U.S. patent application number 12/150513 was filed with the patent office on 2008-10-23 for cancellous bone treated with collagenase and essentially free of blood cells.
Invention is credited to Dayna Buskirk, Earl Fender, Charles Randal Mills, Rodney Monroy, Michelle LeRoux Williams, Robert A. Zambon.
Application Number | 20080262633 12/150513 |
Document ID | / |
Family ID | 41255382 |
Filed Date | 2008-10-23 |
United States Patent
Application |
20080262633 |
Kind Code |
A1 |
Williams; Michelle LeRoux ;
et al. |
October 23, 2008 |
Cancellous bone treated with collagenase and essentially free of
blood cells
Abstract
A bone implant comprising cancellous bone that is essentially
free of blood cells, and which has been treated with at least one
loosening agent, such as collagenase or a digestive enzyme, for a
time and at a concentration to loosen the osteogenic cells in the
cancellous bone matrix. The osteogenic cells in the matrix are
viable cells. The treatment of the cancellous bone with at least
one loosening agent enables the osteogenic cells to be more
available for carrying out their osteogenic function and to provide
for an increased rate of bone formation. Such implant also may
include demineralized bone, such as demineralized cortical bone,
which enhances the bone regenerative capacity of the cancellous
bone.
Inventors: |
Williams; Michelle LeRoux;
(Laurel, MD) ; Mills; Charles Randal; (Finksburg,
MD) ; Monroy; Rodney; (Aberdeen, MD) ; Zambon;
Robert A.; (Rockville, MD) ; Buskirk; Dayna;
(Gainesville, FL) ; Fender; Earl; (Plano,
TX) |
Correspondence
Address: |
Raymond J. Lillie, Esq.;c/o Carella, Byrne, Bain, Gilfillan,
Cecchi, Stewart & Olstein, 5 Becker Farm Road
Roseland
NJ
07068
US
|
Family ID: |
41255382 |
Appl. No.: |
12/150513 |
Filed: |
April 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11799606 |
May 2, 2007 |
|
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|
12150513 |
|
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|
|
60831723 |
Jul 18, 2006 |
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60798474 |
May 8, 2006 |
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Current U.S.
Class: |
623/23.63 ;
623/16.11 |
Current CPC
Class: |
A61F 2/28 20130101; A61L
27/365 20130101; A61F 2310/00359 20130101; A61L 27/3608 20130101;
A61L 27/3687 20130101 |
Class at
Publication: |
623/23.63 ;
623/16.11 |
International
Class: |
A61F 2/28 20060101
A61F002/28 |
Claims
1. A bone implant, comprising: cancellous bone, said cancellous
bone being essentially free of blood cells and having been treated
with at least one loosening agent for a time and at a concentration
to loosen osteogenic cells in the cancellous bone matrix, said
osteogenic cells in the cancellous bone matrix being viable
cells.
2. The implant of claim 1 wherein said at least one loosening agent
is selected from the group consisting of collagenase and digestive
enzymes.
3. The implant of claim 2 wherein said at least one loosening agent
is collagenase.
4. The implant of claim 3 wherein said bone is treated with said
collagenase at a concentration of from about 0.1 mg/ml to about 3
mg/ml.
5. The implant of claim 4 wherein said bone is treated with said
collagenase at a concentration of from about 1.0 mg/ml to about 3
mg/ml.
6. The implant of claim 3 wherein said bone is treated with said
collagenase for a period of time from about 5 min. to about 3
hrs.
7. The implant of claim 6 wherein said bone is treated with said
collagenase for a period of time from about 5 min. to about 30
min.
8. The implant of claim 3 wherein said bone is treated with said
coliagenase at a concentration of about 1.0 mg/ml for a period of
time from about 10 minutes.
9. A bone implant comprising: (i) cancellous bone, said cancellous
bone including cancellous bone which is essentially free of blood
cells and having been treated with at least one loosening agent for
a time and at a concentration to loosen osteogenic cells in the
cancellous bone matrix, said osteogenic cells in the cancellous
bone matrix being viable cells; and (ii) cortical bone which has
been treated with at least one demineralization agent for a time
and at a concentration to expose osteoinductive proteins present in
the cortical bone matrix.
10. The implant of claim 9 wherein said at least one
demineralization agent is hydrochloric acid.
11. The implant of claim 10 wherein said cortical bone is treated
with said hydrochloric acid at a concentration of from about 0.1N
to about 12N.
12. The implant of claim 11 wherein said cortical bone is treated
with said hydrochloric acid at a concentration of about 0.5N.
13. The implant of claim 9 wherein said cortical bone is treated
with said at least one demineralization agent for a period of time
of from about 1 minute to about 72 hours.
14. The implant of claim 13 wherein said cortical bone is treated
with said at least one demineralization agent for a period of time
of from about 50 minutes to about 70 minutes.
15. The implant of claim 9 wherein said cortical bone comprises
particles having a D.sub.90 of less than about 1,500 microns.
16. The implant of claim 15 wherein said particles have a D.sub.90
from about 125 microns to about 1,500 microns.
17. The implant of claim 16 wherein said particles have a D.sub.90
from about 780 microns to about 1,500 microns.
18. The implant of claim 16 wherein said particles have a D.sub.90
from about 125 microns to about 780 microns.
19. The implant of claim 15 wherein said cortical bone comprises a
first portion comprising particles having a D.sub.90 of from about
780 microns to about 1,500 microns and a second portion comprising
particles having a D.sub.90 of from about 125 microns to about 780
microns.
20. The bone implant of claim 19 wherein said bone implant includes
at least 50 vol. % of said cancellous bone, from about 5 vol. % to
about 40 vol. % of said cortical bone particles having a D.sub.90
of from about 780 microns to about 1,500 microns, and from about 5
vol. % to about 20 vol. % of said cortical bone particles having a
D.sub.90 of from about 125 microns to about 780 microns.
21. The implant of claim 20 wherein said cancellous bone is present
in said implant in an amount of from about 55 vol. % to about 85
vol. %.
22. The implant of claim 21 wherein said cancellous bone is present
in said implant in an amount of from about 55 vol. % to about 65
vol. %.
23. The implant of claim 20 wherein said cortical bone particles
having a D.sub.90 of from about 780 microns to about 1,500 microns
are present in an amount of from about 5 vol. % to about 30 vol.
%.
24. The implant of claim 23 wherein said cortical bone particles
having a D.sub.90 from about 780 microns to about 1,500 microns are
present in an amount of from about 24 vol. % to about 28 vol.
%.
25. The implant of claim 20 wherein said cortical bone particles
having a D.sub.90 from about 125 microns to about 780 microns are
present in an amount of from about 8 vol. % to about 18 vol. %.
26. The implant of claim 25 wherein said cortical bone particles
having a D.sub.90 from about 125 microns to about 780 microns are
present in an amount of about 18 vol %.
Description
[0001] This application is a continuation-in part of application
Ser. No. 11/799,606, filed May 2, 2007 which claims priority based
on provisional application Ser. No. 60/831,723, filed Jul. 18,
2006, and provisional application Ser. No. 60/798,474, filed May 8,
2006, the contents of which are incorporated herein by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0002] This invention relates to bone implants comprising
cancellous bone useful in the treatment or prevention of bone
diseases, disorders, defects, or injuries.
