U.S. patent application number 11/433968 was filed with the patent office on 2006-09-28 for composition and device for in vivo cartilagerepair.
Invention is credited to Brent L. Atkinson, James J. Benedict, Pedro Bittman, Donald Chickering, John Ranieri, Marsha L. Whitney.
Application Number | 20060216325 11/433968 |
Document ID | / |
Family ID | 8230340 |
Filed Date | 2006-09-28 |
United States Patent
Application |
20060216325 |
Kind Code |
A1 |
Atkinson; Brent L. ; et
al. |
September 28, 2006 |
Composition and device for in vivo cartilagerepair
Abstract
The composition as described serves for in vivo cartilage
repair. It basically consists of a naturally derived osteoinductive
and/or chondroinductive mixture of factors (e.g. derived from bone)
or of a synthetic mimic of such a mixture combined with a
nanosphere delivery system. A preferred mixture of factors is the
combination of factors isolated from bone, known as BP and
described by Poser and Benedict (WO 95/13767). The nanosphere
delivery system consists of nanospheres defined as polymer
particles of less than 1000 nm in diameter (whereby the majority of
particles preferably ranges between 200-400 nm) in which
nanospheres the combination of factors is encapsulated. The
nanospheres are loaded with the mixture of factors in a weight
ratio of 0.001 to 17% (w/w), preferably of 1 to 4% (w/w) and have a
release profile with an initial burst of 10 to 20% of the total
load over the first 24 hours and a long time release of at least
0.1 per day during at least seven following days. The nanospheres
are composed of e.g. ((D,L)-lactic acid/glycolic acid)-copolymer
(PLGA). The loaded nanospheres are e.g. made by phase inversion.
The composition is advantageously utilized as a device comprising
any biodegradable matrix in which the nanospheres loaded with the
factor combination is contained.
Inventors: |
Atkinson; Brent L.;
(Lakewood, CO) ; Bittman; Pedro; (Zurich, CH)
; Benedict; James J.; (Golden, CO) ; Ranieri;
John; (Austin, TX) ; Whitney; Marsha L.;
(Austin, TX) ; Chickering; Donald; (Framingham,
MA) |
Correspondence
Address: |
WILLIAMS, MORGAN & AMERSON
10333 RICHMOND, SUITE 1100
HOUSTON
TX
77042
US
|
Family ID: |
8230340 |
Appl. No.: |
11/433968 |
Filed: |
May 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10386946 |
Mar 11, 2003 |
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11433968 |
May 15, 2006 |
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09485594 |
Sep 13, 2000 |
6582471 |
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PCT/EP98/05100 |
Aug 12, 1998 |
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10386946 |
Mar 11, 2003 |
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Current U.S.
Class: |
424/425 ;
623/23.61 |
Current CPC
Class: |
A61L 27/54 20130101;
A61F 2002/30677 20130101; A61F 2210/0004 20130101; A61P 19/00
20180101; A61F 2002/30062 20130101; A61L 27/227 20130101; A61L
2430/06 20130101; A61B 17/06166 20130101; A61F 2250/0035 20130101;
A61F 2310/00365 20130101; A61F 2002/30036 20130101; A61F 2/30756
20130101; A61L 27/48 20130101; A61L 2400/12 20130101; Y10S 977/91
20130101; A61L 27/24 20130101; A61L 27/26 20130101; C08L 5/08
20130101; C08L 89/06 20130101; A61K 9/5153 20130101; C08L 89/06
20130101; A61L 27/48 20130101; A61L 27/50 20130101; A61F 2002/2817
20130101; B82Y 5/00 20130101; A61L 27/48 20130101; A61L 2300/30
20130101; A61L 2300/252 20130101; A61L 27/26 20130101; A61L
2300/624 20130101; Y10S 977/914 20130101 |
Class at
Publication: |
424/425 ;
623/023.61 |
International
Class: |
A61F 2/28 20060101
A61F002/28 |
Claims
1-23. (canceled)
24. A composition comprising: a chondroinductive protein mixture;
and a delivery system having an initial first release rate (% total
protein mixture load/time) greater than a subsequent second release
rate (% total protein mixture load/time).
25. The composition of claim 1, wherein the first rate is greater
than 10% per day.
26. The composition of claim 1, wherein the second rate is less
than 1% per day.
27. The composition of claim 1, wherein the delivery system has a
third release rate.
