U.S. patent application number 14/474722 was filed with the patent office on 2015-03-05 for products comprising an extracellular matrix material and osteogenic protein.
The applicant listed for this patent is Muffin Incorporated. Invention is credited to Steven Charlebois, Neal E. Fearnot, Christine M. Steinhart, Amanda F. Taylor, Shelley L. Wallace.
Application Number | 20150065425 14/474722 |
Document ID | / |
Family ID | 52584065 |
Filed Date | 2015-03-05 |
United States Patent
Application |
20150065425 |
Kind Code |
A1 |
Wallace; Shelley L. ; et
al. |
March 5, 2015 |
PRODUCTS COMPRISING AN EXTRACELLULAR MATRIX MATERIAL AND OSTEOGENIC
PROTEIN
Abstract
Osteogenic compositions include a decellularized extracellular
matrix tissue and bone morphogenic protein, preferably BMP-2. The
compositions make beneficial use of the BMP, which can be used at
relatively low doses and can bind to native components (e.g.,
native sulfated glycosaminoglycans such as heparin and/or heparan
sulfate) remaining in the decellularized extracellular matrix
tissue. Methods for preparation and use of such compositions are
also described. The compositions and related methods can be used in
the treatment of diseased or damaged bone tissue.
Inventors: |
Wallace; Shelley L.;
(Athens, GA) ; Taylor; Amanda F.; (West Lafayette,
IN) ; Charlebois; Steven; (West Lafayette, IN)
; Steinhart; Christine M.; (Romney, IN) ; Fearnot;
Neal E.; (West Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Muffin Incorporated |
West Lafayette |
IN |
US |
|
|
Family ID: |
52584065 |
Appl. No.: |
14/474722 |
Filed: |
September 2, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61872827 |
Sep 2, 2013 |
|
|
|
Current U.S.
Class: |
514/7.7 ;
514/8.8 |
Current CPC
Class: |
A61L 27/3633 20130101;
A61L 27/12 20130101; A61P 19/00 20180101; A61L 2300/414 20130101;
A61L 27/54 20130101; A61L 2400/06 20130101; A61L 2430/02
20130101 |
Class at
Publication: |
514/7.7 ;
514/8.8 |
International
Class: |
A61L 27/36 20060101
A61L027/36; A61L 27/54 20060101 A61L027/54; A61L 27/58 20060101
A61L027/58 |
Claims
1. An osteogenic composition, comprising: a collagenous
extracellular matrix tissue material; and bone morphogenic
protein.
2. The composition of claim 1, wherein: the collagenous
extracellular matrix tissue material is a solid; and the bone
morphogenic protein is impregnated in the solid.
3. The composition of claim 1, wherein: the collagenous
extracellular matrix tissue material retains native heparin from a
source tissue for the collagenous extracellular matrix tissue
material; and at least a portion of the bone morphogenic protein is
bound to the native heparin.
4. The composition of claim 1, wherein: the bone morphogenic
protein comprises recombinant human BMP-2.
5. The composition of claim 1, wherein: the collagenous
extracellular matrix tissue material retains native components,
preferably native sulfated glycosaminoglycans, from a source tissue
for the collagenous extracellular matrix tissue material; and at
least a portion of the bone morphogenic protein is bound to the
native components.
6. The composition of claim 1, wherein the bone morphogenic protein
is present at a level in the range of about 75 .mu.g to about 300
.mu.g per gram (dry weight) of the collagenous extracellular matrix
tissue material.
7. The composition of claim 1, wherein the extracellular matrix
tissue material retains native growth factors, glycosaminoglycans,
proteoglycans and glycoproteins from a source tissue for the
extracellular matrix tissue material.
8. The composition of claim 7, wherein the collagenous
extracellular matrix tissue material retains native FGF-2 from a
source tissue for the extracellular matrix tissue material.
9. The composition of claim 8, wherein the native FGF-2 is present
in the collagenous extracellular matrix tissue material at a level
of at least about 50 nanograms per gram of the collagenous
extracellular matrix tissue material.
10. The composition of claim 1, wherein the collagenous
extracellular matrix tissue material comprises submucosal
tissue.
11. The composition of claim 1, wherein the collagenous
extracellular matrix tissue material is a porcine collagenous
extracellular matrix tissue material.
12. The composition of claim 1, also comprising a calcium phosphate
compound.
13. The composition of claim 12, wherein the calcium phosphate
compound includes hydroxyapatite, tricalcium phosphate, or a
combination thereof.
14. The composition of claim 12, wherein the calcium phosphate
compound is in particulate form.
15. The composition of claim 14, wherein the collagenous
extracellular matrix has mineralized native collagen fibers, with
the mineralized native collagen fibers including calcium phosphate
particles adhered to, within, or captured between native collagen
fibers of the extracellular matrix tissue material, and preferably
wherein said native collagen fibers have diameters greater than the
greatest cross-sectional dimension of the calcium phosphate
particles.
16. The composition of claim 12, wherein the bone morphogenic
protein includes amounts bound to native heparin of the
extracellular matrix tissue material and amounts bound to the
calcium phosphate.
17. The composition of claim 12, wherein the bone morphogenic
protein includes amounts bound to native heparin, heparan sulfate
and/or other native components of the extracellular matrix tissue
material and amounts bound to the calcium phosphate.
18. The composition of claim 12, wherein the bone morphogenic
protein is present in an amount not exceeding 3 mg and/or wherein
the bone morphogenic protein is constituted at least 95% by weight
of recombinant human BMP-2.
19-22. (canceled)
23. The composition of claim 1, wherein the collagenous
extracellular matrix tissue material comprises a collagenous
extracellular matrix gel including a mixture of solubilized
extracellular matrix components native to a source tissue for the
collagenous extracellular matrix tissue material.
24. The composition of claim 1, wherein the collagenous
extracellular matrix tissue material comprises particles of the
collagenous extracellular matrix tissue material.
25. The composition of claim 1, which is a flowable
composition.
26. The composition of claim 25, wherein the flowable composition
is settable after delivery to a patient so as to increase the
viscosity of the flowable composition.
27. The composition of claim 26, wherein the flowable composition
is settable to a non-flowable solid after delivery to a
patient.
28. A method for treating a patient, comprising administering to
the patient a composition according to claim 1, preferably wherein
the patient is a human, and/or preferably wherein the bone
morphogenic protein is administered at a total dose of 6 mg or
less, more preferably 4 mg or less.
29-30. (canceled)
31. A method for preparing an osteogenic composition, comprising:
combining bone morphogenic protein and a collagenous extracellular
matrix tissue material.
32. The method of claim 31, wherein the collagenous extracellular
matrix tissue material includes native heparin from a source tissue
for the collagenous extracellular matrix tissue material, and
wherein said combining results in binding of the bone morphogenic
protein to the native heparin.
33. The method of claim 31, wherein the collagenous extracellular
matrix tissue material includes native heparin, heparan sulfate
and/or other native components from a source tissue for the
collagenous extracellular matrix tissue material, and wherein said
combining results in binding of the bone morphogenic protein to the
native heparin, heparan sulfate and/or other native components.