BRIEF SUMMARY OF THE INVENTION
[0003] This invention relates to bone implants which include
cancellous bone. More particularly, this invention relates to bone
implants that include cancellous bone that has been treated with at
least one loosening agent in order to loosen osteogenic cells in
the bone matrix. The cancellous bone also is essentially free of
blood cells.
[0004] This invention also relates to bone implants which include
the cancellous bone hereinabove described, and demineralized bone.
More particularly, this invention also relates to bone implants
which include the cancellous bone hereinabove described, and bone,
such as, for example, cortical bone, which has been treated with at
least one demineralization agent for a time and at a concentration
to expose osteoinductive proteins in the bone matrix.
[0005] Bone implants which include cancellous bone have been used
in a variety of procedures and treatments, including bone fusions
such as spine fusions, disc augmentations in the spine, and bone
fill applications employed in the treatments of diseases,
disorders, or injuries including, but not limited to, avascular
osteonecrosis, osteosarcoma, acute fractures and fracture
non-unions, as well as for bone regeneration for orthopedic
implants. Such bone in general may be harvested from any source of
cancellous bone, including vertebrae, the iliac crest, femur,
tibia, or ribs.
[0006] Cancellous bone implants in general have included, in
addition to osteocytes and osteogenic cells, blood cells including
hematopoietic cells. In some implants, the implants have been
treated in order to preserve the viability of all the cells in the
implant, while in other implants, the viability of the bone cells,
including the osteogenic cells, has been destroyed.
[0007] The present invention provides a bone implant which includes
cancellous bone including viable osteogenic cells which are made
more available for carrying out their osteogenic function.
[0008] In accordance with an aspect of the present invention, there
is provided a bone implant comprising cancellous bone. The
cancellous bone is essentially free of blood cells, and has been
treated with at least one loosening agent for a time and at a
concentration to loosen the osteogenic cells in the cancellous bone
matrix. The osteogenic cells in the bone matrix are viable
cells.
[0009] The term "loosening agent" as used herein, means an agent
which may be contacted with a bone matrix for a time and at a
concentration sufficient to loosen osteogenic cells in the
cancellous bone matrix without releasing the osteogenic cells from
the cancellous bone matrix. An exemplary loosening agent useful in
accordance with the present invention comprises collagenase.
Another exemplary loosening agent useful in accordance with the
present invention comprises a digestive enzyme. In some
embodiments, the digestive enzyme is selected from the group
consisting of trypsin, amylase, lipase, and combinations thereof.
In some embodiments, the digestive enzyme is trypsin. In some
embodiments, the digestive enzyme is amylase. In some embodiments,
the digestive enzyme is lipase. Another exemplary loosening agent
useful in accordance with the present invention comprises a
combination of collagenase and a digestive enzyme for a time and at
a concentration to loosen the osteogenic cells contained in the
bone matrix, but not release the osteogenic cells from the bone
matrix.
[0010] The term "osteogenic cell", as used herein, means any type
of cell having osteoprogenitor potential, that is, any type of cell
that is capable of differentiating into a bone cell.
[0011] The term "D90", as used herein, refers to the size of the
90th percentile (by number) particle in a cumulative size
distribution of all particles sampled.
[0012] Although the scope of the present invention is not to be
limited to any theoretical reasoning, when the bone implant is
essentially free of blood cells and has been treated with a
loosening agent in order to loosen the osteogenic cells in the
cancellous bone matrix, such osteogenic cells become more available
for or are more disposed toward carrying out their osteogenic
function, and provide for an increased rate of bone formation.
Thus, such bone implants are capable of generating or "growing"
bone directly and provide for improved implants vis-a-vis prior art
implants, including previously produced cancellous bone implants,
and ceramic implants.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The compositions of the present invention are useful in the
treatment or prevention of bone diseases, disorders, defects, or
injuries.
[0014] In one embodiment, the present invention comprises a bone
implant that comprises: cancellous bone matrix, said cancellous
bone matrix being essentially free of blood cells, said cancellous
bone matrix having been treated with at least one loosening agent
for a time and at a concentration to loosen osteogenic cells in the
cancellous bone matrix, said osteogenic cells in the cancellous
bone matrix being viable cells.
[0015] In one embodiment, the present invention comprises a bone
implant that comprises: (i) cancellous bone matrix, said cancellous
bone including cancellous bone which is essentially free of blood
cells and having been treated with at least one loosening agent for
a time and at a concentration to loosen osteogenic cells in the
cancellous bone matrix, said osteogenic cells in the cancellous
bone matrix being viable cells; and (ii) cortical bone matrix which
has been treated with at least one demineralization agent for a
time and at a concentration to expose osteoinductive proteins
present in the cortical bone matrix.
[0016] In some embodiments, the at least one demineralization agent
is hydrochloric acid.
[0017] The cancellous bone is treated with the loosening agent to
loosen the osteogenic cells in the cancellous bone matrix. In one
embodiment, the cancellous bone is treated with at least one
loosening agent at a concentration of from about 0.1 mg/ml to about
3.0 mg/ml, and in another embodiment from about 1.0 mg/ml to about
3.0 mg/ml. The cancellous bone is treated with at least one
loosening agent for a period of time to loosen the osteogenic cells
in the cancellous bone matrix, but not release the osteogenic cells
from the cancellous bone matrix. In one embodiment, the cancellous
bone is treated with at least one loosening agent for about 5 min.
to about 3 hrs. In another embodiment, the cancellous bone is
treated with at least one loosening agent for about 5 min. to about
30 min. In yet another embodiment, the cancellous bone is treated
with at least one loosening agent at a concentration of about 1
mg/ml for about 10 minutes.
[0018] Although Applicants do not intend to be limited to any
theoretical reasoning, it is believed that, by treating the bone
with at least one loosening agent as hereinabove described, such
treatment provides for a partial, but not complete, digestion of
the components of the bone matrix (such as collagen, for example).
Such partial digestion of the components of the bone matrix,
loosens, but does not release, osteogenic cells from the matrix,
thereby making such cells, as noted hereinabove, more available for
or more disposed toward carrying out their osteogenic function.
[0019] In general, the cancellous bone is harvested from any
cancellous bone bearing source. Such sources include, but are not
limited to, vertebral bodies in the spine, the iliac crest, femur,
tibia, and ribs. The cortical shell of the bone is removed, and
then the bone is cut or milled into desired pieces or shapes. For
example, the bone may be cut or milled into bone chips, or may be
cut into wedges or plugs, or may be formed into pellets.
[0020] The bone then is washed to remove blood cells, such as red
blood cells and hematopoietic cells. After the bone is washed, the
bone is treated with at least one loosening agent, such as
collagenase or a digestive enzyme. As noted hereinabove, the bone
is treated with a loosening agent for a time and at a concentration
to loosen the osteogenic cells contained in the bone matrix, but
not release the osteogenic cells from the bone matrix. In one
embodiment, the bone is treated with the loosening agent at a
concentration of about 0.1 mg/ml to about 3 mg/ml, and in another
embodiment from about 1.0 mg/ml to about 3.0 mg/ml. In another
embodiment, the bone is treated for about 5 min. to about 3 hrs. In
another embodiment, the bone is treated for about 5 min. to about
30 min. In yet another embodiment, the bone is treated with the
loosening agent at a concentration of about 1.0 mg/ml for about 10
minutes.