28. The composition of claim 4, wherein the third release rate is
greater than 0.1% per day.
29. The composition of claim 4, wherein the third release rate
occurs temporally before the second release rate.
30. The composition of claim 1, wherein the protein mixture
comprises a bone derived protein.
31. The composition of claim 1, wherein the delivery system
comprises nanospheres.
32. The composition of claim 1, further comprising a matrix.
33. The composition of claim 9, wherein the matrix comprises
collagen.
34. A composition comprising: a chondroinductive protein mixture;
and a delivery system having an initial first release rate greater
than 10% total protein mixture load per day and a subsequent second
release rate less than 1% total protein mixture load per day.
35. The composition of claim 10, wherein the delivery system has a
third release rate.
36. The composition of claim 12, wherein the third release rate is
greater than 0.1% per day.
37. The composition of claim 12, wherein the third release rate
occurs temporally before the second release rate.
38. The composition of claim 10, wherein the protein mixture
comprises a bone derived protein.
39. The composition of claim 10, wherein the delivery system
comprises nanospheres.
40. The composition of claim 10, further comprising a matrix.
41. The composition of claim 17, wherein the matrix comprises a
material selected from the group consisting of type I collagen,
type II collagen and hyaluronic acid.
42. The composition of claim 17, wherein the matrix comprises a
material selected from the group consisting of type I collagen.
43. A composition comprising: a bone derived chondroinductive
protein mixture; a biodegradable matrix including collagen; and a
nanosphere delivery system having an initial first release rate
greater than 10% total protein mixture load per day and a
subsequent second release rate less than 1% total protein mixture
load per day.
Description
BACKGROUND OF THE INVENTION
[0001] Articular cartilage, an avascular tissue found at the ends
of articulating bones, has no natural capacity to heal. During
normal cartilage ontogeny, mesenchymal stem cells condense to form
areas of high density and proceed through a series of developmental
stages that ends in the mature chondrocyte. The final hyaline
cartilage tissue contains only chondrocytes that are surrounded by
a matrix composed of type II collagen, sulfated proteoglycans, and
additional proteins. The matrix is heterogenous in structure and
consists of three morphologically distinct zones: superficial,
intermediate, and deep. Zones differ among collagen and
proteoglycan distribution, calcification, orientation of collagen
fibrils, and the positioning and alignment of chondrocytes (Archer
et al., J. Anat. 189(1): 23-35, 1996; Morrison et al., J. Anat.
189(1): 9-22 1996, Mow et al., Biomaterials 13(2): 67-97, 1992).
These properties provide the unique mechanical and physical
parameters to hyaline cartilage tissue.
[0002] In 1965, a demineralized extraction from bovine long bones
was found to induce endochondral bone formation in the rat
subcutaneous assay (Urist Science 150: 893-899, 1965). Seven
individual factors, termed Bone Morphogenetic Proteins (BMPs), were
isolated to homogeneity and, because of significant sequence
homology, classified as members of the TGF.beta. super-family of
proteins (Wozney, et al., Science 242: 1528-34, 1988; Wang et al.,
Proc. Nat. Acad. Sci. 87: 2220-2224, 1990). These individual,
recombinantly-produced factors also induce ectopic bone formation
in the rat model (Luyten et al., J. Biol. Chem. 264: 13377-80,
1989; Celeste et al., Proc. Nat. Acad. Sci. 87: 9843-50, 1990). In
addition, in vitro tests have demonstrated that both BMP-2 and
TGF.beta.-1 induce mesenchymal stem cells to form cartilage
(Denker, et al., Differentiation 59(1): 25-34, 1995; Denker et al.,
41st Ann. Orthop. Res. Society 465: 1995). Both BMP-7 and BMP-2
have been shown to enhance matrix production of chondrocytes in
vitro (Flechtenmacher J. Arthritis Rheum. 39(11): 1896-904, 1996:
Sailor et al., J. Orthop. Res. 14: 937-945, 1996). From these data
we can conclude that not only are the BMPs important regulators of
osteogenesis, but that they also play crucial roles during
chondrogenic development in vitro.
[0003] A partially-purified protein mixture from bovine long bones,
termed BP (Bone Protein), also induces cartilage and bone formation
in the rat subcutaneous assay (Poser and Benedict, WO95/13767). BP
in combination with calcium carbonate promotes bone formation in
the body. In vitro, BP induces mesenchymal stem cells to
differentiate specifically to the cartilage lineage, in high
yields, and to late stages of maturation (Atkinson et al., J.