34-48. (canceled)
49. The composition of claim 1, wherein the composition comprises
the bone morphogenic protein at a level of about 25 micrograms to
about 100 micrograms per cubic centimeter of the extracellular
matrix tissue material and/or wherein the composition further
comprises erythropoietin.
50-55. (canceled)
Description
REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority of U.S.
Patent Application Ser. No. 61/872,827 filed Sep. 2, 2013 and
entitled Products Comprising An Extracellular Matrix Material and
Osteogenic Protein, which is hereby incorporated herein by
reference in its entirety.
BACKGROUND
[0002] The present disclosure pertains to therapeutic compositions
and, in certain forms, to osteogenic compositions that include a
combination of extracellular matrix tissue material and bone
morphogenic protein.
[0003] Many medical procedures today rely on regenerating bone,
which has become deteriorated as a result of a disease or age or
has been damaged (e.g., fractured). While a variety of surgical
procedures are available, the advancement of modern medicine has
allowed for certain techniques to augment, and sometimes even
substitute for these surgeries. For example, a number of genetic
factors have been identified, which can serve this purpose if
delivered to the correct site. While the concept seems easy to
perform, may problems remain.
[0004] It is generally known that successful delivery of
therapeutic factors e.g., osteogenic factors for endochondral bone
formation requires association of the proteins with a carrier.
Currently, there are a number of carriers identified in the prior
art, all of which have their limitations. For example, carriers
include organic substances, such as demineralized bone matrix,
non-collagenous proteins, collagen (e.g., collagen sponge), fibrin,
autolyzed antigen extracted allogenic bone (AAA-bone), polyglycolic
acid, polylactic acid, hydrogels, as well as inorganic materials,
such as hydroxyapatite, tricalcium phosphate, other bioceramics,
bioactive glass, metals, coral, coral-collagen composite, natural
bone mineral, chitin, thermoashed bone mineral, non-demineralized
bone particles, ceramic bone particles, ceramic dentin,
polyphosphate polymer, irradiated cancellous bone chips, calcium
sulfate, and sintered bone. Although these materials are somewhat
effective in delivering a therapeutic factor to a desired tissue,
they have their limitations. For example, some delivery vehicles
fail to retain the therapeutic factor locally for a sufficient
period of time. Other delivery vehicles fail to resorb well in the
host in which they are administered. Still other delivery vehicles
and compositions containing them are lacking in cooperative
interaction among the osteogenic factor and the carrier, to enhance
tissue formation.
[0005] In view of this background, needs remain for improved or
alternative osteogenic compositions that can make highly beneficial
use of an osteogenic factor, and related methods of use and
preparation.
SUMMARY
[0006] It has been discovered that extracellular matrix tissue
materials can serve has highly beneficial carriers or cooperative
components when used with an osteogenic factor to generate hard
tissue such as bone. The extracellular matrix tissue materials can
serve to advantageously bind the osteogenic factor and in preferred
forms contribute additional bioactivity supportive of tissue
formation due to the presence of retained native bioactive
substances from a source tissue for the extracellular matrix tissue
material.
[0007] In certain aspects, provided are osteogenic compositions
that include a collagenous extracellular matrix tissue material and
bone morphogenic protein. Preferred compositional forms are
provided where the collagenous extracellular matrix tissue material
is a solid matrix, and where the bone morphogenic protein is
carried by the solid matrix. The collagenous extracellular matrix
tissue material can retain native heparin, heparan sulfate and/or
other native components from a source tissue for the collagenous
extracellular matrix tissue material, and at least a portion of the
bone morphogenic protein can be bound to the native heparin,
heparan sulfate, and/or other native components. The bone
morphogenic protein can be or comprise any human bone morphogenic
protein, preferably: BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, and/or
BMP-9, even more preferably BMP-2. In some forms, the bone
morphogenic protein comprises a recombinant human bone morphogenic
protein, for example BMP-2 ("rhBMP-2"). The BMP-2 and/or other bone
morphogenic protein can be provided at a relatively low loading in
the composition, for example being present at a level in the range
of about 75 .mu.g to about 300 .mu.g per gram, and/or about 25
.mu.g to about 100 .mu.g per cubic centimeter, of the collagenous
extracellular matrix tissue material; and/or being present at a
total dose of about 6 mg or less, about 4 mg or less, or about 3 mg
or less, for example in the range of about 1 to 6 mg or about 1 to
4 mg. Compositions containing such a level and/or total dose of
rhBMP-2 or other BMP can be for use in a human patient. The
composition can be provided in a non-flowable solid implant form or
a flowable (e.g., injectable) form, and in such flowable forms can
include an extracellular matrix tissue particulate material and/or
a collagenous extracellular matrix gel including a mixture of
solubilized extracellular matrix components native to the source
tissue. In certain aspects, the composition can also include other
bioactive or matrix materials, for example a mineral scaffold
material such as a calcium-containing compound.
[0008] In certain aspects, provided are osteogenic compositions
that include a collagenous extracellular matrix tissue material, a
bone morphogenic protein, and erythropoietin (EPO). Preferred
compositional forms are provided where the collagenous
extracellular matrix tissue material is a solid matrix, and where
the bone morphogenic protein is carried by the solid matrix. The
collagenous extracellular matrix tissue material can retain native
heparin, heparan sulfate and/or other native components from a
source tissue for the collagenous extracellular matrix tissue
material, and at least a portion of the bone morphogenic protein
can be bound to the native heparin, heparan sulfate and/or other
native components. The bone morphogenic protein can be or comprise
any human bone morphogenic protein, preferably: BMP-2, BMP-4,
BMP-5, BMP-6, BMP-7, and/or BMP-9, even more preferably BMP-2. In
some forms, the bone morphogenic protein comprises a recombinant
human bone morphogenic protein, for example BMP-2 ("rhBMP-2"). The
BMP-2 and/or other bone morphogenic protein can be provided at a
relatively low loading in the composition, for example being
present at a level in the range of about 0.1 .mu.g to about 3 .mu.g
per gram, or about 0.1 .mu.g to about 1.5 .mu.g per gram, of the
collagenous extracellular matrix tissue material; and/or being
present at a total dose of about 6 mg or less, about 4 mg or less,
or about 3 mg or less, for example in the range of about 1 to 6 mg
or about 1 to 4 mg. Compositions containing such a level and/or
total dose of rhBMP-2 or other BMP can be for use in a human
patient. In some forms, the EPO comprises a recombinant
erythropoietin (rEPO), in certain embodiments the rEPO comprises
recombinant human erythropoietin (rhEPO). The composition can be
provided in a non-flowable solid implant form or a flowable (e.g.,
injectable) form, and in such flowable forms can include an
extracellular matrix tissue particulate material and/or a
collagenous extracellular matrix gel including a mixture of
solubilized extracellular matrix components native to the source
tissue. In certain aspects, the composition can also include other
bioactive or matrix materials, for example a mineral scaffold
material such as a calcium-containing compound.
[0009] Additional features regarding the components of osteogenic
compositions, including but not limited to their identities,
levels, ratios, and manner of combination or incorporation in the
osteogenic compositions, are provided in the discussions below. It
will be understood that these additional features, alone or in
combination, can be combined with the features described in the
paragraphs above, or elsewhere herein, to form additional
embodiments disclosed herein.