[0021] In one embodiment, subsequent to the treatment with the
loosening agent the bone is treated with one or more antibiotics or
one or more antimycotics in order to reduce the level of bioburden
within the bone. Antibiotics which may be employed include, but are
not limited to, gentamicin; vancomycin; penicillins; macrolide
antibiotics, such as erythromycin; sulfa-based antibiotics, and
combinations thereof. Antimycotics which may be employed include,
but are not limited to, amphotericin, fluconazole, and combinations
thereof.
[0022] After antibiotic or antimycotic treatment, when chipped or
milled bone is employed, the chipped or milled bone, if desired,
may be filtered through sieves in order to retain pieces of the
bone which have a desired size.
[0023] After the bone has been washed, treated with the loosening
agent and treated with an antibiotic or antimycotic, the bone then
may be added to an appropriate preservation medium, such as a
cryopreservation or vitrification medium, in which the bone may be
preserved and stored, and the osteogenic cells contained therein
will remain viable. In one embodiment, the preservation medium may
include one or more of glycerol, dimethylsulfoxide, and DMSO. In
one embodiment, the preservation medium enables the treated bone to
be frozen at temperatures as low as about -140.degree. C. and as
high as about -20.degree. C. while maintaining the viability of the
osteogenic cells. The present invention also contemplates that the
cancellous bone treated with a loosening agent may be combined with
bone that has not been treated with a loosening agent prior to
packaging as a final product. For example, the cancellous bone
treated with the loosening agent may be mixed with bone that has
not been treated with a loosening agent, such as allograft bone
chips, fragments or powder, or nucleus pulposus. For example, in
one embodiment, the bone treated with the loosening agent is
admixed with demineralized bone prior to formulation into a final
product. In another embodiment, the final product includes about 50
vol. % treated bone, and about 50 vol. % untreated bone.
[0024] The bone implant treated with the loosening agent may be
administered to an animal in an amount effective to treat a bone
disease, disorder, defect, or injury in the animal. The animal may
be a mammal, including human and non-human primates. In one
embodiment, the animal is a human.
[0025] Bone diseases, disorders, defects, or injuries which may be
treated by the bone implant treated with the loosening agent
include, but are not limited to, degenerative disc disease,
avascular osteonecrosis, osteosarcoma fractures, and fracture
non-unions. The bone implant treated with the loosening agent also
may be employed in bone fusions, such as spine fusions, as well as
in disc augmentation, and for bone regeneration in orthopedic
implants.
[0026] The bone implant treated with the loosening agent may be
administered directly to the site of the bone disease, disorder,
defect, or injury. Depending upon the form and shape of the
implant, the implant may be injected directly into the site
affected by the bone disease, disorder, defect, or injury, or the
implant may be packed directly into the site affected by the bone
disease, disorder, defect, or injury. The implant has a sufficient
consistency such that the implant will be retained at the
implantation site long enough for initial bone formation,
osteoinductive signaling, and host cell attachment to occur. The
present invention also contemplates that the bone implant may be
employed in conjunction with devices employed in the treatment of
bone diseases, defects, disorders, and injuries, such as, for
example, orthopedic cage devices, ceramics, or plates which may be
employed in the spine or in bones to promote bone growth and
fusion. Furthermore, the bone implant may be used in conjunction
with an autologous bone graft. The bone implant also may be
administered with antibiotics, such as those hereinabove described,
antimycotics, or anti-inflammatory agents. In another embodiment,
the bone implant may be administered in combination with
osteoinductive factors such as, for example, bone morphogenic
proteins, or BMPs, such as BMP-2 and BMP-7, and platelet-derived
growth factor (PDGF), which enhance the osteogenic potential of the
bone implant. It is to be understood, however, that the scope of
the present invention is not intended to be limited to the
treatment of any particular bone disease, defect, disorder, or
injury, or to any particular form or any particular method of
administration of the bone implant.
[0027] As noted hereinabove, the cancellous bone may be admixed
with demineralized bone. Thus, in accordance with another aspect of
the present invention, there is provided a bone implant comprising
(i) cancellous bone, said cancellous bone including cancellous bone
which is essentially free of blood cells, as hereinabove described,
which has been treated with at least one loosening agent for a time
and at a concentration to loosen the osteogenic cells in the
cancellous bone matrix, wherein the osteogenic cells in the bone
matrix are viable cells; and (ii) bone which has been treated with
at least one demineralization agent for a time and at a
concentration to expose osteoinductive proteins present in the bone
matrix.
[0028] In another embodiment, the cancellous bone, in addition to
the treated cancellous bone hereinabove described, further
comprises bone that has not been treated with a loosening
agent.
[0029] Although the scope of the present invention is not to be
limited to any theoretical reasoning, Applicants have discovered
that the demineralized bone enhances the bone regenerative capacity
of the cancellous bone.
[0030] In one embodiment, the bone which is treated with the at
least one demineralization agent is cortical bone.
[0031] In one embodiment, the at least one demineralization agent
which is used to treat the bone, such as, for example, cortical
bone, includes, but is not limited to, any acidic solution. In
another embodiment, the at least one demineralization agent is
hydrochloric acid.
[0032] The bone, such as, for example, cortical bone, is treated
with the at least one demineralization agent for a time and at a
concentration to expose osteoinductive proteins present in the bone
matrix. Such osteoinductive proteins include, but are not limited
to, a heterogeneous mixture of bone morphogenic proteins and growth
factors. In one embodiment, the bone, such as, for example,
cortical bone, is treated with the at least one demineralization
agent, such as hydrochloric acid, at a concentration of from about
0.1 N to about 12 N. In another embodiment, the bone, such as, for
example, cortical bone, is treated with the at least one
demineralization agent, such as hydrochloric acid at a
concentration, for example, of about 0.5 N.
[0033] In one embodiment, the bone, such as, for example, cortical
bone, is treated with the at least one demineralization agent for a
period of time of from about 1 minute to about 72 hours. In another
embodiement, the bone, such as, for example, cortical bone, is
treated with the at least one demineralization agent for a period
of time of from about 15 minutes to 180 minutes. In another
embodiment, the bone, such as, for example, cortical bone, is
treated with the at least one demineralization apart for a period
from about 30 minutes to about 120 minutes. In another embodiment,
the bone, such as, for example, cortical bone, is treated with the
at least one demineralization agent for a period of time of from
about 50 minutes to about 70 minutes.
[0034] In one embodiment, the bone, such as, for example, cortical
bone, comprises particles which have a D.sub.90 of less than about
1,500 microns. In another embodiment, the particles have a D.sub.90
of about 125 microns to about 1,500 microns. In yet another
embodiment, the particles have a D.sub.90 of about 780 microns to
about 1,500 microns. In another embodiment, the particles have a
D.sub.90 of about 125 microns to about 780 microns. In another
embodiment, the particles have a D.sub.90 of about 600 microns to
about 900 microns. In another embodiment, the particles have a
D.sub.90 of about 700 microns to about 800 microns. In another
embodiment, the particles have a D.sub.90 of about 780 microns.
