U.S. patent application number 11/980967 was filed with the patent office on 2009-07-09 for method for in vivo, ex vivo and in vitro repair and regeneration of cartilage and collagen and bone remodeling.
This patent application is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Dennis R. Carter, David J. Schurman, R. Lane Smith.
Application Number | 20090176304 11/980967 |
Document ID | / |
Family ID | 24717364 |
Filed Date | 2009-07-09 |
United States Patent
Application |
20090176304 |
Kind Code |
A1 |
Smith; R. Lane ; et
al. |
July 9, 2009 |
Method for in vivo, ex vivo and in vitro repair and regeneration of
cartilage and collagen and bone remodeling
Abstract
A method for in vivo, ex vivo and in vitro regeneration of
cartilage and collagen. In vivo, ex vivo and in vitro regeneration
and de novo formation of articular cartilage and collagen by
intermittently applied hydrostatic pressure. The application of
external interval loading consisting of repeated periods of applied
hydrostatic pressure followed and interrupted by periods of
recovery. The application of the intermittent hydrostatic pressure
at physiological levels 5-10 MPA for an interval of 4 hours
followed by a recovery period up to about 20 hours, said pressure
applied to the cartilage cells in vitro, explants of cartilage ex
vivo and in vivo to cartilage that remains intact within te joint
space of diarthrotic joints. The interval loading results in the
selective inhibition of matrix degrading enzymes, pro-inflammatory
cytokines and chemokines that attract inflammatory cells into the
joint cavity and in selective decrease of gene expression of growth
factors that are inhibitory to type II collagen expression.
Inventors: |
Smith; R. Lane; (Palo Alto,
CA) ; Carter; Dennis R.; (Stanford, CA) ;
Schurman; David J.; (Stanford, CA) |
Correspondence
Address: |
PETERS VERNY , L.L.P.
425 SHERMAN AVENUE, SUITE 230
PALO ALTO
CA
94306
US
|
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University
|
Family ID: |
24717364 |
Appl. No.: |
11/980967 |
Filed: |
July 11, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10328387 |
Dec 24, 2002 |
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11980967 |
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09677109 |
Sep 29, 2000 |
6528052 |
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10328387 |
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60157337 |
Oct 1, 1999 |
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Current U.S.
Class: |
435/366 ;
435/325 |
Current CPC
Class: |
A61F 2310/00365
20130101; A61F 2/28 20130101; C12N 2500/02 20130101; A61K 35/32
20130101; A61P 19/00 20180101; A61F 2240/001 20130101; C12N 2521/00
20130101; A61K 35/12 20130101; C12N 5/0655 20130101; A61F 2/08
20130101; A61F 2/30756 20130101; A61F 2/3094 20130101 |
Class at
Publication: |
435/366 ;
435/325 |
International
Class: |
C12N 5/08 20060101
C12N005/08; C12N 5/06 20060101 C12N005/06 |
Goverment Interests
[0002] This invention was made with U.S. Government support under
Veteran's Administration Rehabilitation Research and Development
Merit Review Grant No. A857-RC. The U.S. Government has certain
rights in this invention.
Claims
1-73. (canceled)
74. A method of increasing formation of extracellular matrix by
increasing production of aggrecan and Type II collagen, said method
comprising a step of treating adult human chondrocytes in vitro or
ex vivo with an intermittently applied hydrostatic pressure of
between about 0.5 and about 30 MPa, at a frequency of about 0.1 to
about 10 Hz for a period of about 1 to about 8 hours, followed with
a constant atmospheric pressure applied for a period of about 16 to
23 hours, wherein said period of hydrostatic pressure followed by
the recovery period is repeated for about 4 to about 100 days.
75. The method of claim 74 wherein said human chondrocytes are
normal healthy chondrocytes.
76. The method of claim 75 wherein said human chondrocytes are
osteoarthritic chondrocytes.
77. The method of claim 76 wherein said osteoarthritic chondrocytes
are metabolically inactive.
78. The method of claim 77 wherein said osteoarthritic chondrocytes
are isolated from injured, diseased or aged articular
cartilage.
79. The method of claim 78 suitable for treatment of an articular
cartilage injury and repair or for cartilage regeneration.
80. The method of claims 74 wherein said method is used for
increased production of extracellular matrix within a cartilage
graft.
81. The method of claim 80 wherein said graft subjected to
treatment of claim 74 is used as an implant for treatment of
cartilage injury or repair of cartilage.
82. Activated osteoarthritic metabolically inactive chondrocytes
isolated from a diseased, aged or injured articular cartilage
pressure treated in vitro or ex vivo with an intermittently applied
hydrostatic pressure of between about 0.5 and about 30 MPa, at a
frequency of about 0.1 to about 10 Hz for a period of about 1 to
about 8 hours, followed with a constant atmospheric pressure
applied for a period of about 16 to 23 hours, wherein said period
of hydrostatic pressure followed by the recovery period is repeated
for about 4 to about 100 days, said chondrocytes suitable for
treatment of osteoarthritic defects or cartilage injuries.
83. The activated chondrocytes of claim 82 wherein said treatment
increases levels of aggrecan from initial levels between about 0.1
and 1%, wet weight, and type II collagen between about 1 and 10%,
wet weight.
84. The activated chondrocytes of claim 83 wherein said levels of
aggrecan and Type II collagen are increased to levels of about 4
and about 7% and levels of type II collagen are increased to about
10 and 20%.
85. The activated chondrocytes of claim 84 submitted to said
treatment until said levels of aggrecan or Type II collagen reach
levels of active chondrocytes.
Description
[0001] This application is based on and claims priority of the
Provisional Application Ser. No. 60/157,337 filed on Oct. 1,
1999.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention concerns a method for in vivo, ex vivo and in
vitro repair, regeneration, de novo formation and remodeling of
diseased and normal mesenchymal or mesenchymally derived cells,
cartilage, collagen and bone. In particular, this invention
concerns in vivo, ex vivo and in vitro regeneration of articular
cartilage and collagen and bone remodeling by intermittently
applied hydrostatic pressure. The method involves the application
of external interval loading consisting of repeated periods of
applied hydrostatic pressure followed and interrupted by periods of
recovery. The method specifically concerns application of the
intermittent hydrostatic pressure at levels 0.5-30 MPa for an
interval of 1-8 hours followed by a recovery period up to about
16-23 hours, said pressure applied to cartilage and bone cells in
vitro, explants of cartilage and bone graft ex vivo and in vivo to
cartilage that remains intact within the joint space of diarthrotic
joints, or in vivo to the bone. The interval loading results in
significant increase in expression of proteins providing the unique
phenotypic properties of cartilage and bone and in the selective
inhibition of matrix degrading enzymes, pro-inflammatory cytokines
and chemokines that attract inflammatory cells into the joint
cavity and in selective decrease of gene expression of growth
factors that are inhibitory to extracellular matrix repair and
regeneration.
[0005] The invention also concerns methods for treatment of
articular cartilage and collagen regeneration, restoration and
transplantation.
BACKGROUND AND RELATED DISCLOSURES
[0006] Arthritic diseases, particularly osteoarthritis, affect more
people than any other ailment. Osteoarthritis involves loss of
function of cartilage which undergoes a slow progressive
degeneration in many joints. Although osteoarthritis is considered
a non-inflammatory disease a certain degree of inflammation occurs.
Osteoarthritis is distinguished from the rheumatoid arthritis which
is a chronic inflammatory joint disease.
[0007] Osteoarthritis affects most people in late middle age.
Osteoarthritic related conditions decrease personal productivity
and quality of life and in an aging society, increase the morbidity
and mortality for men and women by increasing the incidence of
other chronic conditions, such as, for example, osteoporosis.
Currently, the only successful treatment for end stage joint
disease requires major surgery involving total joint replacement
which is not without associated complications such as infection,
aseptic loosening, and pain. These complications can then lead to
the necessity for revision arthroplasty. Reversal of early onset
osteoarthritis by novel surgical techniques that would abrogate the
necessity for joint replacement is just now being tried in
experimental stages.
[0008] Intra-articular surgical approaches are being developed that
entail transfer of cartilage cells from healthy regions of the
joint to diseased surfaces in order to restore joint function. In
this context, cartilage cells or small regions of cartilage are
placed in partial or full-thickness defects within the joint
surface using an open surgical procedure. The cell construct is
then held in place by periosteal tissue that is sutured in place.
However, implanting cells or resurfacing with autogenous or
allograft cartilage in the absence of an organized extracellular
matrix does not support normal weight bearing. In many cases, these
grafts quickly become fibrillated and degrade. In an alternative
procedure, mosaicplasty involves moving multiple small grafts of
cartilage from one area of the joint surface to another to
facilitate a return to weight bearing. With any type of cartilage
exchange, efficacy of repair will be greatly facilitated following
restoration of an extra-cellular matrix structure of normal
cartilage prior to use.
[0009] Cartilage, collagen and bone diseases, therefore, present a
major medical problem, particularly with an increasing aging
population which is more prone to osteoarthritis and other joint
regenerative diseases, and it would thus be important to have
available a means for regeneration of articulate cartilage and
collagen and bone remodeling.