Cellular Biochem. 65: 325-339, 1997).
[0004] The molecular mechanism for cartilage and bone formation has
been partially elucidated. Both BMP and TGF.beta. molecules bind to
cell surface receptors (the BMP/TGF.beta. receptors), which
initiates a cascade of signals to the nucleus that promotes
proliferation, differentiation to cartilage, and/or differentiation
to bone (Massague Cell 85: 947-950, 1996).
[0005] In 1984, Urist described a substantially pure, but not
recombinant BMP, combined with a biodegradable polylactic acid
polymer delivery system for bone repair (U.S. Pat. No. -4,563,489).
This system blends together equal quantities of BMP and polylactic
acid (PLA) powder (100 .mu.g of each) and decreases the amount of
BMP required to promote bone repair.
[0006] Hunziker (U.S. Pat. No. -5,368,858; U.S. Pat. No.
-5,206,023) describes a cartilage repair composition consisting of
a biodegradable matrix, a proliferation and/or chemotactic agent,
and a transforming factor. A two stage approach is used where each
component has a specific function over time. First, a specific
concentration of proliferation/chemotactic agent fills the defect
with repair cells. Secondly, a larger transforming factor
concentration transforms repair cells into chondrocytes. Thereby
the proliferation agent and the transforming agent may both be
TGF.beta. differing in concentration only. In addition, the patent
discloses a liposome encapsulation method for delivering
TFG.beta.-1 serving as transformation agent.
[0007] Hattersley et al. (WO 96/39170) disclose a two factor
composition for inducing cartilaginous tissue formation using a
cartilage formation-inducing protein and a cartilage maintenance
inducing protein. Specific recombinant cartilage formation inducing
protein(s) are specified as BMP-13, MP-52, and BMP-12, and
cartilage maintenance-inducing protein(s) are specified as BMP-9.
In one embodiment, BMP-9 is encapsulated in a resorbable polymer
system and delivered to coincide with the presence of cartilage
formation inducing protein(s).
[0008] Laurencin et al., (U.S. Pat. No. -5,629,009) disclose a
chondrogenesis-inducing device, consisting of a polyanhydride and
polyorthoester, that delivers water soluble proteins derived from
demineralized bone matrix, TGF.beta., EGF, FGF, or PDGF.
[0009] The results of the approaches to cartilage repair as cited
above are encouraging but they are not satisfactory. In particular,
the repair tissue arrived at is not fully hyaline in appearance
and/or it does not contain the proper chondrocyte organization.
Furthermore, previous approaches to cartilage repair have been
addressed to very small defects and have not been able to solve
problems associated with repair of large, clinically relevant
defects.
[0010] One reason that previous approaches failed to adequately
repair cartilage may be that they were not able to recapitulate
natural cartilage ontogeny faithfully enough, this natural ontogeny
being based on a very complicated system of different factors,
factor combinations and factor concentrations with temporal and
local gradients. A single recombinant growth factor or two
recombinant growth factors may lack the inductive complexity to
mimic cartilage development to a sufficient degree and/or the
delivery systems used may not have been able to mimic the gradient
complexity of the natural system to a satisfactory degree.
[0011] Previous approaches may also have failed because growth
factor concentrations were not able to be maintained over a
sufficient amount of time, which would prevent a full and permanent
differentiation of precursor cells to chondrocytes. The loss of
growth factor could be caused by diffusion, degradation, or by
cellular internalization that bypasses the BMP/TGF.beta. receptors.
Maintaining a sufficient growth factor concentration becomes
particularly important in repair of large sized defects that may
take several days or several weeks to fully repopulate with
cells.
[0012] The object of this invention is to create a composition for
improved cartilage repair in vivo. The inventive composition is to
enable in vivo formation of repair cartilage tissue which tissue
resembles endogenous cartilage (in the case of articular cartilage
with its specific chondrocyte spatial organization and superficial,
intermediate, and deep cartilage zones) more closely than repair
tissue achieved using known compositions for inducing cartilage
repair. A further object of the invention is to create a device for
cartilage repair which device contains the inventive
composition.
[0013] This object is achieved by the composition and the device as
defined by the claims.