[0035] In still another embodiment, the bone, such as, for example,
cortical bone, comprises a first portion comprising particles
having a D.sub.90 from about 780 microns to about 1,500 microns,
and a second portion comprising particles having a D.sub.90 from
about 125 microns to about 780 microns.
[0036] In general, the bone, such as, for example, cortical bone,
is harvested from any bone bearing source. Such sources include,
but are not limited to, the iliac crest, femur, humerus, tibia,
fibula, radius, ulna, and ribs. The bone, such as, for example,
cortical bone, is removed from its source, and then is cut or
milled into particles. The particles then may be separated
according to size, such as by passing the particles through one or
more sieves. For example, the particles may be passed through a
1,500 micron sieve, then through a 780 micron sieve, and then
through a 125 micron sieve. Such sieving provides a portion of bone
particles having a D.sub.90 from 780 microns to 1,500 microns, and
another portion of bone particles having a D.sub.90 from 125
microns to 780 microns.
[0037] The sieved bone then is treated with at least one
demineralization agent for a time and at a concentration to expose
osteogenic proteins present in the cortical bone matrix. In one
embodiment, the bone particles, such as, for example, cortical bone
particles, are treated with hydrochloric acid at a concentration of
about 0.5 N for a period of time of from about 15 minutes to 180
minutes. In one embodiment, the bone particles, such as, for
example, cortical bone particles, are treated with hydrochloric
acid at a concentration of about 0.5 N for a period of time of from
about 30 minutes to 120 minutes. In one embodiment, the bone
particles, such as, for example, cortical bone particles, are
treated with hydrochloric acid at a concentration of about 0.5 N
for a period of time of from about 50 minutes to 70 minutes.
[0038] In one embodiment, subsequent to the treatment with the at
least one demineralization agent, the bone, such as, for example,
cortical bone is treated with one or more antibiotics or one or
more antimycotics in order to reduce the level of bioburden within
the bone. Antibiotics and antimycotics which may be employed
include, but are not limited to, those mentioned hereinabove with
respect to treatment of the cancellous bone.
[0039] After the bone, such as, for example, cortical bone, has
been treated as hereinabove described, it then is combined with the
cancellous bone to provide a bone implant product.
[0040] In one embodiment, the bone implant includes at least about
50 vol. % of the cancellous bone hereinabove described, and up to
about 50 vol. % of the demineralized bone, such as demineralized
cortical bone, as hereinabove described. In another embodiment, the
bone implant includes from about 55 vol. % to about 85 vol. % of
the cancellous bone, and from about 15 vol. % to about 45 vol. % of
the demineralized bone. In yet another embodiment, the bone implant
includes from about 55 vol. % to about 65 vol. % of the cancellous
bone, and from about 35 vol. % to about 45 vol. % of the
demineralized bone.
[0041] In one embodiment, the bone implant includes at least about
50 vol. % of the cancellous bone hereinabove described, from about
5 vol. % to about 40 vol. % of demineralized bone particles, such
as demineralized cortical bone particles, having a D.sub.90 from
about 780 microns to about 1,500 microns, and from about 5 vol. %
to about 20 vol. % of demineralized bone particles, such as
demineralized cortical bone particles, having a D.sub.90 from about
125 microns to about 780 microns.
[0042] In one embodiment, the cancellous bone is present in the
bone implant in an amount of from about 55 vol. % to about 85 vol.
%. In another embodiment, the cancellous bone is present in the
bone implant in an amount of from about 55 vol. % to about 65 vol.
%. In still another embodiment the cancellous bone is present in
the bone implant in an amount of from about 57 vol. % to about 65
vol. %.
[0043] In one embodiment, the demineralized bone particles, such as
demineralized cortical bone particles, having a D.sub.90 from about
780 microns to about 1,500 microns are present in the bone implant
in an amount of from about 5 vol. % to about 30 vol. %. In another
embodiment, the demineralized bone particles having a D.sub.90 from
about 780 microns to about 1,500 microns are present in the bone
implant in an amount of from about 24 vol. % to about 28 vol.
%.
[0044] In one embodiment, the demineralized bone particles, such as
demineralized cortical bone particles, having a D.sub.90 from about
125 microns to about 780 microns are present in the bone implant in
an amount of from about 8 vol. % to about 18 vol. %. In yet another
embodiment, the demineralized bone particles having a D.sub.90 from
about 125 microns to about 780 microns are present in the bone
implant in an amount of about 18 vol. %.
[0045] The bone implant including the cancellous bone and
demineralized bone, such as, for example, demineralized cortical
bone, may be added to an appropriate preservation medium, such as a
cryopreservation medium or vitrification medium as hereinabove
described, in which the bone implant may be stored prior to
use.
[0046] Such bone implant, which includes the cancellous bone and
demineralized bone as hereinabove described, may be used to treat
the bone defects, disorders, and injuries hereinabove described,
and may be administered directly to the site of the bone disease
disorder, defect, or injury as hereinabove described to treat such
disease, disorder, defect, or injury.
[0047] The invention now will be described with respect to the
following examples. It is to be understood, however, that the scope
of the present invention is not intended to be limited thereby.
EXAMPLE 1
[0048] A bone sample was removed from a saline holding solution
after debridement of soft tissue, and swabbed with a wiper. The
bone was placed in a cutting area, and positioned face up. The bone
then was cut with a reciprocating saw to remove the cortical shell.
The remaining bone then was placed into a 500 ml washing bag filled
halfway with saline (0.9%) and anticoagulant citrate dextrose
solution, Formula A (ACD-A), at a ratio of 9 parts 0.9% saline to
one part ACD-A. The bag lid then was closed, and shaken vigorously.
The bone sample then was retrieved from the washing solution, and
then placed into a bone milling chamber. The bone was then milled,
and then the milled bone was spooned out, and placed into a 500 ml
receiver bottle containing up to 300 ml of 0.9% saline and ACD-A at
a volume ratio of 9 parts saline to one part ACD-A.
[0049] The milled bone then was washed. First, the wash solution
was decanted from the receiving bottle into a waste beaker. The
wash solution was replaced with a new wash solution of 0.9% saline
and ACD-A (volume ratio of 9 parts saline to one part ACD-A), the
lid on the receiver bottle was tightened, and the bottle was shaken
vigorously. The above washing was repeated until the wash solution
was clear, and the chips of milled bone were white to off-white in
color. The last wash solution was decanted, and the bone chips were
removed from the receiver bottle with a sampler spoon. Using a
Petri dish and forceps, bone samples then were separated out from
large blood clot pieces and tissues. The bone then was packed into
a 50 ml conical tube, and the total volume was recorded. The total
volume of bone chips was split by separating half for collagenase
treatment and half for fresh chips. The chips designated for
collagenase treatment were placed into a sterile 500 ml receiver
bottle, and the fresh chips were placed into a 500 ml receiver
bottle and covered with 0.9% saline and ACD-A at a volume ratio of
9 parts saline to one part ACD-A.