[0010] Articular cartilage covers the ends of long bones and is
load-bearing tissue that distributes forces across joint surfaces
protects the more rigid underlying bone and provides smooth
articulation and bending of the joints during normal activities of
daily living.
[0011] Attempts are continuously made to regenerate articular
cartilage. U.S. Pat. No. 6,080,194, for example describes a
collagen template formed by combining a porous collagen sponge with
a collagen membrane. U.S. Pat. No. 5,786,217 describes methods and
compositions for the repair of articular cartilage defects. U.S.
Pat. No. 5,206,023 discloses methods and compositions for treatment
and repair of defects or lesions of the cartilage. U.S. Pat. No.
5,041,138 concerns neomorphogenesis of cartilage in vivo from cell
culture for the growth and implantation of cartilaginous
structures. However, none of these patents disclose a method which
would regenerate the diseased cartilage to a functional state and
such method is still lacking.
[0012] Clinical experience in humans and experimental studies with
animal models confirm that mechanical loads provide an essential
stimulus for maintenance of normal articular cartilage homeostasis
(Proc. Soc. Exp. Biol. Med., 190:275 (1989)).
[0013] Alterations in joint loading due to immobilization (Clin.
Orthop. Rel. Res., 219:28 (1987)) ligamentous laxity (Ibid, 213:69
(1986)), excessive impact (J. Biomechanics, 6:51 (1973)) or
increased subchondral bone stiffness (J. Biomechanics, 28:357
(1995)) result in pathological changes in cartilage characteristic
of osteoarthritis.
[0014] The ability of cartilage to change shape rapidly and
reversibly is attributable to a resilient and elastic matrix with a
high content of highly soluble proteoglycans which are entrapped in
collagen, an insoluble fiber network. Proteoglycans, collagen and
other molecules present in the cartilage tissue are produced by
mesenchymally-derived cartilage cells, the chondrocytes.
[0015] In vitro studies confirm that the cartilage cells, the
articular chondrocytes, respond to specific loading conditions
through an anabolic or catabolic reaction that is attributable to
the stress and strain imparted to the cell by the physical stimulus
(Biochem. Biophys. Res. Commun., 240:216 (1997); Spine, 22:1085
(1997) and J. Orthop. Res., 15:189 (1997)).
[0016] Recognition of the role that mechanical loading plays in the
regulation of articular chondrocyte metabolism has been delineated
in part by mathematical analysis of the distribution of forces
across joint surfaces (J. Biomech., 22:853 (1989)).
[0017] Biomechanical analyses described in J. Exp. Physiol., 81:535
(1996) confirm that chondrocytes in the cartilage of a diarthrotic
joint experience levels of hydrostatic pressure in the order of 7
to 10 MPa that result from normal activities of daily living.
Studies examining the influence of mechanical forces on tissue
differentiation revealed that increased cartilage thickness occurs
in regions of the diarthrotic joint exposed to high intermittent
compressive hydrostatic stress. Thinner cartilage coincides with
regions experiencing decreased hydrostatic pressure and having
tensile forces arising tangential to the joint surface (Bone,
11:127 (1990)).
[0018] Experimental studies described in J. Orthop. Res., 9:1-10
(1991) confirmed that hydrostatic pressure influences articular
cartilage matrix metabolism when applied in vitro and established
that hydrostatic pressure at levels of 5-15 MPa modulates
.sup.35SO.sub.4 and .sup.3H-proline incorporation rates into adult
bovine articular cartilage in vitro.
[0019] Organ culture experiments described in J. Biol. Chem.,
262:15490 (1987) demonstrated that sites of proteoglycan production
coincide with regions of pure hydrostatic pressure. Physiological
levels of hydrostatic pressure enhance mRNA signal levels for
aggrecan and type II collagen when measured immediately after
loading as described in J. Orthop. Res., 14:53 (1996). In a study
of load controlled compression of aggrecan mRNA expression in
bovine cartilage explants a transient up-regulation was observed
after 1 hour of loading.
[0020] While the above research describes and recognizes the
importance of the hydrostatic pressure on normal function of
cartilage and type II collagen, such knowledge was nevertheless
impossible to apply clinically because the continuous application
of the hydrostatic pressure leads to exhaustion of the cartilage
metabolic potential and its damage while the brief (<1 hour) of
loading with hydrostatic pressure leads to varied cellular response
which disturbs the chondrocyte metabolism and homeostasis. For
example, a short period of hydrostatic pressure loading results in
increased expression of type II collagen mRNA while the
continuously applied load does not maintain such increased
expression. On the other hand, the aggrecan signal expression
continued to increase throughout the duration of the load. Clearly,
these results disturb the cellular equilibrium between aggrecan and
type II collagen. Cartilage cells respond to multiple stimuli in
unpredictable ways and variability of the response depends on time,
magnitude and frequency of loading. Clearly, this unpredictability
prevents using the continuous long or short periods of
indiscriminate hydrostatic pressure loading for treatment of
osteoarthritis or regeneration of damaged cartilage (J. Rehab. Res.
Dev., 37:153-161 (2000)).
[0021] In view of the severity and disabling effect of
osteoarthritis and other cartilage, collagen or bone diseases, it
would be important to provide a method which would permit a
cartilage or collagen regeneration and bone remodeling.
[0022] Until recently, it was believed that articular cartilage can
no longer repair itself once the arrangement of the supporting
fibers has been disrupted. (Articular Cartilage and Osteoarthritis,
Workshop Conference Hoechst and Werk, Kalle-Albert, Wiesbaden May
12-16, 1991, Eds. Kuettner et al., Rosen Press, New York.
[0023] It has now been found that such regeneration is possible
with a specific regimen of intermittently applied hydrostatic
pressure to mesenchymal or mesenchymally-derived cells, such as
fibroblasts, fibrochondrocytes or chondrocytes and it is,
therefore, a primary objective of the current invention to provide
a method for treatment of osteoarthritis and other cartilage and
collagen diseases by stimulating their regeneration, de novo
formation, and bone remodeling, said method providing a defined
mechanical loading environment which regenerates and repairs adult
cartilage and bone cells.
[0024] All patents, patent applications and publications cited
herein are hereby incorporated by reference.
SUMMARY
[0025] One aspect of the current invention is a method for repair,
regeneration and de novo formation of cartilage, replenishment of
chondrocytes, or deposition of type II collagen and stimulation and
bone remodeling.
[0026] Another aspect of the current invention is a method for
treatment of diseases of cartilage, collagen or bone by stimulating
the cartilage and collagen regeneration and bone remodeling using
an interval loading regimen consisting of repeated periods of
intermittently applied hydrostatic pressure within specified
loading interval followed by a recovery period.
[0027] Still another aspect of the current invention is an in vivo,
ex vivo or in vitro method for repair, regeneration and de novo
formation of cartilage wherein the regeneration is achieved by
applying an interval loading regimen consisting of repeated periods
of applied hydrostatic pressure followed by periods of recovery to
in situ or ex situ cartilage or cartilage cells or to cartilage or
cartilage cells in vitro.
[0028] Still yet another aspect of the current invention is an in
vivo or ex vivo cartilage repair, regeneration and de novo
formation method which involves applying hydrostatic pressure to
the cartilage tissue in need of regeneration in situ or to
cartilage removed from in situ or chondrocytes, fibroblast or
fibrochondrocytes adhered to a matrix and subjected to the regimen
comprising applying intermittently the hydrostatic pressure for
about 1-8 hours followed by about 16-23 hours of recovery
period.
[0029] Still yet another aspect of the current invention is an in
vivo collagen restoration method which involves applying
hydrostatic pressure to the cartilage, cartilage cells, other
mesenchymally-derived cells or collagen tissue in need of repair
and regeneration.
[0030] Still yet another aspect of the current invention is an in
vivo bone restoration method which involves applying hydrostatic
pressure to the bone site in need of regeneration of cartilage as a
progenitor tissue or direct mechanical stimulus using a regimen
comprising applying, intermittently, the hydrostatic pressure to
the bone osteoblast cells for about 1-8 hours, followed by recovery
period of about 16-23 hours.
[0031] Still yet another aspect of the current invention is a
diseased joint regeneration method wherein cartilage grafts that
are submitted to the intermittent hydrostatic pressure loading and
restored to a healthy load-bearing matrix are placed in a diseased
joint to restore its normal function.
[0032] Still yet another aspect of the current invention is a
regenerated functional healthy load bearing cartilage from the
diseased cartilage wherein said diseased cartilage is subjected to
intermittent hydrostatic pressure followed by periods of recovery
until the normal mechanical and biochemical properties are
restored.
[0033] Still yet another aspect of the current invention is a
method for detection of functionality of the cartilage by
determining relative levels of, or levels of expression of,
cartilage or bone degradative enzymes, cytokines and their
inhibitors or growth promoting substances, growth factors and
hormones, following subjecting the diseased cartilage or bone to
intermittent hydrostatic pressure followed by periods of
recovery.
BRIEF DESCRIPTION OF FIGURES
[0034] FIG. 1 is a schematic of a servo-hydraulic loading
instrument suitable for application of intermittent hydrostatic
pressure to articulate chondrocytes.