BRIEF DESCRIPTION OF THE INVENTION
[0014] The inventive composition basically consists of a naturally
derived osteo-inductive and/or chondroinductive mixture of factors
(e.g. derived from bone) or of a synthetic mimic of such a mixture
combined with a nanosphere delivery system. A preferred mixture of
factors is the combination of factors isolated from bone, known as
BP and described by Poser and Benedict (WO 95/13767). The
nanosphere delivery system consists of nanospheres defined as
polymer particles of less than 1000 nm in diameter (whereby the
majority of particles preferably ranges between 200-400 nm) in
which nanospheres the combination of factors is encapsulated. The
nanospheres are loaded with the mixture of factors in a weight
ratio of 0.001 to 17% (w/w), preferably of 1 to 4% (w/w) and have
an analytically defined release profile (see description regarding
FIG. 2) showing an initial burst of 10 to 20% of the total load
over the first 24 hours and a long time release of at least 0.1 per
day during at least seven following days, preferably of 0.1 to 1%
over the following 40 to 60 days. The nanospheres are composed of
e.g. (lactic acid-glycolic acid)-copolymers (Poly-(D,L)lactic
acid-glycolic acid) made of 20 to 80% lactic acid and 80 to 20% of
glycolic acid, more preferably of 50% lactic acid and 50% of
glycolic acid.
[0015] The loaded nanospheres are e.g. made by phase inversion
according to Mathiowitz et al. (Nature, 386: 410-413, 1997) or by
other methods known to those skilled in the art (Landry, Ph.D
Thesis, Frankfurt, Germany).
[0016] The inventive composition is advantageously utilized as a
device comprising any biodegradable matrix including collagen type
I and II, and hyaluronic acid in which matrix the nanospheres
loaded with the factor combination is contained. The matrix can be
in the form of a sponge, membrane, film or gel. The matrix should
be easily digestible by migrating cells, should be of a porous
nature to enhance cell migration, and/or should be able to
completely fill the defect area without any gaps.
[0017] It is surprisingly found that the inventive composition
consisting of an osteo-inductive and/or chondroinductive
combination of factors (e.g. derived from natural tissue)
encapsulated in nanospheres as specified above, if applied to a
defect area of an articular cartilage, leads to the transformation
of virtually all precursor cells recruited to the repair area to
chondrocytes, and furthermore, leads to a homogenous chondrocyte
population of the repair area and to a chondrocyte order and
anisotropic appearance as observed in endogenous hyaline cartilage.
These findings encourage the prospect that the inventive
composition may lead to significant improvements also regarding
repair of large defects.
[0018] As mentioned above, instead of an osteoinductive and/or
chondroinductive mixture of factors derived from bone (BP), the
inventive composition may comprise natural factor mixtures derived
from other tissues (e.g. cartilage, tendon, meniscus or ligament)
or may even be a synthetic mimic of such a mixture having an
osteoinductive and/or chondroinductive effect. Effective mixtures
isolated from natural tissue seem to contain a combination of
proliferation, differentiation, and spatial organizing proteins
which in combination enhance the tissue rebuilding capacity more
effectively than single proteins (e.g. recombinant proteins).
[0019] The specified, analytically defined release profile of such
factor mixtures from nanospheres results in the formation of
concentration gradients of proliferation and differentiation
factors, which obviously mimics the complex gradients of factors
observed during natural development very well. The nanosphere
extended release profile is sufficient to provide growth factor
during the time frame that repair cells arrive into the matrix. The
release profile obviously leads to a homogenous population of a
matrix with precursor cells, to full differentiation of virtually
all of the precursor cells to chondrocytes, and to the formation of
an endogenous hyaline cartilage structure.
[0020] Another advantage of the inventive composition is that when
the nanospheres are placed in a matrix to form a device for
cartilage repair, they are randomly distributed and remain in place
when in a joint cartilage defect. During cellular infiltration and
differentiation, the nanospheres are in the correct position over
the correct time frame.
[0021] Nanospheres have been demonstrated to adhere to the
gastrointestinal mucus and cellular linings after oral ingestion
(Mathiowitz et al., Nature, 386 410-413 1997). We envisage that
nanospheres also adhere to cartilage precursor cells and
furthermore, may also adhere to BMP/TGF.beta. receptors located on
the cell membrane. This property allows localized high-efficiency
delivery to the target cells and/or receptors. Because of the
nanosphere small size and the chemical properties, they are more
effective than liposomes or diffusion controlled delivery systems.
The efficient delivery to the receptors will facilitate
chondrogenesis.