[0050] A collagenase solution then was prepared by mixing a
collagenase powder with 1.times. Phosphate Buffered Saline ("PBS")
in an amount of 1 mg collagenase for each milliliter of PBS. The
collagenase solution was added to the milled bone designated for
the collagenase treatment in an amount of 2 ml collagenase solution
for each cc of bone to be treated. The bottle containing the milled
bone and the collagenase solution then was placed, with the lid
loose, onto a rocker inside a 37.degree. C. incubator for 10
minutes.+-.1 minute. The bottle then was removed from the
incubator, and the collagenase solution was decanted into a
bottles. The collagenase treated milled bone then was rinsed by
adding the same volume of PBS and ACD-A at a volume ratio of 9
parts PBS to one part ACD-A. This rinsing solution then was
decanted into the bottle containing the collagenase solution. The
rinsing then was repeated, the rinsing solution again was decanted,
and the collagenased bone sample was added to the bottle of fresh
bone sample. The resulting combined (collagenase treated plus
fresh) bone sample then was poured through a 1 mm sieve and placed
in a clean bottle. The combined bone sample then was rinsed with
Dulbecco's Minimal Essential Medium (DMEM), low glucose, with
phenol, prior to antibiotic treatment. A demineralized bone sample
also was rinsed with DMEM prior to antibiotic treatment.
[0051] A 1.times. antibiotic solution then was prepared. For each 1
ml of solution, 0.9 ml of DMEM was mixed with 0.005 ml of 10 mg/ml
gentamicin sulfate, 0.05 ml of 50 mg/ml vancomycin HCl, and 0.01 ml
of 250 .mu.g/ml amphotericin B. Thus, the antibiotic solution
included gentamicin sulfate at a concentration of 50 .mu.g/ml,
vancomycin HCl at a concentration of 50 .mu.g/ml, and amphotericin
B at a concentration of 2.5 .mu.g/ml.
[0052] The antibiotic solution then was added to each of the
combined bone sample and the demineralized bone sample in an amount
of 2 ml for each 1 cc of bone sample. The bottles then were placed
onto a rocker inside of a 37.degree. C. incubator, with the lids
loose, for no less than 18 hours. The bottles then were removed
from the incubator, placed under a biological safety container, and
the antibiotic solutions were decanted from each of the combined
bone sample, and the demineralized bone sample, and each of the
bone samples then was washed with the same volume of PBS. The
samples then were shaken vigorously. The rinse solutions from the
PBS wash were retained, and the pH of each solution was determined.
The samples were washed with PBS until the pH of the rinse
solutions was in the range of 5.0 to 7.5. After the PBS washing was
completed, PBS was decanted from each of the bone samples, and the
same volume of Plasma Lyte-A (Baxter) solution, which includes 140
meq/l Na.sup.+, 5 meq/l K+, 98 meq Cl-, 3 meq Mg.sup.2+, and 27
meq/l acetate, was added to each bone sample. The bone samples were
shaken vigorously, and the Plasma Lyte-A washing was repeated twice
for each sample.
[0053] Using a sampler spoon, a 50 ml conical tube was packed
gently with the combined (collagenased and fresh) bone sample, and
a demineralized bone sample was packed gently into a 15 ml conical
tube. 5 ml of a cryopreservation solution was added to the 15 ml
conical tube and shaken vigorously. Each ml of cryopreservation
solution included 0.7 ml of 1.times. Plasma Lyte-A, 0.2 ml of 25%
human serum albumin, and 0.1 ml of 1.times. dimethylsulfoxide
(DMSO) (CryoServ.) The final concentration of human serum albumin,
therefore, was 5% and the final concentration of dimethylsulfoxide
was 10%. After shaking, the demineralized bone sample was
transferred to the 50 ml conical tube holding the cancellous bone,
and then 10 ml of cryopreservation solution were added to the 50 ml
conical tube and shaken vigorously. The entire sample then was
placed in a product dose jar. 5 ml of cryopreservation solution
then were added to the product dose jar. If the bone product were
not covered with solution, up to 5 ml more of cryopreservation
solution were added. The jar then was sealed. After the jar was
sealed, the product dose jar was placed into a packaging bag. The
bag then was sealed. The secondary packaging bag and a package
insert then were placed into a tertiary mailer package, which then
was heat sealed. The mailer package then was placed into an
-80.degree. C. quarantine freezer until the bone product was ready
to be used.
EXAMPLE 2
[0054] A bone product from a single donor was prepared as described
in Example 1, except that, instead of storing 50 ml samples at
-80.degree. C., fourteen 5 cc doses were labeled and stored at
-50.degree. C..+-.5.degree. C. and six 5 cc doses were labeled and
stored at -80.degree. C..+-.5.degree. C. Seventeen days after
freezing, three of the doses stored at -50.degree. C. and three of
the doses stored at -80.degree. C. were thawed.
[0055] The jars containing the frozen bone samples placed in a
37.degree. C. water bath until the entire frozen products were
thawed. The jars were removed from the water bath immediately upon
thawing, and sprayed with 70% isopropanol. The outsides of the jars
then were dried. The jars then were transferred to a biological
safety cabinet, the lids were removed, and the thawed
cryopreservation solutions were aspirated directly from the jars.
25 ml of Dulbecco's Minimal Essential Medium-low glucose (DMEM-lg)
then were added to each of the jars, and the containers were
swirled such that the DMEM-lg covered the entire bone samples
completely. The DMEM-lg then was aspirated from each jar, and the
entire bone samples were transferred to 50 ml conical tubes. An
additional 25 ml of DMEM-lg then were added to each of the conical
tubes containing the bone samples.
[0056] A 1 mg/ml collagenase solution (1 mg/1 ml PBS) was prepared
in a 250 ml receiver bottle. 2 ml of collagenase solution were
prepared per 1 cc of bone sample.
[0057] The DMEM-lg was aspirated from the 50 ml conical tubes
containing the bone samples, and then 25 ml of PBS were added to
each of the conical tubes, and the tubes were swirled to wash away
any remaining DMEM-lg. The washing with PBS was repeated as
necessary to remove any DMEM-lg still visible in the bone
samples.
[0058] After the final PBS wash, the PBS was aspirated off and the
bone samples were transferred to 250 ml receiver bottles containing
the collagenase solutions. The bottles then were placed onto a
rocker inside a 37.degree. C. incubator for 15 minutes.+-.1 minute.
The bottles then were removed from the incubator, and the
collagenase solutions were pipetted over 70 .mu.m cell strainers
into 250 ml conical tubes. The collagenased bone samples then were
rinsed with PBS at volumes equal to the original collagenase
treatments. The PBS rinses were pipetted over the 70 .mu.m cell
strainers into the 250 ml conical tubes. The PBS rinsing was
repeated twice, and the conical tubes were placed in a centrifuge
(Beckman #GS-6R) and spun at 1960 rpm for 8 minutes (at
approximately 878 g). The conical tubes then were transferred to
the biological safety cabinet, and the supernatants were aspirated.
The pellets then were loosened gently by dragging the tubes across
the tube racks. The pellets then were resuspended in appropriate
volumes (1 to 4 ml) of PBS/ACD-A (containing 59 ml ACD-A per 500 ml
PBS).
[0059] Three of the samples that were frozen at -50.degree. C. and
three of the samples that were frozen at -80.degree. C. then were
tested for cell viability.