[0035] FIG. 2 is a graph illustrating effects of interval loading
with different magnitudes of intermittent hydrostatic pressure on
aggrecan (FIG. 2A) and type II collagen (FIG. 2B) expression in
normal human articular chondrocytes.
[0036] FIG. 3 is a graph illustrating the effects of interval
loading for 4 hours for 4 days with 10 MPa of intermittent
hydrostatic pressure on the expression of aggrecan and type II
collagen mRNA signal in human osteoarthritic articular
chondrocytes.
[0037] FIG. 4 is a graph showing the effects of interval loading
with different magnitudes of intermittent hydrostatic pressure on
matrix metalloproteinase-2 release from normal human articular
chondrocytes using ELISA (FIG. 4A) and enzymatic activity using
zymograph for activated (+APMA) and inactivated preparation (FIG.
4B).
[0038] FIG. 5 is a graph showing the effects of interval loading
with different magnitudes of intermittent hydrostatic pressure on
release of transforming growth factor-.beta.(TGF-.beta.) from
normal human articular chondrocytes.
[0039] FIG. 6 is a graph showing the effects of interval loading
with different magnitudes of intermittent hydrostatic pressure on
release of macrophage chemoattractant protein-1 (MCP-1) from normal
human articular chondrocytes.
[0040] FIG. 7 is a graph showing the absence of an effect of
interval loading with different magnitudes of intermittent
hydrostatic pressure on fibroblast growth factor-1 (FGF-1) from
normal human articular chondrocytes.
[0041] FIG. 8 is a graph showing RT-PCR signal levels for type II
and type I collagen and beta-actin expression in unloaded control
cells.
[0042] FIG. 9 shows signal levels for beta actin expression in
cells exposed to IHP and in unloaded control cells.
[0043] FIG. 10 shows RT PCR signal levels for aggrecan following
exposure of high density monolayer cultures to IHP.
[0044] FIG. 11 shows RT PCR signal levels for type II collagen
following exposure of high density monolayer cultures to IHP.
[0045] FIG. 12 shows time course analysis for TGF-.beta.1 release
from MG-63 cells exposed to intermittent hydrostatic pressure (10
MPa).
[0046] FIG. 13 depicts dose response effects of intermittent
hydrostatic pressure on the TGF-.beta.1 release from MG-63
cells.
[0047] FIG. 14 shows time course analysis for MMP-2 release from
MG-63 cells exposed to IHP.
DEFINITIONS
[0048] As used herein:
[0049] "Cancellous" means bone that has a lattice-like or spongy
structure.
[0050] "Mesenchymal", "mesenchymal stem cells" or
"mesenchymally-derived cell" means the cells that are located
within and produce the extracellular matrix of cartilage
(chondrocytes), connective tissue (fibroblasts), fibrocartilage
(fibrochondrocytes), tendon (tenocytes) and bone (osteoblasts and
osteocytes).
[0051] "Loading interval" means a period of applied IHP load, or
stimulus in tissue, that is followed by a recovery period where no
external pressure is applied and where the pressure returns to the
ambient condition.
[0052] "Interval" means a combination of a load and recovery
periods repeated as many times as needed.
[0053] "De novo formation" means production of cartilage connective
tissue, fibrocartilage, tendon and bone as a result of adherence by
chondrocytes, fibroblasts, fibrochondrocytes, tenocytes and
osteoblasts within a support structure (scaffold or collagen
matrix) following exposure to loading interval.
[0054] "Osteoblast" means a bone forming cell derived from
mesenchyme (fibroblast) and forms an osseous matrix in which it
becomes enclosed as an osteocyte.
[0055] "Fibroblast" or "fibrocyte" means a stellate or
spindle-shaped cell with cytoplasmic processes present in
connective tissue capable of forming collagen fibers.
[0056] "Aggrecan" means a large aggregating proteoglycan that plays
a role in imparting compressive resilience to the articular
cartilage and enable load bearing. Aggrecan is the abundant
proteoglycan which represents about 85% of all total
proteoglycans.
[0057] "Type II collagen" means a homopolymeric molecule of
.alpha.1 (11) chains which is the product of a single gene COL2A1.
Type II collagen is the most abundant of all other collagen types
and represents about 95% of the total collagen.
[0058] "Matrix metalloproteinase" or "MMP" means a protease which
causes and is associated with cartilage degeneration in a diseased
joint. MMP may be further distinguished as MMP-1, MMP-2, MMP-9,
etc.
[0059] "Macrophage chemoattractant protein-1" or "MCP-1" means a
pro-inflammatory mediator that influences the immune state of
tissue. Its increased level is associated with the beginning or
increase of tissue destruction, its decreased level is associated
with decrease of tissue damage or healing.
[0060] "Transforming Growth Factor-.beta." or "TGF-.beta." means a
factor released by the cells upon applying intermittent hydrostatic
pressure and signifies a change in the cell metabolism.
[0061] "Fibroblast growth factor 1" or "FGF-1" means a growth
factor which is not known to be involved in chondrocytes or
osteoblast-like cells proliferation.
[0062] "MPa" means MegaPascal. One MPa is equal to 145 psi.
[0063] "GAG" means glycosaminoglycans.
[0064] "IHP" means intermittent hydrostatic pressure.
[0065] "GAPDH" means glyceraldehyde-3-phosphate dehydrogenate.
[0066] "APMA" means 4-aminophenylmercuric acetate, an activator of
latent proenzyme to the active enzyme.
DETAILED DESCRIPTION OF THE INVENTION
[0067] The invention described herein concerns a discovery that
intermittently and repeatedly applied hydrostatic pressure during
interval loading periods influences articular chondrocyte gene
expression, elicits load-dependent collagen type II expression,
decreases a matrix metalloproteinase expression, results in
regeneration of diseased or damaged cartilage and collagen, permits
the de novo formation of mesenchymal or mesenchymally-derived cells
within a matrix and alters bone remodeling. The discovery is
suitable for repair and treatment of degenerative joint diseases
such as osteoarthritis, arthrosis, injuries to the joint cartilage,
degenerative joint disease, joint replacement, bone restructuring
and other diseases of cartilage, collagen and bone.
[0068] I. Method for Cartilage and Collagen Regeneration and Bone
Remodeling
[0069] The method for cartilage and collagen regeneration and bone
remodeling for treatment of the above listed diseases comprises, in
its broader scope, the application of interval loading regimen
according to the invention to a site of injury in vivo, to a
transplantable full-thickness grafts of human osteoarthritic or
diseased cartilage or bone ex vivo or to the cells isolated from
the healthy or diseased cartilage, collagen and bone in vitro.
[0070] A. Therapeutic Regimen for Cartilage Regeneration General
Conditions
[0071] The therapeutic regimen consists of stimulation of treated
tissue or isolated cells with repeated periods of applied
hydrostatic pressure followed by periods of recovery (loading
interval). Pressure is preferably applied intermittently within
each loading interval. Method parameters, such as, pressure, a
frequency, length of intervals, intermittence and recovery period,
are selected based on the chosen method for regeneration, that is,
in vivo, in situ, ex vivo, or in vitro, as well as on the disease
and on the seriousness of the cartilage degeneration.
[0072] 1. Hydrostatic Pressure
[0073] The loading regimen suitable for repair, regeneration or de
novo formation of intact cartilage, intact bone, cartilage and bone
graft or cells generally comprises the application of hydrostatic
pressure between 0.5 MPa and 30 MPa, preferably between 1 MPa and
20 MPa, and most preferably between 5 MPa and 10 MPa in
intermittent intervals of 1 Hz frequency.
[0074] Typically, pressures higher than about 30 MPa are not used
or recommended as they can lead to cell damage, while pressures
lower than 0.1 MPa may not be effective for the regeneration of
cells or tissue. Pressures around 5-10 MPa correspond to normal
physiological levels encountered by articular cartilage in
vivo.
[0075] 2. Duration of IHP and Recovery Periods
[0076] The duration of the high pressure intervals generally range
from minutes to about 8 hours, and are preferably between about 1
and 8 hours, most preferably about 4 hours.
[0077] During the recovery period, the tissue/cells of interest are
exposed to atmospheric or sufficiently low constant pressure. The
recovery periods can generally range from minutes to tens of hours,
and are preferably between about 16 to about 23 hours, preferably
about 20 hours.
[0078] 3. Frequency
[0079] Within each intermittent hydrostatic pressure interval, the
pressure is applied intermittently with a frequency between 0.1 Hz,
and 10 Hz, preferably on the order of 1 Hz. In the preferred
embodiment for cartilage cells, the interval loading regimen is
applied for at least 4 consecutive days, typically for about 4
hours of loading at 10 MPa applied at 1 Hz followed by about 20
hours of recovery at constant atmospheric pressure.