[0010] Further embodiments disclosed herein relate to methods of
use of osteogenic compositions as disclosed herein. These methods
can be for the formation of hard tissue such as bone, which can be
for the purpose of treating diseased or damaged bone (e.g., for
therapeutic or prophylactic treatment).
[0011] Still further embodiments disclosed herein relate to methods
of preparation of osteogenic compositions as disclosed herein.
[0012] Additional embodiments, as well as features and advantages
thereof, will be apparent to those skilled in the field upon
reviewing the following descriptions.
BRIEF DESCRIPTION OF THE FIGURE
[0013] FIG. 1 shows digital images of X-ray and microCT images of
NOD/SCID mice 2 weeks after injecting the right thigh muscle with
either flowable ECM containing 5 .mu.g rhBMP-2 (FIG. 1,A) or
flowable ECM alone (FIG. 1,B), as described further in Example 2
below. Mice receiving injections of a flowable ECM containing 5
.mu.g rhBMP-2 showed abundant, de novo bone formation within the
thigh muscle (FIG. 1,A). Animals injected with flowable ECM alone
displayed no detectable ectopic bone formation at 2 weeks post
injection (FIG. 1,B).
DETAILED DESCRIPTION
[0014] Reference will now be made to certain embodiments, and
specific language will be used to describe the same. It will
nevertheless be understood that no limitation of the scope of the
invention is thereby intended. Any alterations and further
modifications in the described embodiments and any further
applications of the principles of the present invention as
described herein are contemplated as would normally occur to one
skilled in the art to which the invention relates.
[0015] As disclosed above, certain aspects of the present invention
relate to osteogenic compositions including bone morphogenic
protein, erythropoietin, and extracellular matrix tissue material,
and to methods for preparation or use of such compositions.
[0016] A variety of osteogenic bone morphogenic proteins are known
and can be used in embodiments herein either alone or in
combinations of bone morphogenic proteins. Recombinant human bone
morphogenetic proteins (rhBMPs) are preferred. Most preferably, the
bone morphogenetic protein is rhBMP-2, rhBMP-4, or a heterodimer
thereof. rhBMP-2 and rhBMP-7 are commercially available and such
commercial forms can be used herein. The bone morphogenic protein
can be or comprise any bone morphogenic protein, preferably: BMP-2,
BMP-4, BMP-5, BMP-6, BMP-7, and/or BMP-9, even more preferably
BMP-2. In some forms, the bone morphogenic protein comprises a
recombinant human bone morphogenic protein, for example BMP-2
("rhBMP-2"). These or other rhBMPs may also be prepared using
materials and methods known to those skilled in the art, for
example as described in U.S. Pat. Nos. 5,187,076; 5,366,875;
5,108,922; 5,116,738; 5,013,649; 6,352,972 and International PCT
Applications WO93/00432; WO94/26893; WO94/26892. The bone
morphogenic protein(s) may be provided as a freeze-dried powder,
which can be reconstituted during product manufacture or
intra-operatively in sterile water for injection or another liquid
vehicle for administration or otherwise as a part of manufacture of
a composition herein.
[0017] Erythropoietin (EPO) is a hormone produced by the kidney and
liver in response to hypoxia. EPO binds to the EPO receptor (EpoR)
to increase red blood cell production, increase VEGF expression,
and stimulate angiogenesis. EPO has also been demonstrated to
induce a bone remodeling response through direct stimulation of
mesenchymal stem cells and/or bone marrow stromal cells (e.g., by
increasing osteoblastogenesis). EPO may indirectly further induce a
bone remodeling response by increasing the number of hematopoietic
progenitor cells (e.g., increasing osteoclastogenesis). In addition
EPO has been shown to induce BMP production by hematopoietic stem
cells. The EPO for use in the present invention can be native or
recombinant forms of human EPO (rhEPO).
[0018] Accordingly, in certain aspects, compositions of the present
disclosure include an ECM, a BMP, and EPO. The EPO can be effective
to stimulate new blood vessel formation and/or to stimulate the
recruitment of mesenchymal stem cells (MSC) to the implant site,
and the BMP can be effective to promote the development of
osteogenic cells from the mesenchymal stem cells.
[0019] The collagenous extracellular matrix (ECM) material used
herein can be a decellularized animal tissue layer including ECM
tissue. In this regard, "decellularized" as used herein refers to a
state of the ECM tissue in which all or substantially all of the
cells native to the ECM tissue have been removed; thus, other
(non-native) cells can be present on or in the ECM tissue, which is
nonetheless referred to as decellularized. The ECM tissue layer can
be obtained from a source tissue of a warm-blooded vertebrate
animal, such as an ovine, bovine or porcine animal. The source
tissue layer is preferably a nonmineralized (i.e. soft tissue)
source tissue. For example, suitable ECM tissue include those
comprising submucosa, renal capsule membrane, dermal collagen, dura
mater, pericardium, amnion, abdominal fascia, fascia lata, serosa,
peritoneum or basement membrane layers, including liver basement
membrane. Suitable submucosa materials for these purposes include,
for instance, intestinal submucosa including small intestinal
submucosa, stomach submucosa, urinary bladder submucosa, and
uterine submucosa. ECM tissues comprising submucosa (potentially
along with other associated tissues) useful in the present
invention can be obtained by harvesting such tissue sources and
delaminating the submucosa-containing matrix from smooth muscle
layers, mucosal layers, and/or other layers occurring in the tissue
source. Porcine tissue sources are preferred sources from which to
harvest ECM tissues, including submucosa-containing ECM
tissues.
[0020] ECM tissue used in the invention is preferably
decellularized and highly purified, for example, as described in
U.S. Pat. No. 6,206,931 to Cook et al. or U.S. Patent Application
Publication No. US2008286268 dated Nov. 20, 2008, publishing U.S.
patent application Ser. No. 12/178,321 filed Jul. 23, 2008, all of
which are hereby incorporated herein by reference in their
entirety. Preferred ECM tissue material will exhibit an endotoxin
level of less than about 12 endotoxin units (EU) per gram, more
preferably less than about 5 EU per gram, and most preferably less
than about 1 EU per gram. As additional preferences, the submucosa
or other ECM material may have a bioburden of less than about 1
colony forming units (CFU) per gram, more preferably less than
about 0.5 CFU per gram. Fungus levels are desirably similarly low,
for example less than about 1 CFU per gram, more preferably less
than about 0.5 CFU per gram. Nucleic acid levels are preferably
less than about 5 .mu.g/mg, more preferably less than about 2
.mu.g/mg, and virus levels are preferably less than about 50 plaque
forming units (PFU) per gram, more preferably less than about 5 PFU
per gram. These and additional properties of submucosa or other ECM
tissue taught in U.S. Pat. No. 6,206,931 or U.S. Patent Application
Publication No. US2008286268 may be characteristic of any ECM
tissue used in the present invention.
[0021] In certain embodiments, the ECM tissue material used herein
will be a membranous tissue with a layer structure as isolated from
the tissue source. The ECM tissue can, as isolated, have a layer
thickness that ranges from about 50 to about 250 microns when fully
hydrated, more typically from about 50 to about 200 microns when
fully hydrated, although isolated layers having other thicknesses
may also be obtained and used. These layer thicknesses may vary
with the type and age of the animal used as the tissue source. As
well, these layer thicknesses may vary with the source of the
tissue obtained from the animal source.