[0060] Using a 2-20 .mu.l Pipetman pipette (Gilson, No. P20), 20
.mu.l aliquots of the resulting cell suspensions were added to a
microcentrifuge tube. 20 .mu.l aliquots of 0.4% Trypan Blue then
were added to each of the microcentrifuge tubes, and the contents
were mixed by finger tapping. 10 .mu.l of the cell suspensions then
were transferred into each side of hemacytometers (Hausser
Scientific, Model No. Steri-Cult 200) with a cover slip in place.
The cells then were allowed to settle in the counting chambers
before starting counts of viable and non-viable cells. The cells
were not exposed to the Trypan Blue for more than 10 minutes, to
prevent viable cells from absorbing the dye.
[0061] With the 10.times. objective of the microscope, the grid
lines on each of the chambers' center 1 mm.times.1 mm squares were
focused. The slides were positioned such that the central areas of
the grids were seen. The objective of the microscope then was
changed to the 20.times. objective. The microscope then was
focused, and viable and non-viable cells were counted. Viable and
non-viable cells in two chambers for each cell suspension were
counted. Viable and non-viable cell counts then were determined,
from which were calculated the concentration of viable cells, the
concentration of all cells, and the % viability of the cells.
[0062] In order to be acceptable for in vivo administration, the
samples must have a viability of at least 70%, and have a cell
density of at least 1.times.10.sup.3 cells/cc.
[0063] The viability results for each of the samples are given in
Table 1 below.
TABLE-US-00001 TABLE 1 Storage temperature % Viabilitv Cells/cc 50
.+-. 5.degree. C. 78.6 8.80 .times. 10.sup.5 50 .+-. 5.degree. C.
77.8 7.00 .times. 10.sup.5 50 .+-. 5.degree. C. 79.3 5.84 .times.
10.sup.5 80 .+-. 5.degree. C. 76.1 5.60 .times. 10.sup.5 80 .+-.
5.degree. C. 77.0 5.36 .times. 10.sup.5 80 .+-. 5.degree. C. 81.1
6.16 .times. 10.sup.5
[0064] The above data shows that acceptable viability data were
obtained for each sample tested, and that cancellous bone prepared
in accordance with the present invention can be stored at
-80.degree. C..+-.5.degree. C. at least for seventeen days.
[0065] With respect to some applications, surgeons may need to
store the cancellous bone at temperature higher than -80.degree.
C., such as -50.degree. C. The above data shows that the cancellous
bone may be stored at -50.degree. C. and still retain acceptable
viability.
EXAMPLE 3
[0066] 23 samples of collagenase-treated bone were prepared as
described in Example 1, except that the bone samples were treated
with collagenase at concentrations from 0.167 mg/ml to 3 mg/ml as
given in Table 2 below, and for a period of time from 5 min. to 24
hrs., also given in Table 2 below, and the bone samples were not
frozen. The volume of each bone sample and the total volume of
collagenase used to treat each bone sample also are given in Table
2 below.
[0067] The fresh bone samples were tested for % viability and cell
density as described in Example 2. The results are given in Table 2
below.
TABLE-US-00002 TABLE 2 Volume of Carrier for Volume of Collagenase
Sample Donor Collagenase Conc. Duration Bone (cc) (ml) Viability
Cell Yield Cells/cc Bone 1 020505 DMEM 0.5 mg/ml 24 hrs 18 50 91.0%
6.12E+06 3.40E+05 2 020805 DMEM 0.5 mg/ml 24 hrs 12.5 30 83.5%
1.68E+05 1.34E+04 3 121404 DMEM 0.5 mg/ml 3 hrs 5 30 84.0% 4.80E+05
9.60E+04 4 121404 DMEM 1.5 mg/ml 3 hrs 10 30 81.0% 4.40E+06
4.40E+05 5 012105 DMEM 3 mg/ml 3 hrs 10 30 86.0% 6.24E+06 6.24E+05
6 020805 DMEM 3 mg/ml 3 hrs 12.5 30 91.0% 1.62E+06 1.30E+05 7
011305 DMEM 3 mg/ml 3 hrs 10 30 86.0% 1.53E+05 1.53E+04 8 020505
DMEM 3 mg/ml 3 hrs 10 50 77.6% 9.35E+06 9.35E+05 9 121804 DMEM
0.167 mg/ml 3 hrs 20 60 74.0% 4.77E+05 2.39E+04 10 121804 DMEM
0.833 mg/ml 3 hrs 20 60 78.0% 2.65E+06 1.33E+05 11 B-031505 DMEM 3
mg/ml 1 hr 20 60 n/a 1.00E+06 5.00E+04 12 022405 DMEM 3 mg/ml 3 hrs
25 50 76.0% 1.20E+05 4.80E+03 13 030905 DMEM 3 mg/ml 3 hrs 20 50
94.0% 1.40E+07 6.99E+05 14 022405 DMEM 3 mg/ml 3 hrs 10 25 94.7%
3.63E+05 3.63E+04 15 030905 DMEM 3 mg/ml 24 hrs 10 30 92.8%
3.59E+06 3.59E+05 16 040705 Saline 3 mg/ml 15 min 10 30 66.0%
1.30E+05 1.30E+04 17 040705 PBS 3 mg/ml 10 min 5 30 90.4% 2.08E+05
4.16E+04 18 040705 PBS 3 mg/ml 20 min 10 25 83.0% 1.83E+06 1.83E+05
19 040705 PBS 3 mg/ml 30 min 10 30 95.0% 3.58E+06 3.58E+05 20
041305 PBS 3 mg/ml 5 min 15 30 92.1% 2.05E+06 1.37E+05 21 041305
PBS 3 mg/ml 10 min 15 30 96.2% 1.64E+06 1.09E+05 22 041305 PBS 3
mg/ml 30 min 15 30 91.9% 3.83E+06 2.55E+05 23 041305 PBS 3 mg/ml 1
hr 15 30 90.9% 1.78E+07 1.18E+06
[0068] The above data shows that cancellous bone treated with
collagenase at various concentrations and for various periods of
time in accordance with the present invention retain acceptable
viability.
EXAMPLE 4
[0069] In this example, two samples of cells obtained from
cancellous bone samples treated with collagenase are tested for
their ability to differentiate into osteogenic cells.
[0070] A stock of a standard culture medium was prepared by
pipetting 111 ml of FBS and 11 ml of antibiotic-antimycotic
(Invitrogen Cat. No. 15240-062) into 1,000 ml of DMEM-lg to provide
a medium having a final concentration of 10% FBS and 1%
antibiotic-antimycotic.
[0071] A stock of a culture medium including osteogenic supplements
(OS medium) was prepared by mixing 246 ml of the standard medium
with 25 .mu.l of 1 mM dexamethasone solution, 2.5 ml of 1 M
.beta.-glycerophosphate (.beta.GP) solution, and 1.25 ml of 10 mM
ascorbic acid-2-phosphate (AsAP) solution in a 500 ml sterile
bottle. The materials are mixed by swirling the bottle gently for
30 seconds. The medium then is poured into the reservoir of a 500
ml 0.2.mu. filter with storage system. A vacuum line then is
attached to the 500 ml 0.2.mu. filter, and the medium is filter
sterilized. The reservoir then is removed and replaced with a cap.