[0080] 4. Length of Treatment
[0081] The length of the treatment of cartilage depends entirely on
the degree and seriousness of cartilage degeneration, extent of
collagen loss, severity of bone disease, or degree and seriousness
of joint injury. Typically, the improvement in cartilage
functionality and its regeneration is observed after about four
treatments with IHP loading, that is, typically after 4 days of
treatment. In more degenerate, diseased or injured cartilage, such
treatment is continued for as long as 100 days without any negative
consequences and may be continued indefinitely when cartilage
and/or collagen function is chronically impaired. Preferable
treatment will last and be successful in between 7 and 30 days.
[0082] For de novo formation, the collagen matrix is laden with
healthy or diseased cells to be treated and the treatment is
continued until the new tissue is formed.
[0083] 5. Functionality Testing
[0084] The length of treatment depends on the rapidity of
functional recovery. Functionality of cartilage depends on the
recovery and/or rebuilding and/or formation of load-bearing matrix.
Degeneration of cartilage results from deformation of cells, from
inappropriate levels of shear stress loading to matrix, and
degeneration due to metabolic shifts.
[0085] These metabolic shifts affect genetic expression of
cartilage cell with respect to aggrecan and collagen, particularly
type II collagen, and certain growth factor such as TGF-.beta.. The
shifts and chondrocyte metabolism also lead to expression or over
expression of proteins which are not normally expressed or are less
expressed in healthy functional chondrocytes, such as
metalloproteinases MMP-1, MMP-2, MMP-9 and pro-inflammatory
cytokines, such as IL-1 and IL-6.
[0086] Consequently, during and particularly after the IHP loading
treatment, some or all of the above factors are tested and their
presence is evaluated for increased activity. Absence or decreased
activities are correlated with the regeneration of cartilage
integrity and load-bearing function.
TABLE-US-00001 TABLE 1 Functionality Testing Type II Aggrecan
Collagen MMP-2 MMP-1 MMP-9 IL-6 IL-1 MCP-1 Physiological 4-7%
10-29% low low nd low nd low Levels Wet wt wet wt. Pathological
0.1-1% 1-10% High high elevated high elevated high Levels wet wt.
wet wt. or high Regenerated 4-7% 10-20% low low nd low nd low
Levels nd = not detectable or trace
[0087] Functionality testing disclosed herein involves
determination of relative values of extracellular matrix components
such as aggrecan and type II collagen, proteases such as MMP-1,
MMP-2, MMP-9 and cytokines such as IL-6, IL-1 and MCP1. These
values differ depending on the tissue preparation and the method
used but the trend remains the same, namely, levels of
extracellular matrix proteins decrease and levels of proteases and
cytokines increase in injured or degenerated cartilage and
collagen. During regeneration, the levels are returning to their
normal physiological ranges.
[0088] The loading regimen applied according to the invention was
found to stimulate the regeneration of articular cartilage tissue,
collagen tissue, bone tissue and/or their respective isolated
cells. The interval loading regimen was also found to increase gene
expression for proteins that form the functional extracellular
matrix of articular cartilage. After application of an interval
loading regimen of the present invention, staining of the matrix
with cationic dyes confirmed the increased presence of
extracellular matrix.
[0089] Additionally, the interval loading regimen was found to
result in the selective inhibition of matrix-degrading enzymes,
pro-inflammatory cytokines and chemokines that attract inflammatory
cells into the joint cavity. Furthermore, the interval loading
regimen was found to selectively decrease gene expression for
growth factors inhibitory to type II collagen expression and was
also found to affect expression of transforming growth
factor-.beta.1 (TGF-.beta.1), a matrix metalloproteinases such as,
for example, MMP-1, MMP-2, MMP-9 and tissue inhibitor of
metalloproteinase (TIMP) in human osteoblast-like cells. These and
other factors may be conveniently used for assessment of cartilage
and bone functionality as shown in Table 1.
[0090] The methods which are used for detection of the parameters
for determination of functionality include but are not limited to
RT-PCR, zymography, biochemistry, staining, ELISA, gene array
techniques and proteomics.
[0091] B. In Vitro Treatment
[0092] For in vitro treatment, damaged cartilage tissue is removed
from a patient by surgical means. The interval loading regimen can
be applied to the intact tissue such as osteochondro cartilage
graft for ex vivo treatment. For in vitro treatment, the normal or
diseased cartilage matrix is degraded and the interval loading
regimen is applied to the resulting cartilage cells cultured in
suspension within scaffold/support or as monolayers. After the
application of the loading regimen, the resulting de novo formed
tissue or collection of cells is re-implanted into a patient.
Preferably, but not necessarily, the transplant is autologous.
[0093] The surgical procedure generally follows the technique that
has been developed and used for arthroscopic intervention using
osteochondral grafting. In this procedure, a full-thickness sample
of cartilage is removed from peripheral regions of the joint
surface and is then transferred into a circular defect. The host
site typically has a circumference that is smaller than the
material to be inserted so that the union between the host site and
the replacement tissue is a resistance fit. In the material to be
produced in response to interval loading, the restored cartilage
material is adjusted in size to match the surface contours of the
joint. This is different from the usual procedure of osteochondral
grafting where the oversized graft is left 2-3 mm above the surface
of the surrounding cartilage. The formation of a type II
collagen-based extracellular matrix that is capable of resisting
normal joint loads permits press-fit grafts to match the normal
joint surface thickness that coincide with the natural thickness
that corresponds to the regional variation of the normal joint.
[0094] In vitro treatment of cells, cell monolayers or cell
cultures is essentially as described in section II.
[0095] C. Ex Vivo Treatment
[0096] For ex vivo treatment, which is particularly suitable for
treatment of joint cartilage injury, such as shredded or torn
meniscus where only a part of the cartilage may be damaged and the
rest of the cartilage is healthy and functioning, the torn or
shredded cartilage is surgically removed as a cartilage graft and
subjected to IHP loading regimen. Functionality of the cartilage
graft is periodically tested until the criteria reach normal
healthy cartilage levels as shown in Table 1. Then, the graft which
retains its original shape and size is re-implanted into the joint.
The IHP ex vivo treatment, testing, surgical removal and
re-implantation is performed under sterile conditions.
[0097] The advantage of the ex vivo treatment is that the tissue is
autologous, only the damaged tissue is subjected to treatment and
the explant shape and size remains the same so that there is no
less or more cartilage tissue added. Additionally, the tissue is
retransplanted only if and when it is fully regenerated. The
disadvantage of this approach is a double surgery. However, in
extensive articular joint injury, this approach is still preferable
to joint replacement or leaving the joint without cartilage or a
part of the cartilage altogether.
[0098] D. In Vivo Treatment
[0099] Suitable in vivo cartilage restoration or bone remodeling
methods include applying described hydrostatic pressures to the
cartilage tissue or bone of the patient's joints and/or limbs,
according to an interval loading regimen of the present invention.
The manipulation may be done manually by a physical therapist, or
automatically by a powered device.
[0100] E. Apparatus for Intermittent Hydrostatic Pressure
Loading
[0101] Instruments and apparatuses for the regeneration of
articular cartilage matrix and/or cells, in in vivo, ex vivo and in
vitro treatments essentially comprise of a hydrostatic pressure
generator, a frequency counter, a timer and a temperature control
device.
[0102] A suitable in vitro apparatus comprises a pressurization
chamber for holding the tissue, cells of interest or cell cultures,
a hydraulic loading instrument (pressurization device) in fluid
communication with the pressurization chamber, for pressurizing the
pressurization chamber to predetermined pressures of interest, and
control electronics for frequency control in electrical
communication with the loading instrument for controlling the
loading instrument to apply a predetermined interval loading
regimen to the tissue or cells of interest.
[0103] A suitable instrument for application of intermittent
hydrostatic pressure to isolated articular chondrocytes for
treatment in vitro is seen in FIG. 1. The particular instrument
seen in FIG. 1 is a commercially available stainless steel pressure
vessel interfaced to a servo-hydraulic loading instrument. This
design provides for the complete evacuation of air from the system
resulting in application of purely hydrostatic pressure.
[0104] The apparatus seen in FIG. 1 is exemplary only and it should
be understood that any instrument and apparatus, regardless of how
modified, comprising similar components and providing similar
results, is intended to be within the scope of this invention. A
suitable in vivo instrument comprises a holder for holding a
patient's joint or limb or attaching means to the patient's tissue,
a motor coupled with the holder for moving the holder along a
predetermined path, and control electronics electrically connected
to the motor, for controlling the motor to move the patient's limb
so as to apply an interval loading regimen of the present invention
to the cartilage tissue of interest.
[0105] The in vivo loading of the joint is carried out by a device
that spans the diarthrodial joint in question. For example, for the
knee, the apparatus spans the distance from the hip to the bottom
of the foot, with corresponding restraints to maintain the leg in
extension. The device includes a contracting mechanism by which the
femoral condylar cartilage can be juxtaposed on the tibial plateau
cartilage across the meniscus at frequency between 0.1 and 10 Hz
and will generate pressures in the range of 1 to 20 MPa for
prescribed intervals between 1 and 8 hours, more closely
approximating 4 hours per day at normal daily activity.
[0106] II. Interval Loading of Human Normal and Osteoarthritic
Chondrocytes
[0107] The major component of mechanical loads to which an
articular chondrocytes are exposed within the extracellular matrix
of joint cartilage is hydrostatic pressure. The normal loading of
joints during daily activities causes the articular cartilage to be
exposed to high levels of intermittent hydrostatic pressure.