[0022] The ECM tissue material utilized desirably retains a
structural microarchitecture from the source tissue, including
structural fiber proteins such as collagen and potentially also
elastin that can form native fibers. Such fibers can in certain
embodiments be non-randomly oriented, as can occur in the source
tissue for the decellularized ECM tissue material. Such non-random
collagen and/or other structural protein fibers can in certain
embodiments provide an ECM tissue that is non-isotropic in regard
to tensile strength, thus having a tensile strength in one
direction that differs from the tensile strength in at least one
other direction.
[0023] The decellularized ECM tissue material may include one or
more bioactive agents native to the source of the ECM tissue
material and retained in the ECM tissue material through
processing. For example, a submucosa or other ECM tissue material
may retain one or more native growth factors such as but not
limited to basic fibroblast growth factor (FGF-2), transforming
growth factor beta (TGF-beta), epidermal growth factor (EGF),
cartilage derived growth factor (CDGF), and/or platelet derived
growth factor (PDGF). As well, submucosa or other ECM materials
when used in the invention may retain other native bioactive agents
such as but not limited to proteins, glycoproteins, proteoglycans,
and glycosaminoglycans. For example, decellularized ECM tissue
materials may include native heparin, heparin sulfate, hyaluronic
acid, fibronectin, cytokines, and the like. Thus, generally
speaking, a submucosa or other ECM tissue material may retain from
the source tissue one or more bioactive components that induce,
directly or indirectly, a cellular response such as a change in
cell morphology, proliferation, growth, protein or gene
expression.
[0024] Submucosa-containing ECM materials or other ECM materials
used in the present invention can be derived from any suitable
organ or other tissue source, usually a soft tissue source
(non-bone, non-cartilage) containing connective tissue. The ECM
materials processed for use in the invention will typically include
abundant collagen, most commonly being constituted at least about
80% by weight collagen on a dry weight basis. Such
naturally-derived ECM materials will for the most part include
collagen fibers that are non-randomly oriented, for instance
occurring as generally uniaxial or multi-axial but regularly
oriented fibers. When processed to retain native bioactive factors
(e.g., as discussed above), the ECM material can retain these
factors interspersed as solids between, upon and/or within the
collagen fibers. Particularly desirable naturally-derived ECM
materials for use in the invention will include significant amounts
of such interspersed, non-collagenous solids that are readily
ascertainable under light microscopic examination with appropriate
staining. Such non-collagenous solids can constitute a significant
percentage of the dry weight of the ECM material in certain
inventive embodiments, for example at least about 1%, at least
about 3%, and at least about 5% by weight in various embodiments of
the invention.
[0025] The submucosa-containing or other ECM tissue material used
in the present invention may also exhibit an angiogenic character
and thus be effective to induce angiogenesis in a host engrafted
with the material. In this regard, angiogenesis is the process
through which the body makes new blood vessels to generate
increased blood supply to tissues. Thus, angiogenic materials, when
contacted with host tissues, promote or encourage the formation of
new blood vessels into the materials. Methods for measuring in vivo
angiogenesis in response to biomaterial implantation have recently
been developed. For example, one such method uses a subcutaneous
implant model to determine the angiogenic character of a material.
See, C. Heeschen et al., Nature Medicine 7 (2001), No. 7, 833-839.
When combined with a fluorescence microangiography technique, this
model can provide both quantitative and qualitative measures of
angiogenesis into biomaterials. C. Johnson et al., Circulation
Research 94 (2004), No. 2, 262-268.
[0026] Decellularized ECM tissue layers can be used in the
invention as single layer implants, but in certain embodiments will
be used in multilaminate constructs. In this regard, a variety of
techniques for laminating layers together are known and can be used
to prepare multilaminate constructs used for the graft in the
present invention. For example, a plurality of (i.e. two or more)
layers of collagenous material, for example submucosa-containing or
other ECM material, can be bonded together to form a multilaminate
structure. Illustratively, two to about two hundred decellularized
collagenous ECM tissue layers can be bonded together to provide a
multilaminate construct for use in the present invention. In
certain embodiments, two to eight decellularized collagenous ECM
tissue layers are bonded together to form a multilaminate construct
for use herein. Preferably submucosa-containing ECM tissue layers
are isolated from intestinal tissue, more preferably small
intestinal tissue. Porcine-derived tissue is preferred for these
purposes. The layers of ECM tissue can be bonded together in any
suitable fashion, including dehydrothermal bonding under heated,
non-heated or lyophilization conditions, using adhesives, glues or
other bonding agents, crosslinking with chemical agents or
radiation (including UV radiation), or any combination of these
with each other or other suitable methods. For additional
information as to multilaminate ECM constructs that can be used in
the invention, and methods for their preparation, reference may be
made for example to U.S. Pat. Nos. 5,711,969, 5,755,791, 5,855,619,
5,955,110, 5,968,096, and to U.S. Patent Publication No.
20050049638 A1 published Mar. 3, 2005. These constructs can be
perforated or non-perforated, and when perforated may include an
array of perforations extending substantially across the surface of
the construct, or may include perforations only in selected
areas.
[0027] Osteogenic compositions of embodiments herein can
incorporate xenograft ECM tissue material (i.e., cross-species
material, such as tissue material from a non-human donor to a human
recipient), allograft ECM material (i.e., interspecies material,
with tissue material from a donor of the same species as the
recipient), and/or autograft ECM material (i.e., where the donor
and the recipient are the same individual). Further, BMP and/or
other exogenous bioactive substances incorporated into an ECM
material may be from the same species of animal from which the ECM
material was derived (e.g., autologous or allogenic relative to the
ECM material) or may be from a different species from the ECM
material source (xenogenic relative to the ECM material). In
certain embodiments, the ECM tissue material will be xenogenic
relative to the patient receiving the graft, and any added cells or
other exogenous material(s) will be from the same species (e.g.,
autologous or allogenic) as the patient receiving the graft.
Illustratively, human patients may be treated with xenogenic ECM
materials (e.g., porcine-, bovine- or ovine-derived) that have been
modified with exogenous human BMP(s) such are rhBMP(s) as described
herein.
[0028] ECM tissue materials used in embodiments herein can be free
or essentially free of additional, non-native crosslinking, or may
contain additional crosslinking. Such additional crosslinking may
be achieved by photo-crosslinking techniques, by chemical
crosslinkers, or by protein crosslinking induced by dehydration or
other means. However, because certain crosslinking techniques,
certain crosslinking agents, and/or certain degrees of crosslinking
can destroy the remodelable properties of a remodelable material,
where preservation of remodelable properties is desired, any
crosslinking of the remodelable ECM material can be performed to an
extent or in a fashion that allows the material to retain at least
a portion of its remodelable properties. Chemical crosslinkers that
may be used include for example aldehydes such as glutaraldehydes,
diimides such as carbodii mides, e.g.,
1-ethyl-3-(3-dimethylaminopropyl)carbodii mide hydrochloride,
ribose or other sugars, acyl-azide, sulfo-N-hydroxysuccinamide, or
polyepoxide compounds, including for example polyglycidyl ethers
such as ethyleneglycol diglycidyl ether, available under the trade
name DENACOL EX810 from Nagese Chemical Co., Osaka, Japan, and
glycerol polyglycerol ether available under the trade name DENACOL
EX 313 also from Nagese Chemical Co. Typically, when used,
polyglycerol ethers or other polyepoxide compounds will have from 2
to about 10 epoxide groups per molecule.