The OS medium is stored at 2.degree. to 8.degree. C.
[0072] A Fast Violet B solution was prepared by placing a Sigma
(RTM) Fast Violet B Salt Capsule into 48 ml of distilled water. The
resulting solution then was aliquoted into two 50 ml conical tubes
with 12 ml of solution being added to each tube. The tubes then
were stored at 4.degree. C.
[0073] A citrate solution was prepared by diluting 2 ml of
Sigma.RTM. Citrate Concentrated Solution to 100 ml with distilled
water. The citrate solution was stored at 4.degree. C.
[0074] Two collagenase-treated cancellous bone samples were
prepared as described in Example 1, and collagenase released (CR)
cells from such samples were obtained as described in Example
2.
[0075] CR cells were plated at a density of 0.5-5.0.times.10.sup.6
cells per well in a 6-well plate using 3 ml of the standard medium
hereinabove described. The plate was labeled "PO", and covered with
a lid, and transferred to a 37.degree. C. incubator set at 5%
CO.sub.2 and 90%.+-.5% humidity. Medium changes were performed
twice weekly with a medium volume of 3 ml per well.
[0076] Starting on day 7, the plate was examined for cell growth.
Confluent cultures were trypsinized for cells between day 14 and
day 21.
[0077] An appropriate volume of trypsin was warmed to 20.degree.
C.-37.degree. C. in a water bath. The plate that is to be
trypsinized was removed from the incubator. The medium was
aspirated off, and 1.5 ml of trypsin was added to each well to be
trypsinized. The plate was placed back in the 37.degree. C.
incubator for 5 minutes. The plate then was removed from the
incubator, and the sides and bottom of the plate were tapped to
dislodge attached cells. 3 ml of standard medium then was added to
the wells, the cell suspensions then were mixed, and the entire
volumes from the wells were transferred into a 5.0 ml conical tube.
The wells then were rinsed with 3 ml of standard medium, and the
rinse medium was added to the tube. The total volume of the tube
then was brought to 45 ml.
[0078] The tube then was placed in a centrifuge, and spun at 1,960
rpm for 8 minutes (about 878 g). The supernatant then was aspirated
off, and the pellet was loosened gently by dragging the tube across
a tube rack. The pellet then was re-suspended in an appropriate
volume of standard medium (about 1 to about 3 ml) for cell
counting.
[0079] A 20 .mu.l aliquot of the cell suspension then was tested
for % viability and cell density as described in Example 2.
[0080] The cells then were plated at a density of 30.times.10.sup.3
cells (.+-.15.times.10.sup.3 cells) per well in a 6-well plate
using 3 ml standard medium. The plate then was labeled "OS",
covered with a lid, and transferred to a 37.degree. C. incubator
set at 5% CO.sub.2 and 90%.+-.5% humidity. The plate was incubated
for 24 to 48 hours.
[0081] The OS medium was warmed to 20.degree.-37.degree. C. in a
water bath. The standard medium then was aspirated off from each
well of the plate, and 3 ml of OS medium then was added to each
well. The OS medium was changed every third or fourth day, and the
cultures were assayed for alkaline phosphatase expression between
days 7 and 14.
[0082] Alkaline phosphatase staining then was conducted. Each well
was rinsed twice with 1 ml of PBS. A fixative solution then was
prepared by mixing two volumes of citrate working solution with
three volumes of acetone. Each well then was fixed for one minute
with 1 ml of the fixative solution.
[0083] 0.5 ml of Sigma (RTM) alkaline solution naphthol AS-MX
phosphate was added to 12 ml of Fast Violet B solution. The
solution then was covered with aluminum foil to protect it from
light. 1 ml of the Fast Violet B/naphthol solution then was added
to each well.
[0084] The plate then was incubated at room temperature, in the
dark for one hour. The wells then were aspirated, and rinsed twice
with 1 ml of distilled water. Cell cultures that exhibited a
distinct pink stain in some or all of the cells were positive. Cell
cultures from both samples of cells gave positive results.
EXAMPLE 5
[0085] Cancellous bone, which included cancellous bone treated with
collagenase and cancellous bone that was untreated, was prepared as
described in Example 1. The cortical bone was processed, milled,
and washed according to normal standard operating procedure for
tissue products. The cortical bone then was sieved through a
1,500.mu. sieve stacked atop a 780.mu. sieve stacked atop a 125.mu.
sieve stacked onto a receiving pan with WFI (water for injection)
quality water. Larger fragments, herein referred to as cortical
bone chips, which were above the 780.mu. sieve and below the
1,500.mu. sieve were transferred to 50 mL conical tubes. Smaller
fragments, herein referred to as cortical bone powder, which were
above the 125.mu. sieve and below the 780.mu. sieve, were
transferred to another set of 50 mL conical tubes. After sieving,
the cortical bone samples were processed separately using the same
methods described below. The samples are referred to hereinbelow as
cortical bone samples.
[0086] Cortical bone samples were treated with 0.5 M hydrochloric
acid for 70 minutes, at a ratio of 1 cc of cortical bone fragments
to 17 cc of 0.5 M hydrochloric acid (HCl) at 4.degree. C. in roller
bottles under constant rolling. The HCl was decanted off each
sample and the samples were neutralized with a 5% Sodium
Phosphate/PBS solution. The sample was shaken vigorously, and the
5% Sodium Phosphate/PBS solution was decanted off. The sample was
then rinsed with 1.times.PBS, shaken vigorously, and the pH of the
PBS was taken. If the pH of the PBS were outside a 5-7.5 pH range,
the sample was rewashed with 1.times.PBS until the pH was between 5
and 7.5 pH range, the sample was rewashed with 1.times.PBS until
the pH was between 5 and 7.5 pH. The bone sample then was rinsed
through a 125 .mu.M sieve and placed into a clean bottle. The
sample was then treated with a 1.times. antibiotic solution
consisting of 0.9 mL of DMEM mixed with 0.005 mL of 10 mg/ml
gentamicin sulfate, 0.05 ml of 50 mg/ml vancomycin HCl, and 0.1 ml
of 250 .mu.g/ml amphotericin B resulting in a final antibiotic
solution of gentamycin sulfate (50 .mu.g/ml), vancomycin HCl (50
.mu.g/ml) and amphotericin B (2.5 .mu.g/ml). The bottles then were
capped with a gas permeable lid and transferred into a 37.degree.
C. incubator for 18+ hours. The bottles then were removed from the
incubator, placed into a BSC (biological safety cabinet), and the
antibiotic solution was decanted off. The bone samples were then
washed with the same volume of PBS, shaken vigorously, and the PBS
decanted off. The bone samples then were washed three times in the
same fashion with PlasmaLyte-A. After washing, the bone samples
were transferred to 50 mL conical tubes using sampler spoons.
[0087] Using a sampler spoon and tweezers, 7 cc cancellous bone, 3
cc of cortical chips, and 2 cc of cortical powder processed as
described above were added to a product dose jar. Cryopreservation
solution, consisting of 1.times. PlasmaLyte-A, 0.2 mL of 25% human
serum albumin, and 0.1 mL of 1.times. dimethylsulfoxide (DMSO)
(CryoServ) per mL, then was added to cover the material within the
jar. The jar then was sealed tightly and shaken vigorously. After
the jar was sealed, the product dose jar was placed into a
packaging bag and the bag was then sealed. The secondary packaging
bag then was placed into a storage containing and placed into an
-80.degree. C. quarantine freezer until the product was used for
further study.