Studies described in this section show that osteoarthritic
chondrocytes maintained in vitro respond to applied hydrostatic
pressure by increasing a positive metabolic activity and decreasing
of expression of destructive enzymes.
[0108] Effect of intermittent hydrostatic pressure on cartilage
matrix protein synthesis in isolated adult human articular
chondrocytes focused on the mRNA expression of type II collagen,
aggrecan and other degradative and growth promoting proteins, such
as, MMP-2, TGF-.beta., MCP-1 and FGF-1. Results of these studies
are seen in FIGS. 2-7.
[0109] In this series of studies, mRNA expression of aggrecan, type
II collagen, MMP-2, TGF-.beta., MCP-1 and FGF-1 of normal healthy
and osteoarthritic human chondrocytes stimulated with 1 MPa or 10
MPa hydrostatic pressure for 5 minutes or 4 hours for 4 days was
compared to the mRNA expression in controls. When the loading was
performed as dose response manner ranging from 1, 5 and 10 MPA
vis-a-vis the hydrostatic pressure limited either to intermittent 5
minutes or intermittent 4 hours per day at 1 MPA and 10 MPA
pressure, aggrecan mRNA signal was observed to increase with loads
at 1 MPa, 5 MPa and 10 MPA, as seen in FIG. 2.
[0110] The results seen in FIG. 2 clearly confirm that the current
interval loading regimen results in increased genetic expression of
aggrecan and type II collagen. Aggrecan is large and most abundant
aggregating proteoglycan that plays a fundamental role in imparting
compressive resilience to the articular cartilage and enables load
bearing to persist. Type II collagen provides the tissue with
tensile strength. The treatment of the invention thus leads to
increased resilience of cartilage and ability to bear larger
loads.
[0111] FIG. 3 shows the effects of 4 hour interval loading for 4
days with 10 MPA of intermittent hydrostatic pressure (IHP) on the
expression of aggrecan and type II collagen mRNA signal expressed
as a ratio aggrecan or type II collagen to intracellular reference
protein .beta.-actin of in human osteoarthritic articular
chondrocytes.
[0112] These results clearly confirm that expression of both
aggrecan and type II collagen, the major macromolecular proteins in
the cartilage extracellular matrix, was increased in diseased
osteoarthritic human cartilage cells following IHP treatment.
[0113] As seen in FIGS. 3A and 3B, application of intermittent
hydrostatic pressure resulted in increased ratio of production of
aggrecan to .beta.-actin signal from about 0.1 to about 0.5, that
is, about 5 times increase. Ratio of type II collagen to
.beta.-actin signal increased even more from about 0.01 to about
0.12 as seen in FIG. 3B.
[0114] Immunohistochemical analysis of the effect of hydrostatic
pressure on chondrocytes was also investigated.
Immunohistochemistry provides an index of the extracellular matrix
response to mechanical loads. With loading conditions described
above, immunohistochemical analysis showed that application of
hydrostatic pressure increased extracellular matrix deposition of
proteoglycan and collagen (data not shown).
[0115] To investigate whether IHP might also have effects on
expression of molecules deletions to cartilage, a series of
experiments were carried out to evaluate the release and effective
interval loading of proteases and growth factors.
[0116] The effect of interval loading with intermittent hydrostatic
pressure on inhibition of matrix metalloproteinase mRNA expression
was determined by testing the effects of hydrostatic pressure under
the same conditions as described in FIG. 2.
[0117] Results are seen in FIG. 4 which shows the effects of
interval loading with different magnitudes of intermittent
hydrostatic pressure on matrix metalloproteinase-2 release from
normal human articular chondrocytes.
[0118] Matrix metalloproteinase-2 (MMP-2) degrades the
extracellular matrix collagen and is one of the several enzymes
that are known to be associated with cartilage degeneration in a
diseased joint. Reduction in levels of this enzyme following the
application of IHP at 10 MPa for four hours for 4 days shows that
repair and regeneration of the extracellular matrix is in progress.
Zymographic analysis, seen in FIG. 4B, of neutral metalloproteinase
expression showed that APMA subjected to intermittent hydrostatic
pressure for 4 hours followed by 20 hours recovery for 4 days
decreased the levels of gelatinolytic activity.
[0119] Transforming growth factor .beta.1 (TGF-.beta.1) is known to
be involved in metabolic changes associated with the accelerated
matrix resorption which occurs, for example, in osteoarthritis
where both anabolic and catabolic pathways of aggrecan metabolism
are activated. TGF-.beta.1 thus affects cartilage homeostasis.
[0120] The role of TGF-.beta.1 is most pronounced on progenitor
cells, such as, for example, the surrounding periosteal region of
bone. The direct effects of TGF-.beta.1 on isolated cartilage cells
vary depending on the level of produced protein since it exhibits
pleiotrophic actions of different cells. The addition of
TGF-.beta.1, to articular cartilage cells in culture decreases type
II collagen expressions which is counter productive to matrix
production.
[0121] FIG. 5 shows the effects of interval loading with different
magnitudes of intermittent hydrostatic pressure on TGF-.beta.1
release from normal human articular chondrocytes.
[0122] The effect of intermittent hydrostatic pressure on the
release of TGF-.beta. seen in FIG. 5 means that the cell metabolism
is modulated by the mechanical stimulus. The decreased production
of this growth factor is clearly coupled to other changes in the
cell metabolism.
[0123] As seen in FIG. 5, the application of IHP for 4 hours at 10
MPa results in significant decrease released TGF-.beta. from normal
chondrocytes showing that protein synthesis decreases with higher
pressure (10 MPa) applied intermittently for longer periods. These
results establish that IHP stimulation differentially influences
the metabolic state of chondrocytes in a time and dose-dependent
manner. As will be seen below, similar results were observed
following the IHP stimulation of osteoblasts-like cells.
[0124] FIG. 6 shows the effects of interval loading with different
magnitudes of intermittent hydrostatic pressure on macrophage
chemoattractant protein-1 (MCP-1) release from normal human
articular chondrocytes.
[0125] MCP-1 represents one of a number of pro-inflammatory
mediators that influence the immune state of tissues. A decrease in
MCP-1 in chondrocytes is significant as a marker of a change in
cell metabolism and in termination of inflammatory processes
accompanying the cartilage degeneration or damage. The release of
MCP-1 is normally associated with the recruitment of monocytes and
macrophages, the cells of the immune system, that play a role in
tissue destruction. Its decreased expression signals amelioration
of cartilage damage.
[0126] As seen in FIG. 6, releases of MCP-1 from normal human
articular chondrocytes is significantly decreased, approximately 8
times, following the application of IHP at 10 MPa for 4 hours.
[0127] Decrease observed in release of MCP-1 clearly shows that
anti-inflammatory or anti-injury mechanism of the cartilage is less
activated thus resulting in lesser degree of cartilage degeneration
and destruction.
[0128] To determine whether the decrease in the release of MMP-1,
MMP-2 or TGF-.beta. following IHP is selective for those proteins
involved in cartilage degeneration, studies were performed with
fibroblast growth factor 1 which is not involved in such processes
and thus should not react to the IHP. Results are seen in FIG.
7.
[0129] FIG. 7 shows the absence of an effect of interval loading
with different magnitudes of intermittent hydrostatic pressure on
fibroblast growth factor-1 (FGF-1) from normal human articular
chondrocytes.
[0130] The FIG. 7 shows that not all growth factors respond to
intermittent hydrostatic pressure but only those factors which are
involved in cartilage degeneration or regeneration. The FIG. 7 also
shows that the chondrocytes were not being damaged by the IHP and
that the decreased release of MMP-2, TGF-.beta. and MMP-1 was due
to damaged cartilage.
[0131] The objective of the studies described in FIGS. 2-7 was to
investigate whether the application of intermittent hydrostatic
pressure on cartilage results in changes of matrix protein
synthesis in isolated adult human articular chondrocytes.
[0132] FIGS. 2-7 cumulatively show that excessive or insufficient
(5 minutes at 10 MPa or 4 hours at 1 MPa) loading of joint
cartilage does not provide sufficient stimulus for cartilage repair
and regeneration and in some cases, may even lead to increased
degeneration and loss of function. Results seen in FIGS. 2-7
confirm that there is a correlation between IHP and between
occurrence of beneficial changes in chondrocyte metabolic processes
leading to cartilage regeneration.
[0133] Rehabilitation of joint function depends on repair and
regeneration of diseased cartilage. A variety of experimental
approaches described above confirm that mechanical loading
influences synthesis of articular chondrocyte extracellular matrix
components, i.e., aggrecan and type II collagen. However, until now
no clear connection was shown to exists for mechanically-induced
modulation of cartilage matrix gene expression establishing that
mechanical IHP stimulation serves as impetus for repair and
regeneration of cartilage. The above described results make the
therapeutic intervention in vivo, in vitro and ex vivo
feasible.