[0022] Derived from the above findings, we envisage the following
mechanism for cartilage repair using the effect of the inventive
composition. During the first 24 hours (initial burst) 10 to 20% of
the total load of the factor mixture is released from the
nanospheres into the matrix and diffuses into the synovial
environment. Following the initial burst, the nanospheres begin to
release factors at a slow rate, which produces gradients of
proliferation, differentiation, and spatial organizing proteins. In
response to such gradients, precursor cells migrate to the defect
site. The loaded nanospheres adhere to cartilage precursor cells
and to the BMP and TGF.beta. receptors to provide localized highly
efficient delivery. The precursor cells become differentiated to
chondrocytes and secrete type II collagen and cartilage-specific
proteoglycans. The composition of the present invention stimulates
differentiation of virtually all of these cells to overt
chondrocytes and induces an ordered cartilage structure which
closely resembles hyaline cartilage. Furthermore, we envisage that
this release system will allow homogenous repair of large defect
sites and repair of defects from patients with low quantities of
precursor cells.
[0023] For in vivo cartilage repair, the inventive device
consisting of a matrix and the loaded nanospheres is placed in a
chondral lesion that was caused by trauma, arthritis, congenital,
or other origin. The damage can result in holes or crevices or can
consist of soft, dying, or sick cartilage tissue that is removed
surgically prior to implantation of the device. Because of the
unique properties of the inventive device precursor cells populate
the matrix, differentiate to chondrocytes, and form hyaline
cartilage.
[0024] Application of the inventive composition (without matrix)
e.g. by injection can be envisaged also, in particular in the case
of small defects. Thereby at least 2 .mu.g of the composition per
ml of defect size is applied or at least 20 ng of the
osteoinductive and/or chondroinductive mixture encapsulated in the
nanospheres is applied per ml defect size.
[0025] The inventive composition and the inventive device are
suitable for repair of cartilage tissue in general, in particular
for articular cartilage and for meniscus cartilage.
BRIEF DESCRIPTION OF THE FIGURES
[0026] The following figures illustrate the physical and chemical
parameters of the inventive composition, the in vitro cartilage
inductive activity of BP released from nanospheres and in vivo
repair of an articular cartilage defect using the inventive
device.
[0027] FIG. 1 shows a scanning electron micrograph of BP-loaded
nanospheres;
[0028] FIG. 2 shows the release profile (cumulative release vs.
time) of the inventive composition;
[0029] FIG. 3 shows the release profile of the inventive
composition compared with release profiles of nanosphere delivery
systems loaded with other proteins;
[0030] FIG. 4 shows the volume of a cartilage defect vs. the days
required for populating the defect with repair cells;
[0031] FIG. 5 shows micromass cultures in the presence or absence
of nanospheres loaded with BP;
[0032] FIG. 6 shows cartilage marker analyses for in vitro cultures
containing BP only and for similar cultures containing the
inventive composition;
DETAILED DESCRIPTION OF THE INVENTION
[0033] FIG. 1 shows a scanning electron micrograph of BP-loaded
nanospheres. The microparticle sizes range from 100-1000 nm with
the majority of individual particles ranging between 200-400
nm.
[0034] The release rate profile of the inventive composition was
determined by in vitro analysis of BP delivered from nanospheres.
These nanospheres were made by phase inversion according to the
method as disclosed by Mathiowitz et al. (Nature 386, 410-414,
1997) of ((DL)lactic acid/glycolic acid)-copolymer containing the
two acids in a weight ratio of 50:50 and they were loaded with 1%
and with 4% of BP.
[0035] For determination of the release rate profile, the
nanospheres were placed in a sterile saline solution and incubated
at 37.degree. C. BP released into the supernatant was measured
using a BCA assay (Pierce). BP released from the nanospheres as
specified shows two successive and distinct profile parts: a fast
release (initial burst) of approximately 10 to 20% of the loaded BP
over the first 24 hours and a slow release of 0.1 to 1% per day
(cumulative 40% to 50%) over 40 to 60 days (FIG. 2).
[0036] The release is intermediate between zero-order and
first-order kinetics. Both the 1% and 4% encapsulated BP have
similar release profiles.
[0037] For attaining release rate profiles as specified above and
as necessary for the improved results in cartilage repair the
nanospheres are to be adapted accordingly when using factor
mixtures other than BP. Thereby, e.g the composition of the
nanosphere copolymer, the molecular weight of the polymer molecules
and/or the loading percentage of the nanospheres may be changed.