[0029] In additional embodiments, osteogenic compositions herein
can incorporate ECM tissue material that has been subjected to a
process that expands the tissue material. In certain forms, such
expanded materials can be formed by the controlled contact of an
ECM material with a denaturing agent such as one or more alkaline
substances until the material expands, and the isolation of the
expanded material. Illustratively, the contacting can be sufficient
to expand the ECM tissue material to at least 120% of (i.e. 1.2
times) its original bulk volume, or in some forms to at least about
two times its original volume. Thereafter, the expanded material
can optionally be isolated from the alkaline medium (e.g., by
neutralization and/or rinsing). The collected, expanded material
can be used in any suitable manner in the preparation of a material
for administration to a patient. The expanded material can be
enriched with bioactive components, comminuted, dried, and/or
molded, etc., in the formation of an implantable body of a desired
shape or configuration. In certain embodiments, a dried implant
body formed with an expanded ECM tissue material can be
compressible.
[0030] Treatment of an ECM tissue material with a denaturant, such
as an alkaline material, can cause changes in the physical
structure of the material that in turn cause it to expand. Such
changes may include denaturation of the collagen in the material.
In certain embodiments, it is preferred to expand the material to
at least about three, at least about four, at least about 5, or at
least about 6 or even more times its original bulk volume. It will
be apparent to one skilled in the art that the magnitude of the
expansion is related to several factors, including for instance the
concentration or pH of the alkaline medium, the exposure time of
the alkaline medium to the material, and temperature used in the
treatment of the material to be expanded, among others. These
factors can be varied through routine experimentation to achieve a
material having the desired level of expansion, given the
disclosures herein.
[0031] A collagen fibril is comprised of a quarter-staggered array
of tropocollagen molecules. The tropocollagen molecules themselves
are formed from three polypeptide chains linked together by
covalent intramolecular bonds and hydrogen bonds to form a triple
helix. Additionally, covalent intermolecular bonds are formed
between different tropocollagen molecules within the collagen
fibril. Frequently, multiple collagen fibrils assemble with one
another to form collagen fibers. It is believed that the addition
of an alkaline substance to the material as described herein can be
conducted so as to not significantly disrupt the intramolecular and
intermolecular bonds, but denature the material to an extent that
provides to the material an increased processed thickness (e.g., at
least twice the naturally-occurring thickness). ECM materials that
can be processed to make expanded materials for use as substrates
can include any of those disclosed herein or other suitable ECM's.
Typical such ECM materials will include a network of collagen
fibrils having naturally-occurring intramolecular cross links and
naturally-occurring intermolecular cross links. Upon expansion
processing as described herein, the naturally-occurring
intramolecular cross links and naturally-occurring intermolecular
cross links can be retained in the processed collagenous matrix
material sufficiently to maintain the collagenous matrix material
as an intact collagenous sheet material; however, collagen fibrils
in the collagenous sheet material can be denatured, and the
collagenous sheet material can have an alkaline-processed thickness
that is greater than the thickness of the starting material, for
example at least 120% of the original thickness, or at least twice
the original thickness. The expanded ECM material can then be
processed to provide foam or sponge substrates for use as or in the
graft body, e.g. by comminuting, casting, and drying the processed
material. Additional information concerning expanded ECM materials
and their preparation is found in United States Patent Application
Publication No. US20090326577 published Dec. 31, 2009, publishing
U.S. patent application Ser. No. 12/489,199 filed Jun. 22, 2009,
which is hereby incorporated herein by reference in its
entirety.
[0032] In certain embodiments herein, the osteogenic composition
can consist or consist essentially of the decellularized ECM tissue
and the BMP, preferably rhBMP-2. Additionally or alternatively, the
osteogenic composition can be predominantly comprised of the
decellularized ECM tissue and the BMP, preferably rhBMP-2, for
example at least 80% by weight, at least 90% by weight, or at least
95% by weight, on a dry weight basis.
[0033] In other forms, in addition to ECM tissue materials,
compositions herein can include other organic carrier materials.
Illustrative materials include, for example, synthetically-produced
substrates comprised or natural or synthetic polymers. Illustrative
synthetic polymers are preferably biodegradable synthetic polymers
such as polylactic acid, polyglycolic acid or copolymers thereof,
polyanhydride, polycaprolactone, polyhydroxy-butyrate valerate,
polyhydroxyalkanoate, or another biodegradable polymer or mixture
thereof. Preferred implant bodies comprised of these or other
materials (e.g., ECM materials as discussed herein) will be porous
matrix materials configured to allow cellular invasion and ingrowth
into the matrix.
[0034] Inorganic scaffolding materials can also be incorporated in
the compositions herein. In certain embodiments, the compositions
can incorporate one or more mineral-containing materials along with
the ECM tissue material and bone morphogenic protein. Such mineral
material(s) can serve as scaffolding to support the generation of
hard tissue such as bone. Many mineral-containing materials for
such purposes are known and can be used, for example in particulate
form. Suitable materials include for instance hydroxyapatite,
tricalcium phosphate, bioglass, calcium phosphate, calcium sulfate,
bone, or combinations thereof.
[0035] A mineral-containing material and the ECM tissue material
can be combined in any suitable manner. In some variants, the
mineral-containing material is a particulate material, such as a
powder or granular material, and the ECM tissue material is also a
particulate material. In these forms, the mineral-containing
particulate and the ECM tissue particulate can be in admixture with
one another, preferably in a substantially homogenous admixture.
Such admixtures can be provided in dry form for later combination
with bone morphogenic protein, or can have bone morphogenic protein
in dry (e.g., lyophilized) form included in the admixture. In still
other forms, the ECM tissue material can provide an ECM matrix, and
particles of the mineral-containing material can be embedded in the
ECM matrix; or, the mineral-containing material can provide a
mineral matrix, and particles of the ECM tissue material can be
embedded in the mineral matrix.
[0036] As well, the mineral-containing material and the ECM tissue
material can be combined in the form of a mineralized ECM tissue
matrix, in which mineral particles are adhered to native structural
fibers of the ECM tissue, such as collagen and/or elastin fibers,
and/or entrapped between the native structural fibers, and/or
entrapped within the native structural fibers. Such a mineralized
ECM tissue matrix can be prepared by a method in which the mineral
particles are precipitated from solution onto the native structural
fibers of the ECM tissue matrix, into the native structural fibers
of the ECM tissue matrix, between the native structural fibers of
the ECM tissue matrix, or combinations thereof. For example, the
mineralization process can include mixing, within the porous matrix
of the ECM tissue, a first solution containing solvated ions of a
first component of the mineral particles to be formed, and at least
a second solution containing solvated ions of a second component of
the mineral particles to be formed. The first and second component
can thereby interact (e.g., ionically or otherwise) in the
formation of the mineral particles of the mineralized ECM tissue
matrix. In other preparative modes, the ECM tissue matrix can be
alternately contacted with at least the first and second solutions
to result in the formation of the mineral particles within the ECM
tissue matrix. In certain embodiments the first solution can
include dissolved amounts of a soluble calcium salt and the second
solution can include dissolved amounts of a soluble phosphate salt,
and the resulting precipitated mineral particles can contain
calcium and phosphate. Other cationic or anionic species may also
be present in the reagent solutions such as carbonate, chloride,
fluoride, sodium or ammonium, and the mineral particles can be
comprised of calcium hydroxyapatite, calcium hydroxy/fluorapatite,
brushite, dahlite, monetite, phosphated calcium carbonate
(calcite), octacalcium phosphate, or tri calcium phosphate, as
examples. It will be understood that the choice of stoichiometry of
the calcium and the phosphate, as well as the presence of other
ions, will result in a particular composition for the formed
mineral particles. For additional information regarding
mineralizing solutions and techniques, reference can be made to
U.S. Pat. Nos. 5,455,231, 5,508,267, 6,187,047, 6,384,196 and
6,764,517.