[0134] III. Time-Dependent Effects of IHP on mRNA Expression of
Type II Collagen and Aggrecan
[0135] Initial studies leading to this invention have shown that
the hydrostatic pressure applied continuously does not lead to
cartilage repair and regeneration measured by the increase or
decrease metabolic activities of relevant protein synthesis. Such
pressure applied for a short period of time resulted in increased
expression of type II collagen mRNA while the continuously applied
load did not maintain such increased expression. On the other hand,
the aggrecan signal expression continued to increase throughout the
duration of the load.
[0136] Studies documented in Section II have shown that there is a
correlation between IHP and chondrocyte metabolic processes.
[0137] The studies described in this section investigated
time-dependent effects of IHP on type-II collagen and aggrecan mRNA
expression in normal adult bovine articular chondrocytes in
vitro.
[0138] The purpose of this study was to test whether the
intermittent pressure applied to chondrocytes enhances type II
collagen and aggrecan mRNA levels without stimulating type I
collagen mRNA. The experimental approach relied on use of RT-PCR
for relative quantification of collagen and aggrecan mRNA levels
with respect to beta-actin mRNA as an internal reference
signal.
[0139] Specifically, this study examined the effects of two types
of loading regimens of intermittent hydrostatic pressure on
expression of type II collagen and aggrecan mRNA in normal adult
articular chondrocytes. The chondrocytes were isolated according to
Example 1. A system for quantitative loading of adult articular
chondrocytes used a high-density monolayer culture
(1.75.times.10.sup.5 cells/cm.sup.2) or aggregated cell
clusters.
[0140] The loading of the cells with intermittent hydrostatic
pressure was carried out either in a continuous pattern over a
period of 24 hours or as interval loading with the load being
applied for four hours per day according to Example 3. The
metabolic response of the articular chondrocytes was determined
using semi-quantitative reverse transcription polymerase chain
reaction (RT-PCR) assays for type II collagen, aggrecan and
beta-actin mRNA signal. Results are seen in FIGS. 8-12.
[0141] FIG. 8 is a graph showing RT-PCR signal levels for type II
and type I collagen and beta-actin expression in unloaded high
activity chondrocyte cultures maintained throughout designated
testing periods 2-24 hours.
[0142] FIG. 9 shows signal levels for M-actin expression in cells
exposed to IHP and in unloaded control cells.
[0143] FIG. 8 shows signal levels determined by RT-PCR for type II
collagen and .beta.-actin expression in control cells obtained
without stimulation at 2, 4, 8, 12 and 24 hours according to
Example 3. RT-PCR conditions were as described in Example 4.
[0144] FIG. 8 shows RT-PCR signal levels for type II and type I
collagen and .beta.-actin expression in unloaded control cells is
clearly detectable and does not change.
[0145] Articular chondrocytes plated and maintained as high-density
monolayer cultures at atmospheric pressure (unloaded control
cultures) did not exhibit significant variation with respect to
signal levels for type II collagen mRNA or .beta.-actin mRNA. These
observations remained true over a time course of 2, 4, 8, 12 and 24
hours as seen in FIG. 8.
[0146] To determine a reference mRNA signal that would not change
in response to applied IHP and thus would provide a reference point
for comparison, .beta.-actin was selected due to its relative
abundance of mRNA and based on the location as an intracellular
cytoskeletal protein.
[0147] FIG. 9 shows RT-PCR signal levels for .beta.-actin
expression in cells exposed to intermittent hydrostatic pressure
and in unloaded control cells.
[0148] As seen in FIG. 9, exposure of chondrocytes to intermittent
hydrostatic pressure did not alter .beta.-actin mRNA signal levels
over a time course of 2, 4, 8, 12 and 24 hours when compared to the
unloaded control cells.
[0149] In contrast, application of IHP to normal chondrocyte in
monolayer culture or aggregate culture demonstrated that type II
collagen mRNA signal was not pronounced at loading periods of 4 and
8 hours and decreased thereafter. On the other hand, aggrecan mRNA,
under the same conditions have shown different profile and
continued to increase for 24 hours.
[0150] The results pertaining to type II collagen expression
prompted examination of interval loading where IHP was applied for
4 hour periods followed by a period of recovery.
[0151] The chondrocyte culture aggregates were exposed to
intermittent hydrostatic pressure using continuous loading through
a twenty-four hour period. Results are seen in FIGS. 10 and 11.
[0152] FIG. 10 shows RT-PCR signal levels for aggrecan following
exposure of high-density monolayer cultures to intermittent
hydrostatic pressure using interval loading at 4 hours per day
followed by 20 hours recovery for 4 days.
[0153] FIG. 11 shows RT-PCR signal levels for type II collagen
following exposure of high-density monolayer cultures to
intermittent hydrostatic pressure using interval loading at 4 hours
per day followed by 20 hours recovery for 4 days.
[0154] Under these loading conditions, signal level of the aggrecan
mRNA increased approximately four times relative to the unloaded
controls (FIG. 10). The change in loading protocol resulted in a
about seven-fold increase in the type II collagen mRNA signal
relative to unloaded controls (FIG. 11).
[0155] The results of the studies described above and illustrated
in FIGS. 8-11 demonstrated that the chondrocytes responded to
changed loading protocol to a pattern that included exposure to
intermittent hydrostatic pressure for four hours followed by a
twenty hour period of recovery, the RT-PCR signal attributable to
type II mRNA was significantly elevated relative to signal
representing .beta.-actin mRNA. The aggrecan mRNA signal was also
elevated.
[0156] These data show that mechanical IHP loading with an
appropriate type of stimulus serves to modulate articular
chondrocyte matrix macromolecule expression. The obtained results
confirm that application of specific loading regimens facilitates
the repair and regeneration of articular cartilage and contributes
to the successful production of composite transplantable cartilage
grafts.
[0157] The documented effects of hydrostatic pressure on the
chondrocytes shows that the specific loading regiments provide an
effective stimulus to modulated cartilage extracellular matrix
macromolecules synthesis and to initiate reactions connected with
repair and regeneration of damaged cartilage and type II
collagen.
[0158] IV. Effects of Intermittent Hydrostatic Pressure on
TGF-.beta.1 and MMP-2 Expression in Osteoblast-like Cells
[0159] Mechanical stress generated during normal weight bearing
activities contributes to bone structure and function through a
specific cycle involving osteoblastic formation and osteoclastic
resorption. Maintenance of normal bone homeostasis in response to
loading history involves a number of hormones, cytokines and growth
factors.
[0160] Following the above described findings, this study
investigated whether intermittent hydrostatic pressure (IHP)
modulates expression of the pleiotrophic growth factor, namely,
transforming growth factor-beta 1 (TGF-.beta.1), in bone cells
MG-63, HOS TE85, and whether it functions as a mechanical signal
for modulation of bone cell metabolism. To this end, the effect of
IHP on osteoblast expression of TGF-.beta.1, which is known to
influence osteoblast proliferation and phenotypic expression of
bone specific proteins was tested. Additionally, expression of
matrix metalloproteinase MMP-2 was investigated.
[0161] Results are seen in FIGS. 12-14. FIGS. 12-14 illustrate the
effect of IHP on TGF-.beta. protein production or MMP-2 expression
in osteoblast-like cells MG-63.
[0162] FIG. 12 shows time course analysis for TGF-.beta.1 release
from MG-63 cells exposed to IHP at 10 MPa. FIG. 13 shows dose
response effects of IHP on the TGF-B1 release from MG-63 cells. The
bar and the vertical line in FIGS. 12 and 13 represent the
means.+-.SD, respectively (n=3), p<0.05 compared with
control.
[0163] The concentration of TGF-.beta.1 protein in the conditioned
medium of MG-63 was investigated by ELISA. With MG-63 cells, no
significant change in TGF-.beta.1 release was observed following a
12 hour exposure to IHP as shown in FIG. 12. However, as also seen
in FIG. 12, application of IHP for 24 and 48 hours decreased
release of TGF-.beta.1 from the MG-63 cells by 65% (p<0.05) and
53% (p<0.05), respectively, relative to the unloaded cells.
Similar results were observed for HOS TE85 cells (data not
shown).
[0164] A second series of studies seen in FIG. 13 examined the dose
response effect of IHP on TGF-.beta.1 expression with MG-63 cells.
A 12 hour exposure to IHP at a level of 1 and 10 MPA did not alter
TGF-.beta.1 release. After 24 hours application of IHP at either 1
MPA or 10 MPA, as seen in FIG. 13, TGF-.beta.1 release into the
medium was inhibited by 23% and 31% respectively, when compared to
unloaded cells.
[0165] FIG. 14 depicts a time course analysis for MMP-2, release
(pg/ml) from MG-63 cells exposed to IHP as quantified by ELISA.
[0166] FIG. 20 shows quantification of the release of MMP-2 in the
conditioned medium using ELISA. A similar pattern was observed in
the zymographic analysis (data not shown). As seen in FIG. 20,
following 24 and 48 hours of exposure to IHP, MMP-2 release was
significantly inhibited by 46% and 28%, respectively, relative to
controls. Application of IHP under the same conditions also
decreased release of TIMP-1 in the conditioned medium of the
osteoblast-like cells. Following 24 and 48 hours of exposure to
IHP, TIMP-1 release was significantly inhibited by 63% and 29%,
respectively, relative to the unloaded cells. MMP-1 and MMP-9 were
not detected in medium samples from cells exposed to IHP or
unloaded cells at any time period (data not shown).