The optimum nanosphere character for each specific case has to be
found experimentally whereby the release rate profile is analyzed
in vitro as described above.
[0038] In the same way, the nanosphere delivery system can be
modified regarding the percentage of BP to be released in the first
24 hours, percentage of BP to be released after 24 hours and/or
length of time after the first 24 hours during which the remainder
of BP is released. In addition, the percentage of BP loaded to the
nanospheres is of course variable too, whereby for obtaining the
results as described for the specified composition. all the
modifications are to be chosen such that the resulting delivery
keeps within the range as specified.
[0039] All of the above parameters can be modified to account for
the patient's age, sex, diet, defect location, amount of blood
present in the defect, and other clinical factors to provide
optimal cartilage repair. For example, nanospheres with longer
release rates are used for treating larger defects and/or for
patients with fewer precursor cells (e.g. older patients or
patients with degenerative symptoms). In contrast, patients with
larger quantities of progenitor cells and/or smaller defects may
require a shorter release rate profile.
[0040] FIG. 3 shows the release profile as shown in FIG. 2 for
nanospheres as specified above loaded with BP and with other
proteins (same loading percentages) such as BSA (bovine serum
albumin) or lysozyme. The drastically different release
characteristics shows that the profile is dependent on the protein
type also. Tne same is valid for a more hydrophobic mixture of
bovine bone derived proteins (PIBP).
[0041] FIG. 3 illustrates the singularity of the inventive
combination consisting of the specific delivery system (nanospheres
as specified above encapsulating the factors) and the specific
protein mixture (BP) which is obviously the key to the improved
results in cartilage repair as observed when using the inventive
composition or device.
[0042] To determine the length of time required for precursor cell
repopulation of different sized defects, the following calculation
was performed. We estimate that approximately 50,000 cells are
recruited to the defect/day. Since the cellular density of
cartilage is about 4.times.10.sup.7 cells/ml, a 10 .mu.l volume
defect will take approximately 8 days to fill with cells. FIG. 4
plots the number of days required to fill different volume defects
with cells. The Figure assumes an infinite supply of cells and a
constant rate of cell attraction to the defect site, The graph
demonstrates that the larger a defect size is, the more time is
required to completely fill it with cells. Since a 60 .mu.l volume
defect will take over 45 days to fill, this Figure demonstrates the
necessity for a long term release of factors to induce
differentiation of the precursor cells over up to a two month
period.
[0043] To determine whether BP bioactivity is harmed by the
encapsulation process and to determine whether the released BP was
fully bioactive, the following assay was performed. Previously, it
was demonstrated that 10T1/2 micromass cultures exposed to BP
induce formation of a three dimensional spheroid structure that can
be observed macroscopically in tissue culture wells (Atkinson et
al., J. Cellular Biochem. 65: 325-339, 1997). BP concentrations
equal or greater than 20 ng/ml were required for spheroid
formation. No spheroid forms in the absence of BP or at
concentrations less than 10 ng/ml (see following table). In this
assay, 10T1/2 mesenchymal stem cells act as in vitro models for the
precursor cells recruited to a natural defect.
[0044] We employed the same assay to test the bioactivity of BP
released from 1% loaded nanospheres. BP was eluted from nanospheres
at 37.degree. C. in a 5% CO.sub.2 humidified incubator. After 24
hours 16% BP is released; and between 24 hours and 7 days, 7% BP
was released (FIG. 2). The supernatant was collected, serial
dilutions were made, and the supernatant was added to 10T1/2
micromass cultures. BP released from nanospheres at both time
points formed spheroids at concentrations greater than 20 ng/ml,
but not at concentrations between 0 and 10 ng/ml (see following
table). Non-encapsulated BP also formed spheroids at concentrations
greater than 20 ng/ml, but not at concentrations between 0 and 10
ng/ml. We conclude that both nanosphere encapsulation and release
of BP does not inhibit BP bioactivity.
[0045] Spheroid formation (-=no spheroid formation; +=spheroid
formation): TABLE-US-00001 BP concentration (ng/ml) state of used
BP 0-10 20-1000 non-encapsulated BP - + released from nanospheres
(24 h) - + released from nanospheres (168 h) - +
[0046] To determine the effect of BP slow release in the direct
presence of micromass cultures, the following assay was performed.