[0037] In mineralized ECM tissue materials herein or in other
compositions incorporating a mineral scaffolding material, the
mineral scaffolding material can constitute any suitable percentage
by weight of the overall composition. In certain embodiments, the
mineral scaffolding material constitutes about 5% to about 90% by
weight, or about 5% to about 60% by weight, or about 5% to about
40% by weight, of the overall composition on a dry weight
basis.
[0038] Further in regard to mineralized ECM tissue, as discussed
above, preferred decellularized ECM tissue materials used herein
can retain native bioactive substances from a source tissue for the
ECM tissue material. The mineralization of such bioactive ECM
tissue materials can be conducted so as to result in an ECM tissue
matrix that not only has mineral particles adhered to, within,
and/or between the collagen and/or elastin fibers of the ECM
tissue, but that also retains amounts of such native bioactive
substances from the source tissue, which can include one or more
growth factors (e.g., FGF-2), glycosaminoglycans, proteoglycans
and/or glycoproteins. As discussed above, these non-collagenous
native bioactive materials can be present as solids interspersed
between collagen fibers of the ECM tissue material. Thus,
mineralized ECM tissue materials can in certain forms include
native collagen and/or elastin fibers, mineral particles adhered,
within and/or between those fibers with mineral particles
preferably having maximum cross-sectional dimensions smaller than
the fibers, and non-collagen bioactive solids interspersed between
the fibers and including one or more growth factors (e.g., FGF-2),
glycosaminoglycans, proteoglycans, and/or glycoproteins from the
source tissue for the ECM tissue material. Embodiments disclosed
herein include those in which such interspersed non-collagen native
bioactive solids can constitute at least 1% by weight, or at least
3% by weight, of the ECM tissue material on a dry weight basis
(excluding the mineral material).
[0039] The ECM tissue material used herein can optionally be in
particulate form, for example as incorporated into flowable
compositions for administration. Such ECM particulate materials can
have particles or random and/or regular shape. Illustratively,
random ECM tissue particulates can be prepared by crushing,
grinding or chopping a larger decellularized ECM tissue sheet
material. On the other hand, a regular ECM tissue particulate can
be prepared by controlled cutting of shapes such as circular, ovoid
or polygonal shapes from a larger decellularized ECM tissue layer
material (e.g., to provide disk form particles). Such regular ECM
particles can retain a sheet form, and can in certain embodiments
have maximum sheet dimensions (across the face of the sheet
particles) in the range of about 0.1 to about 1 mm, or about 0.1 to
about 5 mm, or about 0.1 to about 2 mm. In addition or
alternatively, the regular ECM particles can be multilaminate
constructs containing multiple bonded decellularized ECM layers,
for example as can be prepared by controlled cutting, as mentioned
above, of corresponding larger multilaminate decellularized ECM
tissue constructs. Methods of laminating multiple layers of
decellularized ECM layers are described herein and can be used in
the generation of the larger multilaminate decellularized ECM
tissue constructs to be cut to generate the regular ECM
particulate. An ECM particulate can be incorporated with a flowable
liquid carrier, typically an aqueous carrier, along with other
components herein, to form an injectable or otherwise flowable
composition for administration.
[0040] The BMP can be combined with a solid ECM tissue in any
suitable fashion. For example, the BMP can be dissolved in a liquid
carrier such as distilled water or a buffered aqueous solution, and
the liquid carrier can be contacted with the ECM tissue. Any
suitable period of contact can be used. In certain modes, the ECM
tissue and the liquid carrier containing the BMP are contacted with
one another for a period of at least 1 minute, at least 5 minutes,
or at least 10 minutes, for example in the range of about 5 minutes
to 60 minutes. As discussed above, in some embodiments herein, the
ECM tissue material will retain native sulfated glycosaminoglycans
such as heparin and/or heparan sulfate, and potentially also other
native components, from its source tissue. Contact of the liquid
carrier with the ECM tissue material over these periods of time can
allow the BMP to bind to this native heparin, heparan sulfate
and/or other native components. The BMP component(s) may also be
modified, encapsulated, or chemically and/or covalently bound to
the ECM to promote a longer half-life of biological activity. The
contact of the liquid BMP formulation and the ECM tissue can occur
at the point of care; alternatively, this can occur during
commercial product manufacture, for example where the resulting BMP
impregnated ECM tissue material is thereafter lyophilized to form a
dry construct, which can be sterilely packaged for storage and
later use. In addition, after contact of the liquid BMP formulation
with the ECM tissue, in some embodiments, the resulting
BMP-impregnated ECM tissue can be rinsed with water or another
appropriate rinse liquid to remove at least some of the unbound BMP
from the composition, and in certain aspects to remove at least 70%
of the unbound BMP. This can provide a composition in which a
predominant amount of the BMP administered to the patient is bound
to the carrier material and thus more effectively localized to the
implant site.
[0041] The ECM tissue matrix and BMP can also be combined in a
flowable implant composition. For these purposes, the ECM tissue
can be in particulate and/or gel form. The flowable carrier
material in such compositions can include a gel form of the ECM
tissue and/or another material, and will typically be an aqueous
carrier material. The flowable carrier in some embodiments can be
or include an inorganic flowable carrier, for instance a hardenable
inorganic flowable carrier such as a paste that is settable to form
a calcium phosphate-containing or calcium sulfate-containing
cement. Illustratively, reactants that include a calcium source and
a phosphate source can be combined with the ECM tissue material in
particulate and/or gel form, and the BMP, to produce a flowable
composition that sets into a non-flowable calcium phosphate solid.