[0167] Zymographic analysis illustrated showed that MMP-2 was the
prominent matrix metalloproteinase released by the MG-63. In the
presence of IHP zymography demonstrated an inhibition in the
release of the latent 72 kD form of MMP-2 and both of the activated
forms, 68 kD and 62 kD, in medium samples from cells exposed to IHP
for 24 and 48 hours when compared to samples from unloaded
cells.
[0168] VI. Therapeutic Utility
[0169] The therapeutic method of the invention is useful in the
fields of orthopedic surgery, rheumatology, sport and
rehabilitation medicine. The method of the invention permits
formation of de novo and regeneration of diseased or injured
cartilage, particularly articular cartilage. The methods permit
external treatment of cartilage in situ by applying externally a
device administering hydrostatic pressure to a diseased or injured
joint intermittently for several hours followed by the periods of
recovery. The method also permits ex vivo regeneration of cartilage
or usable cartilage grafts removed from the diseased or injured
joint, regenerating such cartilage or graft to a degree where both
the mechanical and biochemical properties of the cartilage are
restored to normal levels and replacing the graft into the joint
once cartilage collagen matrix is restored. Additionally, the
method permits the in vitro treatment of cartilage and bone cells
and cell cultures into functional tissue suitable for
transplantation and de novo formation and production of healthy
normally functioning cartilage and other mesenchymally-derived
cells.
Example 1
Chondrocyte Isolation
[0170] This example describes procedure used for isolation of
chondrocytes.
[0171] Adult bovine articular chondrocytes were isolated from full
thickness cartilage dissected from radiocarpal joints obtained
fresh from a local abattoir.
[0172] The cartilage cells were released from the matrix by
incubation in 15 ml of Dulbecco's modified Eagle's medium (DMEM)
containing gentamicin (50 ug/ml) and a mixture of bacterial
collagenases, type II and type IV, (Worthington, Freehold, N.J.) at
a final concentration of 0.6 mg/ml each in 15 ml of Dulbecco's
Modified Eagle's Medium/Ham's F12 (DMEM/F12, Gibco BRL, Grand
Island, N.Y.) containing 25 .mu.g/ml gentoamicin (Sigma, St. Louis,
Mo.). The cartilage samples were incubated for a total of 18 hours
at 37.degree. C. in 7.5% CO.sub.2 and 100% humidity to ensure
complete digestion. Chondrocytes released from matrix were filtered
through a nylon mesh filter to isolate single cells. The cells were
subsequently collected by repeated centrifugation at 600.times.g
with the cells being resuspended and collected in Dulbecco's
phosphate buffered saline (3.times.50 ml).
[0173] A final cell pellet was suspended in the serum-free DMEM/F12
medium and the cells counted in a hemacytometer with viability
assessed by Trypan Blue exclusion. Normal viability obtained for
chondrocytes under these conditions was greater than 95%.
[0174] Chondrocytes were then plated on 60 mM tissue plates and the
culture were maintained at 37.degree. C. in a humidified atmosphere
of 7.5% CO.sub.2 in air. For attachment in serum free conditions,
the individual plates were pre-treated overnight with poly-D-lysine
(Sigma, 0.1 mg/ml) and washed twice with PBS without calcium or
magnesium. Cells were plated at a density of 1.times.10.sup.5 cell
s/cm.sup.2.
Example 2
Serum Containing or Serum-Free Medium
[0175] This example describes a composition of serum containing or
serum-free medium.
[0176] Serum containing Dulbecco's modified Eagle's medium (DMEM)
contained dialyzed heat-inactivated fetal bovine serum at a
concentration of 10% v/v. Serum-free medium consisted of a 1:1
mixture of Ham's F12/DMEM supplemented with selenium, and
liposomes. Liposomes were prepared by dissolving lecithin,
cholesterol, sphingomyelin, and vitamin E acetate in 1 ml of 2:1
chloroform/methanol (vol/vol) which is dried under N.sub.2. One ml
of DMEM/F12 was added and the lipid mixture was then sonicated
3.times. for intervals of 3 minutes each, using a microtip with a
70% duty cycle. This liposome stock was made up at 1,000.times. the
final concentration, kept under N.sub.2, and stored at 4.degree. C.
In some experiments, ascorbate was added to the medium at a
concentration of 50 .mu.g/ml.
Example 3
Mechanical Loading with Intermittent Hydrostatic Pressures
[0177] This example describes loading protocol and conditions for
applying intermittent hydrostatic pressure.
[0178] Hydrostatic pressure was cyclically applied at a loading
dose of 10 MPA and at a frequency of 1 Hz. The intermittent
hydrostatic pressure was applied continuously with cells removed at
periods of 2, 4, 8, 12 and 24 hours, or through an interval loading
protocol with the cells removed after a 4 day period during which
intermittent hydrostatic pressure was applied for and limited to 4
hours per day followed by 20 hours of recover. This was repeated
daily for four days or more. Each experimental time point was
tested in triplicate and each experiment was carried out for a
minimum of three independent trials.
[0179] The pressure was generated with a commercially available
stainless steel pressure vessel interfaced to a servo-hydraulic
loading instrument seen in FIG. 1. The design provided for the
complete evacuation of air from the system so the application of
pressure was purely hydrostatic. The culture plates were loaded
within sterile heat-sealed bags containing forty-five ml of culture
medium (DMEM/F12 1:1 with 30 mM HEPES adjusted to pH 7.4 for pH
stability in the absence of carbon dioxide).
[0180] Temperature control was achieved by partial immersion of
hydrostatic loading vessel within a circulating water bath and
maintained at 37.degree. C. No measurable change in temperature
occurred over loading periods up to 96 hours. Control cultures were
maintained under identical conditions in heat sealed bags and
placed in an identical container placed in the same temperature
controlled water bath as the loaded cultures.
Example 4
Analysis of Aggrecan and Type II Collagen mRNA Signal Levels
(RT-PCR)
[0181] This example describes the procedure used for analysis of
mRNA signal levels of aggrecan and type II collagen by RT-PCR.
[0182] To permit multiple samples to be tested for each loading
condition, an experimental approach using semi-quantitative RT-PCR
was used for analysis of aggrecan and type II collagen mRNA signal
levels as described in J. Orthop. Res., 15:94 (1997) for MMP-9
expression. Immediately at the cessation of loading, total RNA from
the cells exposed to intermittent hydrostatic pressure and from the
unloaded cells was extracted from the cells by the quanidinium
isothiocynate method described in Biochemistry, 18:5296 (1979) with
a commercially available tri-reagent (Sigma, St. Louis, Mo.). A
typical yield of cellular RNA per 60-mm plate was 5 micrograms.
[0183] All RNA preparations were routinely screened on agarose gels
for integrity of ribosomal RNA. Total RNA concentrations was
determined by spectrophotometry and adjusted to 200 ng/ul for
reverse transcription using random hexamer priming. The mRNA sample
was converted to single stranded cDNA using m-MLV reverse
transcriptase (Gibco-BRL) in the presence of RNase inhibitor
(5-Prime, 3-Prime, Inc., Boulder, Co.) and in the presence of 500
uM dNTPs (Perkin Elmer Cetus, Norwalk, Conn.). The reaction was
carried out at 37.degree. C. for 15 minutes, 42.degree. C. for 10
minutes, 47.degree. C. for 10 minutes and finally raised to
99.degree. C. to inactivate the reverse transcriptase. The reaction
mixture was diluted 10.times. and used for PCR.
[0184] The target sequences in the reverse-transcripted cDNA
samples were amplified by PCR, using sequence-specific
oligonucleotide primers designed to yield approximately 200-bp
sequences that span different exons within the aggrecan (Anal.
Biochem., 225:356 (1995)) and type II collagen (Arch. Biochem.
Biophys., 314:90 (1994)) genes. The primer sets for aggrecan and
type II collagen were based on published sequence data for these
genes.
[0185] DNA size analysis and DNA sequencing of the specific
products determined the validity of the products generated using
the primer sets.
[0186] PCR was carried out with 1.0 ul of cDNA in a 0.5 ml reaction
tube containing 1.5 ul of PCR master mix; the reaction was
initiated at 65.degree. C. to avoid nonspecific annealing. The PCR
master mix contained 125 mM Tris HCl, 50 mM ammonium sulfate, 3.75
mM magnesium chloride, 62.5 mM dNTPs, 300 nM of each downstream and
upstream primers, and 0.625 U/ml Tfl DNA polymerase (Epicentre
Technologies, Madison, Wis.). .sup.32P-.alpha.-dCTP at 3,000
Ci/mmol (Amersham NEN-Corp.) was added to the master mix to make
0.1 mCi/ml final concentration for random radiolabelling of
amplified products. The total reaction volume at the start of PCR
was 2.5 ml.
