic ADAS cells are successful for spinal
fusion if the following outcomes are achieved: minimal evidence of
fusion (manual manipulation fusion score of 0 in 90% of animals, no
radiographic evidence of ectopic bone, and less than 5% of the area
of the surgical site occupied by bone matrix in 10 sections per
specimen based on histology and CT analysis) in Cohort A (no
treatment) at the 6 and 12 week time points; detection of HA/TCP
scaffold in histologic analysis of all animals in Cohorts B and D
at 6 and 12 week time points; minimal evidence of fusion (manual
manipulation fusion score of 0 in 90% of animals, no radiographic
evidence of ectopic bone, and less than 5% of the area of the
surgical site occupied by bone matrix in 10 sections per specimen
based on histology and radiographic analysis) in Cohort B (HA/TCP
alone) at the 6 and 12 week time points; minimal evidence of fusion
(manual manipulation fusion score of 0 in 90% of animals, no
radiographic evidence of ectopic bone, and less than 5% of the area
of the surgical site occupied by bone matrix in 10 sections per
specimen based on histology and CT analysis) in Cohort B (HA/TCP
alone) at the 6 and 12 week time points; detection of transplanted
ADAS cells in Cohorts D for up to 6 weeks following surgery based
on .beta.-galactosidase enzyme activity on histologic analysis;
superior spinal fusion in the presence of ADAS cells (Cohorts D)
(manual manipulation fusion score of "1" in 90% of animals,
radiographic evidence of ectopic bone at the surgical site, and
greater than 30% of the area of the HA/TCP implant occupied by bone
matrix in 10 sections per specimen based on histology and by CT
analysis) relative to scaffold alone (Cohort B) or empty lesion
(Cohort A) controls at the 6 and 12 week time points; less than a
1.5-fold increase in the level of anti-ADAS antibodies in Cohorts C
and D (ADAS cell implants) relative to Cohorts A and B (no cell
treatment); and no evidence of enhanced responder cell
proliferation stimulated by allogeneic derived spleen cells as
compared to medium alone or syngeneic derived spleen cells in the
one-way mixed lymphocyte reaction when comparing Cohorts A, B and
D. The mixed lymphocyte reaction positive controls will display at
proliferative response of at least 10,000 cpm.
Example 7
Compare and Contrast the Relative Effectiveness of Syngeneic and
Allogeneic ADAS Cells in a Spinal Fusion Model
[0190] The disclosure presented herein provides data allowing for
the determination of whether allogeneic (HLA mismatched) and
syngeneic (HLA compatible) ADAS cells display equal function in
achieving a spinal fusion. The experimental design is summarized in
Table 6. Without wishing to be bound by any particular theory, it
is believed that the two cell populations are equivalent, based on
the previous studies that achieved a successful repair of a
critical sized bone defect in dogs using allogeneic MSCs (Arinzeh
et al., 2003, J. Bone Joint Surg. Am. 85-A:1927-35). The comparison
of syngeneic and allogeneic ADAS cells provides significant medical
and commercial implications. The disclosure presented herein
provides for the use of allogeneic ADAS cells for tissue
regeneration therapy. TABLE-US-00006 TABLE 6 Comparison of Spinal
Fusion with Allogeneic vs. Syngeneic ADAS Cells Parameter at 6
& 12 wks Syngeneic (Cohort C) Allogeneic (Cohort D) Percentage
of implant N = 8 per time point N = 8 per time point composed of
bone based on histological analysis Manual manipulation N = 16 per
time point N = 16 per time point fusion scores Radiographic
measures N = 16 per time point N = 16 per time point of fusion
Biomechanical testing of N = 8 per time point N = 8 per time point
fusion
[0191] Without wishing to be bound by any particular theory, it is
believed that allogeneic ADAS cells are comparable to syngeneic
ADAS cells for success in spinal fusion if all the parameters in
Table 6 do not show a statistically significant difference between
the allogeneic and syngeneic ADAS cell Cohorts (p>0.05,
preferably p>0.30).
[0192] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0193] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
* * * * *