The calcium source and phosphate source may be present as a single
compound or present as two or more compounds. As such, a single
calcium phosphate present in the dry reactants may be the calcium
source and the phosphate source. Alternatively, two or more
compounds may be present in the dry reactants, where the compounds
may be compounds that include calcium, phosphate or calcium and
phosphate. Calcium phosphate sources of interest that may be
present in the dry reactants include: MCPM (monocalcium phosphate
monohydrate or Ca(H.sub.2PO.sub.4).sub.2.H.sub.2O); DCPD (dicalcium
phosphate dihydrate, brushite or CaHPO.sub.4.2H.sub.2O), ACP
(amorphous calcium phosphate or Ca.sub.3(PO.sub.4).sub.2H.sub.2O),
DCP (dicalcium phosphate, monetite or CaHPO.sub.4), tricalcium
phosphate, including both .alpha.- and .beta.-
(Ca.sub.3(PO.sub.4).sub.2, tetracalcium phosphate
(Ca.sub.4(PO.sub.4).sub.2O, etc. Calcium sources of interest
include: calcium carbonate (CaCO.sub.3), calcium oxide (CaO),
calcium hydroxide (Ca(OH).sub.2 and the like. Phosphate sources of
interest include: Phosphoric acid (H.sub.3PO.sub.4), soluble
phosphate salts, and the like. In certain forms, the above calcium
containing and phosphorous containing reactants can be in dry form
(e.g., with ECM tissue particles admixed therewith) and these dry
reactants can be combined with a liquid medium, for example
distilled water, an aqueous acid solution (e.g., phosphoric acid),
or an aqueous solution containing a soluble orthophosphate or
monocalcium phosphate monohydrate, to form a flowable, settable
composition. The settable composition can set, as examples, to a
non-stoichiometric calcium-deficient hydroxyapatite or brushite
material. This set material can entrain the particulate and/or gel
form ECM tissue material. As well, the BMP in such settable
compositions, or set materials, can be bound to the ECM tissue
material (e.g., through binding to native sulfated
glycosaminoglycans such as heparin and/or heparan sulfate and/or
other native components therein), included within the flowable
carrier and resulting set inorganic matrix material, or a
combination thereof. In certain forms, at least 50%, at least 70%,
or at least 90% by weight of the BMP will be bound to the ECM
tissue material in solid carrier form (e.g., particulate form),
which will thus be enriched in the BMP as compared to the flowable
liquid carrier material of the composition.
[0042] The BMP can be incorporated into the flowable composition
using any suitable technique. It can be impregnated into the ECM
tissue material, which can then be incorporated into the flowable
composition (e.g., by suspension or mixture with an aqueous medium
or other flowable carrier material). Alternatively or in addition,
the BMP can be incorporated into a liquid medium to serve as the
flowable carrier medium or at least a portion thereof, and this
liquid medium can then be combined with the ECM tissue material.
Still further, the BMP in dry powder (e.g., lyophilized) form can
be combined with the ECM material in dry form, to form a dry
mixture. This dry mixture can then be combined with a liquid medium
to form the flowable composition. These and other modes of
preparation of the flowable composition including the ECM tissue
material and the BMP will be apparent to those skilled in the
pertinent field from the descriptions herein. Likewise, kits
containing these components in separately packaged form, for
combination to prepare a flowable composition, for example at the
point of care, provide additional embodiments herein.
[0043] In compositions herein, the BMP-2 and/or other BMP can be
provided at a relatively low loading in the composition and/or used
at a relatively low dose in the patient. For example, the BMP-2 or
other BMP can be present at a level in the range of about 75 to
about 300 micrograms per gram of the collagenous extracellular
matrix tissue material in the composition (dry weight basis), at a
level in the range of about 25 to about 300 micrograms per cubic
centimeter of the collagenous extracellular matrix tissue material
in the composition, and/or at a total BMP-2 or other BMP dose of
about 4 mg or less or about 3 mg or less, for example in the range
of about 1 to 4 mg. These values for loading and dosing can be used
when the patient is human.
[0044] In some forms, the present disclosure includes a method for
preparing an osteogenic composition comprising saturating a
collagenous extracellular matrix tissue material in a solution
containing BMP. In accordance with certain inventive variants, the
solution comprises about 0.1 .mu.g/ml BMP to about 10 .mu.g/ml BMP.
In preferred embodiments the solution comprises about 0.1 .mu.g/ml
BMP to about 1 .mu.g/ml BMP.
[0045] As disclosed above, in some embodiments, the compositions
containing ECM and BMP will also include EPO. In this regard, the
EPO can be combined with the ECM together with, or separately from,
the BMP. For example, a liquid medium (e.g., solution) containing
both the EPO and BMP can be combined with the ECM in certain
embodiments. In others, separate solutions or other liquid media
containing, respectively, the BMP and the EPO, can be combined with
the ECM. As disclosed above, EPO can be present in an amount
effective to stimulate new blood vessel formation and/or to
stimulate the recruitment of mesenchymal stem cells (MSC) to the
implant site. In conjunction therewith, the BMP can be effective to
promote the development of osteogenic cells from mesenchymal stem
cells. The EPO, when used, can be native or recombinant form of
human EPO (rhEPO).
[0046] The compositions disclosed herein may also be seeded with
cells, which can in some forms be autologous or allogenic to the
recipient of the composition. The cells employed may be primary
cells, explants, or cell lines, and may be dividing or non-dividing
cells. Cells may be expanded ex-vivo prior to introduction into the
inventive cement compositions. Autologous cells are preferably
expanded in this way if a sufficient number of viable cells cannot
be harvested from the host. The cells may be non-genetically
engineered (not having been subjected to introduction of genetic
material to genetically alter the cells), or may be genetically
engineered, for example to produce a protein or other factor that
it useful in a particular application. The cells may be combined
into the compositions herein during preparation (before
administration to a patient) or may be administered to the patient
separately from the compositions herein so as to seed the
administered composition in situ in the patient.
[0047] The compositions disclosed herein can be used in a variety
of applications. In preferred uses, the compositions are used in
the treatment of skeletal defects such as diseased or damaged bone
or other defects that require bone growth, for example to fuse
adjacent vertebrae. For example, the diseased or damaged bone can
occur in any of the bones in an animal, especially a mammal such as
a human, including flat bones (e.g., ribs and the frontal and
parietal bones of the cranium), long bones (e.g., bones of the
extremities), short bones (e.g., wrist and ankles bones), irregular
bones (e.g., vertebrae and the pelvis), and sesamoid bones (e.g.,
the patella). Damaged bone to be treated can include fractured
bone. Diseased bone to be treated can in some embodiments include
osteopenic bone, osteoporotic bone, or necrotic bone. Combined
diseased and damaged bone can also be treated, for example in the
case of fractured osteopenic bone or fractured osteoporotic bone.
The fusion of adjacent vertebrae can involve the implantation of
compositions disclosed herein between first and second adjacent
vertebral bodies, potentially in combination with one or more
fusion cages or other spacer implants configured to support the
vertebral bodies in spaced condition from one another.
[0048] For the purpose of promoting a further understanding of
embodiments herein and features and advantages thereof, the
following specific Examples are provided. It will be understood
that these Examples are illustrative, and not limiting, of the
scope of embodiments otherwise described herein.
Example 1
Preparation of 1 .mu.g BMP-2 Samples
[0049] rhBMP-2 was obtained from -20.degree. C. freezer. A 4 mM
solution of HCl in water was made up and sterilized by passing it
through a 0.2 .mu.m syringe filter. 100 .mu.l of the 4 mM HCl
solution was added to the BMP-2 vial making a 100 .mu.g/ml
solution. The solution was briefly spun in a microfuge. A 10 .mu.l
(1 .mu.g) aliquot was placed into a sterile tube and stored at
-20.degree. until day of implant.