Nanospheres were washed for 24 hours and the supernatant was
discarded. The nanospheres were then added to micromass cultures at
a quantity such that 10 or 25 ng/ml of BP would be released over 24
hours. Release of 25 ng/ml resulted in spheroid formation whereas
release of 10 ng/ml did not form spheroids (FIG. 5). Similarly, the
addition of 10 ng of non-encapsulated BP per ml did not form a
spheroid whereas the addition of 25 ng of non-encapsulated BP per
ml did form a spheroid. Regarding the specific in vitro set-up, we
conclude that slow release of BP over 24 hours is as effective as a
single dose of BP.
[0047] To determine whether the BP released from nanospheres was as
chondrogenic as non-encapsulated BP, spheroids were analyzed for
type II collagen and proteoglycan content. 10T1/2 spheroids from
the above assay that had formed with 1 .mu.g of released BP per ml
or 1 .mu.g of non-encapsulated BP per ml were tested histologically
with Azure and H+E stains and immunocytochemically with antibodies
to type II collagen after 7 days. Both encapsulated and
non-encapsulated BP induced cartilage markers such as type II
collagen, proteoglycan, and round cell shape (FIG. 6). In addition,
no qualitative differences were observed between encapsulated and
non-encapsulated BP with respect to cell quantity, viability,
morphology, or organization (FIG. 6). We conclude that BP retains
full chondrogenic capacity after release from nanospheres.
[0048] The in vitro models used for determining the
chondroinductive effect of BP differ from the in vivo case by the
fact that in the in vitro case the precursor cells are present in
an appropriate number and in an appropriate distribution whereas in
the in vivo case the precursor cells first have to populate the
defect and for this reason have to migrate into the defect. Only in
the latter case and for achieving repair cartilage which resembles
natural cartilage to a high degree, it is essential for the BP to
be released over a prolonged time period according to a specific
release profile.
EXAMPLE
[0049] The following example shows that BP released from
nanospheres induces cartilage repair in chondral defects in vivo
whereby virtually all cells recruited to the defect become
chondrocytes, whereby the cell structure obtained is ordered, and
whereby a hyaline matrix is built up.
[0050] Using a sheep model, unilateral defects of 0.5 mm width, 0.5
mm depth and 8 to 10 mm length were created in the trochlear groove
of the patella. The defects did not penetrate the subchondral bone.
The sheep employed in this study were seven years old and displayed
degenerative symptoms, including brittle bones, chondromalacia, and
subchondral cysts. Because of their advanced age and degenerative
symptoms, these amimals probably have decreased numbers of
precursor cells. The defects were then dressed according to
Hunziker and Rosenberg (J. Bone Joint Surg. 78A(5): 721-733, 1996)
with minor changes. Briefly, after enzymatic proteoglycan removal
with Chondroitinase AC, 2.5 .mu.l of a solution containig 200 units
Thrombin per ml was placed in the defect. Then, a paste was filled
into the defect, the paste containing per ml: 60 mg Sheep
Fibrinogen (Sigma), 88 mg Gelfoam (Upjohn) and either 10 .mu.g of
BP-nanospheres or 10 .mu.g of BP-nanospheres plus 80 ng rhIGF-1
(R+D Systems).
[0051] The nanospheres used were the nanospheres as specified in
the description regarding FIG. 2 and they were loaded with 1% (w/w)
of BP.
[0052] Assuming that the in vitro determined release rate is
approximately the same as for the in vivo case, 10 to 20 ng BP per
ml were released during the first 24 hours and approximately 0.1 to
1 ng per day for the following approximately 60 days.
[0053] After eight weeks, necropsies were performed. The repaired
cartilage histology showed that virtually all of the precursor
cells were differentiated to chondrocytes throughout the defect. In
addition, there was an ordered cartilage appearance with cells on
the top being more flattened morphologically than cells in the
center and with the presence of ordered, stacked chondrocytes in
the lowest zone. The repaired cartilage was fully integrated into
the endogenous tissue. In addition, the cartilage repaired with
only BP-nanospheres was not significantly different from the
cartilage repaired using BP-nanospheres plus IGF-1.
[0054] In conclusion, these results demonstrate that BP released
from nanospheres is sufficient for cartilage repair and that no
addintional factor is required (such as e.g recombinant factor
IGF-1). Using the inventive device constitutes a one step method
for cartilage repair, whereby the nanosphere release of BP is
sufficient for differentiation of virtually all of the precursor
cells to chondrocytes and for induction of an ordered cartilage
structure.
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