[0187] For comparison of relative expression, 0.5 ml of a primer
solution containing 900 nM of an oligonucleotide primer set for
amplification of the 3' untranslated region of .beta.-actin, 50 mM
Tris HCL, 20 mM ammonium sulfate, 1.5 mM magnesium chloride was
added at the tenth cycle and amplified in the same reaction tube.
.beta.-actin mRNA signal served as an internal control to monitor
for tube-to-tube variations in amplification conditions and
differences in the initial concentration or loading of cDNA. The
thermocycle program included one cycle of 95.degree. C. for 3
minutes for initial heating, followed by repeated cycles of
95.degree. C. for 1 minute and 65.degree. C. for 1 minute. Final
extension was carried out at 72.degree. C. for 5 minutes. The total
cycle number employed in this study was 30 cycles for aggrecan and
type II collagen and 26 cycles for .beta.-actin.
[0188] The amplified products from PCR were separated on 5%
polyacrylamide gels and the gels were directly analyzed using the
PhosphoImager (Molecular Dynamics, Sunnyvale, Calif.) Relative
expression of the mRNA was expressed as specific signal levels and
as a ratio of signal of the aggrecan and type II to the
.beta.-actin signal.
Example 5
Statistical Methods
[0189] This example describes the details of statistical methods
used for evaluation of results obtained in Examples 3 and 4.
[0190] Significance of differences between loaded and unloaded
samples were examined using the general linear method for one-way
analysis of variance (NOVA) with the addition of Tukey's correction
for multiple comparison testing (SAS, Gary, N.C.).
[0191] Determination of the mRNA signal levels by semi-quantitative
RT-PCR techniques provided sufficient differences between treated
and untreated samples so that a power level was achieved to
determine significance at p<0.05 with five independent trials.
In the case of the mRNA quantification, the hypothesis being tested
was that a change in matrix gene expression occurred relative to
expression of .beta.-actin. The .beta.-actin expression was
determined not to change in response to intermittent hydrostatic
pressure. This permitted paired t-test to be used to test for
significance from the different culture samples.
Example 6
Human Osteoblast Cell Culture
[0192] This example describes procedure used to prepare human
osteoblast cell culture.
[0193] Human osteoblast-like cells, MG-63 and HOS TE85, were
purchased from the American Type Culture Collection (Manassas,
Va.).
[0194] The cells were cultured in 60 mM dishes with alpha minimal
essential medium (alpha-MEM) (Gibco, Grand Island N.Y.) containing
10% fetal bovine serum (Gibco) and antibiotic-antimycotic (Gibco;
100 U/ml penicillin, 100 .mu.g/ml streptomycin, 0.25 .mu.g/ml
amphotericin B), at 37.degree. C. in an atmosphere of air
containing 5% CO.sub.2.
[0195] Confluent cultures were incubated for an additional 24 hours
in the absence of serum to establish growth arrest. Culture medium
was removed and each culture plate was placed into a heat sealed
bag containing 40 ml of serum-free alpha-MEM supplemented with 0.1%
BSA, 15 mM HEPES, ascorbic acid (50 .mu.g/ml) and
Na-.beta.-glycerolphosphate (10 mM). The heat sealed bags were
immersed in a high pressure vessel filled with water and IHP (1 or
10 MPA at a frequency of 1 Hz) was applied for 12, 24 and 48
hours.
[0196] Control cultures were maintained at atmospheric pressure.
Both the pressure vessel and unloaded control cultures were
maintained at atmospheric pressure. Both the pressure vessel and
unloaded control cultures were maintained in the water bath during
testing period to maintain the temperature at 37.degree. C. Culture
medium samples were collected at the time periods specified in the
figure legends of FIGS. 13-18 and stored at -20.degree. C. until
use.
[0197] To study the mRNA stability, actinomycin D (2.5 mg/ml) was
dissolved in methanol and applied to cultures at a final
concentration of 5 .mu.M.
[0198] Human chondrocytes were obtained in the same way but were
pretreated with trypsin 0.1 mg/ml to remove protease and collagen
inhibitors.
Example 7
Measurement of TGF-.beta. Protein Level in the Conditioned
Medium
[0199] This example describes a method used for measurement of
TGF-.beta. protein level in the medium.
[0200] Matched pair antibodies against TGF-.beta.1 were purchased
from R & D Systems (Minneapolis, Minn.). Latent TGF-.beta.1 in
the conditioned medium was activated by 1N HCl and conditioned
medium samples were neutralized by the addition of 1.2 M NaOH/0.5 M
HEPES buffer. Total TGF-.beta.1 was measured using enzyme-linked
immunosorbent assays (ELISAs). All samples were analyzed in
triplicate. The optical densities were determined on an ELISA
reader at 450 nM with background correction at 595 nM.
Example 8
Northern Blot Analysis for TGF-.beta.
[0201] This example describes conditions used for TGF-.beta.
Northern blot analysis.
[0202] Total RNA was extracted from the cells by the method
described in Anal. Biochem., 162:156 (1987).
[0203] For a Northern blot analysis, 10 .mu.g of total RNA was
denatured and fractioned on a 1% agarose 1.1 M formaldehyde gel and
stained with ethidium bromide to determine the integrity of the 28S
and 18S bands. Transfer to a nylon membrane was performed by
standard methods. The membranes were hybridized to .sup.32P-labeled
cDNA probes for TGF-.beta.1 (ATCC) and GAPDH (ATCC).
[0204] The radioactivity of the each hybridized probe was analyzed
using a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).
Example 9
Method for Statistical Analysis
[0205] This example describes the method for statistical analysis
of studies described in Examples 7 and 8.
[0206] The significance between treated and control groups was
determined using ANOVA and unpaired Student's two-sample t-test
(two-tailed) using Bonferroni approximation for multiple
comparisons. All values are given as the means.+-.SD. Northern
blotting results were expressed as a ratio of the target mRNA
signal (TGF-.beta.1) over the housekeeping mRNA signal (GAPDH).
Differences in mRNA ratios observed with and without IHP treatment
were assessed using the Mann-Whitney U-test with a p<0.05
representing significance.
Example 10
Zymography
[0207] This example describes conditions used for zymography.
[0208] Medium samples were activated by incubating in 1.0 mM
4-aminophenylmercuric acetate (APMA) for one hour at 37.degree. C.
The samples were subsequently mixed with sample buffer and run in
10% SDS-polyacrylamide gels impregnated with 1 mg/ml gelatin. The
gels were washed with water and soaked for one hour in 2.5% Triton
X-100 in water. After a sixteen hour exposure at 37.degree. C. in
substrate buffer (0.05 M Tris-HCl, pH 8.0, 5 mM CaCl.sub.2, and
0.02 NaN.sub.3) the gels were stained in a solution of 0.5%
Commassie Blue R-250 in 30% ethanol and 10% acetic acid, then
destained in water to visualize gelatinolytic activity.
Example 11
Measurement of Peptide of MMPs and TIMP-1 in the Conditioned
Medium
[0209] This example describes a method used for determination of
presence of MMP-1, MMP-2, MMP-9 and TIMP-1 proteins in medium.
[0210] At the completion of loading, the medium samples were
collected from each bag and the concentration of MMP-1, MMP-2,
MMP-9 and TIMP-1 proteins were measured using enzyme-linked
immunosorbent assay (ELISA) kits (Oncogene, Cambridge, Mass.)
according to the manufacturer's instructions.
Example 12
Northern Blot Analysis for MMP-2 and TIMP-1
[0211] This example describes the conditions for Northern blot
analysis for MMP-2 and TIMP-1.
[0212] Total RNA was extracted from the cells by the same method as
described in Example 8. For a Northern blot analysis, 10 .mu.g of
total RNA was denatured and fractionated on a 1% agarose 1.1 M
formaldehyde gel and stained with ethidium bromide to determine the
integrity of the 28S and 18S bands. Transfer to a nylon membrane
was performed by standard methods.
[0213] The membranes were hybridized to .sup.32P-labeled cDNA
probes for MMP-2 and TIMP-1 (ATCC, Manassas, Va.). The
radioactivity of the each hybridized probe was analyzed using a
Phosphor Imager (Molecular Dynamics, Sunnyvale, Calif.).
Example 13
Determination of mRNA Stability
[0214] This example describes a method used for determination of
mRNA stability.
[0215] The stability of the mRNA following a mechanical loading
test period of 24 hours was determined by the addition of
actinomycin D to the cells according to Example 6.
[0216] Actinomycin D was added at a concentration of 2 micrograms
per milliliter in four milliliters of serum free culture medium.
The cells were maintained for test periods of 0, 1, 2 and 4 hours
prior to isolation of total cellular RNA. The analysis of mRNA
signal levels was as described in Example 11 for Northern blotting.
Stability of mRNA in control cultures was determined as described
with cells not exposed to IHP.
Example 14
Statistical Analysis
[0217] This example describes a method for statistical analysis of
results obtained in studies described in Examples 10-12.
[0218] The significance between treated and control groups was
determined using ANOVA and unpaired Student's two-sample t-test
(two-tailed) using Bonferroni approximation for multiple
comparisons. All values are given as the means.+-.SD.
* * * * *