Preparation of 0.3 .mu.g BMP-2 Samples
[0050] rhBMP-2 was obtained from -20.degree. C. freezer. A 4 mM
solution of HCl in water was made up and sterilized by passing it
through a 0.2 .mu.m syringe filter. 100 .mu.l of the 4 mM HCl
solution was added to the BMP-2 vial making a 100 .mu.g/ml
solution. The solution was briefly spun in a microfuge. A 30
.mu.l/ml solution was made by adding 30 .mu.l of the rhBMP-2
solution and 70 .mu.l of the 4 mM HCl. The 30 .mu.g/ml solution was
briefly spun in the microfuge. A 10 .mu.l (0.3 .mu.g) aliquot was
placed into a sterile tube and stored at -20.degree. until day of
implant.
Preparation of 0.1 .mu.g BMP-2 Samples
[0051] rhBMP-2 was obtained from -20.degree. C. freezer. A 4 mM
solution of HCl in water was made up and sterilized by passing it
through a 0.2 .mu.m syringe filter. 100 .mu.l of the 4 mM HCl
solution was added to the BMP-2 vial making a 100 .mu.g/ml
solution. The solution was briefly spun in a microfuge. A 10
.mu.l/ml solution was made by adding 10 .mu.l of the rhBMP-2
solution and 90 .mu.l of the 4 mM HCl. The 10 .mu.l/ml solution was
briefly spun in the microfuge. A 10 .mu.l (0.1 .mu.g) aliquot was
placed into a sterile tube and stored at -20.degree. until day of
implant.
[0052] Implant Preparation and Surgery
[0053] A 4 mm diameter disk was cut from an ECM sheet material (a
4-layer laminate of renal capsule, "RC") with a 4-mm biopsy punch.
The RC disk implant was added to a 10 .mu.l aliquot of rhBMP-2
solution prepared as described in this Example above, ensuring the
sheet was fully saturated in the solution. The vial with the
saturated RC disk implant was placed in an incubator at 37.degree.
C. for a minimum of 30 minutes. The hydrated RC disk was removed
from the vial, and then passed into the surgical field to allow the
surgeon to implant into a prepared defect of a known
immunodeficient (SCID) mouse calvarial defect model. For the model,
bilateral 4 mm diameter defects were drilled in each mouse. One of
the bilateral defects was used as a control (receiving no treatment
material) and the other received the treatment material. Two
lengths of titanium wire were tied to the outer edge of the implant
disc on opposed sides as imageable references. Sutures were routed
through cranial and caudal suture holes drilled in the treatment
defects and through mated holes in the implant disk. The implant
disk was secured to parietal bone with a suture knot. Control
(void) treatment sites were similarly tied with sutures but
received no implant. The incisions were closed, and the mouse was
fitted with an Elizabethan collar (worn for 1 week after surgery).
The mice in the study were imaged in vivo with microCT (small scale
computed tomography) at 2, 4, 8, and 12 weeks post implant. The
microCT scans were used the change in bone coverage for the treated
and untreated defects.
[0054] Results:
[0055] For defects treated with the ECM implant disks soaked in the
1, 0.3 and 0.1 microgram solutions the percent bone coverage at 12
weeks post implant averaged 88.8% (n=7), 87.1% (n=6) and 68.5%
(n=7), respectively. For the corresponding untreated defects in
these groups, the percent bone coverage at 12 weeks post implant
averaged 19.5%, 18.6% and 24.5% (n=7), respectively. Also, in
experiments similarly conducted but carried out to 16 weeks instead
of 12 weeks, a group of mice (n=7) receiving just the ECM implant
disk (no added rhBMP-2) on one side and no treatment on the other
side averaged about 35% bone coverage on the treated side and about
22% bone coverage on the untreated side at 16 weeks post
implant.
Example 2
Preparation of 10 .mu.g rhBMP-2 Samples
[0056] rhBMP-2 was obtained from -20.degree. C. freezer. A 4 mM
solution of HCl in water was made up and sterilized by passing it
through a 0.2 .mu.m syringe filter. 500 .mu.l of the 4 mM HCl
solution was added to the BMP-2 vial making a 100 .mu.g/mL
solution. The solution was briefly spun in a microfuge. A 100 .mu.l
(10 .mu.g) aliquot was placed into a sterile tube and stored at
-80.degree. C. until day of implant.
[0057] Implant Preparation and Surgery:
[0058] A flowable implant composition of ECM material was produced
by rehydrating micronized small intestine submucosa, (SIS) in PBS.
The rehydrated, flowable ECM formulation was mixed well, and 100
.mu.l was transferred to a sterile syringe and connected to a
second syringe containing the 100 .mu.l aliquot of rhBMP-2 solution
prepared as described in this Example above. The rehydrated,
flowable ECM formulation and rhBMP-2 were mixed together using a
syringe connector and then placed in an incubator at 37.degree. C.
for a minimum of 30 minutes.
[0059] The rehydrated, flowable ECM containing rhBMP-2 was removed
from the incubator and then passed into the surgical field.
Formulations were briefly mixed, and 100 .mu.l of the rehydrated,
flowable ECM containing approximately 5 .mu.g of rhBMP-2 was
transferred to a sterile syringe and promptly injected through a
23G needle into the hind thigh muscle of a known immunodeficient
(NOD/SCID) mouse model of ectopic bone formation. Control
(negative) treatments were prepared and injected into the thigh
muscle as described in the example above with the exception that
the rehydrated, flowable ECM formulation was mixed with 100 .mu.l
of PBS. Post injection mice were recovered and returned to the
housing unit. The mice in the study were imaged in vivo with x-ray
at 1 and 2 weeks post injection. The x-rays were used to detect de
novo bone formation in the thigh muscle. The mice in the study were
sacrificed at 2 weeks post injection and imaged ex vivo with
micro-computed tomography (microCT). The microCT scans were used to
image and quantify de novo bone formation in the thigh muscle.
[0060] Results:
[0061] Mice treated with the flowable ECM containing 5 .mu.g
rhBMP-2 showed abundant de novo bone formation within the thigh
muscle at 2 weeks post injection (FIG. 1,A). Control animals that
received an injection of 100 .mu.l of flowable ECM exhibited no
intramuscular bone formation at 2 weeks follow-up (FIG. 1,B).
[0062] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural reference unless
the context clearly dictates otherwise. Unless defined otherwise
all technical and scientific terms used herein have the same
meaning as commonly understood to one of ordinary skill in the art
to which this invention belongs.
[0063] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower
limit, unless the context clearly dictates otherwise, between the
upper and lower limit of that range and any other stated or
intervening value in that stated range, is encompassed within the
invention. The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and such
embodiments are also encompassed within the invention, subject to
any specifically excluded limit in the stated range. Where the
stated range includes one or both of the limits, ranges excluding
either or both of those included limits are also included in the
invention.
[0064] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing components
that are described in the publications that might be used in
connection with the presently described invention.
[0065] While the invention has been illustrated and described in
detail in the drawings and foregoing description, the same is to be
considered as illustrative and not restrictive in character, it
being understood that only the preferred embodiment has been shown
and described and that all changes and modifications that come
within the spirit of the invention are desired to be protected. In
addition, all publications cited herein are indicative of the
abilities of those of ordinary skill in the art and are hereby
incorporated by reference in their entirety as if individually
incorporated by reference and fully set forth.
* * * * *