EXAMPLE 6
[0088] Five lots of "test" bone samples, prepared as described in
Example 5, and five lots of "control" bone samples, prepared as
described in Example 1, were obtained from five donors. From each
donor, a test bone sample and a control bone sample was
prepared.
[0089] Prior to implantation, the bone samples were thawed either
at room temperature, or in a water bath at a temperature no greater
than 39.degree. C. Once the bone samples were thawed completely, as
much cryoprotectant was decanted off and discarded. Sterile saline
then was added to the jars to cover the bone samples. The bone
samples were implanted within 4 hours of being thawed. The samples
then were removed from the sterile saline, weighed, and were placed
gently in sterile syringes.
[0090] 25 athymic male nude rats, each weighing at least 100 grams
at the beginning of the study, were divided into five groups of 5
rats each. Each rat of each group received a test bone sample and a
control bone sample from the same donor.
[0091] Each rat was given one pre-operative injection of
buprenorphine (0.1 mg/kg IP), and then anesthetized with inhalant
isoflurane gas. The upper portion of the back then was clipped and
scrubbed for surgery. Hemostasis was employed as necessary. Doses
of anesthesia were given as needed to maintain the proper plane of
unconsciousness.
[0092] Incisions of approximately 1 cm were made in the skin over
each scapula. Pockets were made in the subcutaneous tissue by blunt
and sharp dissection. Approximately 300.+-.25 mg of either the test
bone sample or the control bone sample were placed into each
pocket. The subcutaneous tissue and skin then were closed with
sutures. After the rats recovered from the anesthesia, they were
returned to their cages. The rats received one post-operative
injection of buprenorphine (0.1 mg/kg IP) for analgesia on the day
after surgery.
[0093] The rats were weighed and then euthanized on Day 28 with
CO.sub.2. The implant sites then were opened and observed in order
to confirm the retention of the implanted bone samples. The bone
samples with adjacent soft tissue were excised, fixed, and
processed for pathology.
[0094] Gross necropsy findings, and most critically, the
microscopic analyses were the main study endpoints determining the
presence or absence of osteoinduction at the implant sites.
[0095] Evaluation of osteoinduction was based on the successful
implantation of all bone samples and the survival of sufficient
animals until the study endpoint. Also, at recovery of the
implants, the implant sites must grossly be free from bacterial
contamination as evidenced by the absence of exudates,
inflammation, or frank tissue destruction (necrosis). Sites that
were associated with local infection that resolved during the
course of the study were considered suspect. Microscopic evidence
of the bone sample must be present at the implant site.
[0096] The bone samples and the surrounding tissue were fixed in
10% neutral buffered formalin (NBF) prior to routine histology,
decalcification, and processing. The samples then were processed
into paraffin, and sections from 3 to 6 microns thick were cut and
mounted onto glass slides and stained with hematoxylin and eosin.
Blocks were faced as necessary. Sections were taken from at least
three levels within the block. The slides were evaluated for
evidence of osteoinduction. More particularly, the slides were
evaluated for the presence of the following: [0097] 1.
chondroblasts; [0098] 2. chondroytes; [0099] 3. osteoblasts; [0100]
4. osteocytes; [0101] 5. cartilage; [0102] 6. osteoid tissue;
[0103] 7. new bone; and, [0104] 8. new bone marrow.
[0105] Evidence of osteoinduction was graded as follows: [0106]
0--no evidence of new bone formation [0107] 1--1-25% of field shows
evidence of new bone formation [0108] 2--26-50% of field shows
evidence of new bone formation [0109] 3--51-75% of field shows
evidence of new bone formation [0110] 4--76-100% of field shows
evidence of new bone formation
[0111] Samples having a score of 0 were considered
non-osteoinductive while samples having a score of from 1 to 4 were
considered osteoinductive.
[0112] The results for the test and control samples from each donor
for each rat are given in Table 3 below. As shown in Table 3, an
"X" indicates the presence of the element, while a minus sign "-",
indicates that the element was not present.
TABLE-US-00003 TABLE 3 Site Rat L OR CHONDROBLASTS/ OSTEOBLASTS/
CARTILAGE/ NEW BONE ORIGINAL GRADE Number R CYTES CYTES OSTEOID
BONE MARROW DBM (0-4) TEST ARTICLE- DONOR 1 OBSERVED ELEMENTS OF
NEW BONE FORMATION 1 R -- X -- X X X 1 2 -- -- -- -- -- X 0 3 -- --
-- -- -- X 0 4 -- -- -- -- -- X 0 5 -- X -- X -- X 1 TEST ARTICLE-
DONOR 2 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 R -- X -- X -- X
1 2 X X X X X X 1 3 X X X X X X 1 4 X X X X -- X 1 5 -- X -- X X X
1 TEST ARTICLE- DONOR 3 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 R
-- -- -- -- -- X 0 2 -- -- -- -- -- X* 0 3 X X X X X X 4 4 X X X X
X X 1 5 -- -- -- -- -- X 0 TEST ARTICLE- DONOR 4 OBSERVED ELEMENTS
OF NEW BONE FORMATION 1 R X X -- X -- X 1 2 -- -- -- -- -- X 0 3 X
-- X -- -- X 1 4 -- -- -- -- -- X 0 5 X X X X X X 1 TEST ARTICLE-
DONOR 5 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 R -- -- -- -- --
X 0 2 X X X X X X 1 3 -- X -- X -- X 1 4 X X X X X X 1 5 -- -- --
-- -- X 0 CONTROL ARTICLE- DONOR 1 OBSERVED ELEMENTS OF NEW BONE
FORMATION 1 L -- -- -- -- -- X 0 2 -- -- -- -- -- X 0 3 -- -- -- --
-- X 0 4 -- -- -- -- -- X 0 5 -- -- -- -- -- X 0 CONTROL ARTICLE-
DONOR 2 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 L -- X -- X -- X
1 2 -- X -- X X X 1 3 X X -- X X X 1 4 -- X X X X X 1 5 -- -- -- --
-- X 0 CONTROL ARTICLE- DONOR 3 OBSERVED ELEMENTS OF NEW BONE
FORMATION 1 L -- -- -- -- -- X 0 2 -- -- -- -- -- X 0 3 -- -- -- --
-- X 0 4 -- -- -- -- -- X 0 5 -- -- -- -- X 0 CONTROL ARTICLE-
DONOR 4 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 L -- X -- X X X 1
2 -- -- -- -- -- X 0 3 -- X -- X X X 1 4 -- -- -- -- -- X 0 5 -- --
-- -- -- X 0 CONTROL ARTICLE- DONOR 5 OBSERVED ELEMENTS OF NEW BONE
FORMATION 1 L -- X -- X -- X 1 2 -- -- -- -- -- X 0 3 -- -- -- --
-- X 0 4 -- -- -- -- -- X 0 5 -- -- -- -- -- X 0
[0113] The results for each of the test and control articles are
summarized as follows:
[0114] Test Article from Donor 1--Evidence of osteoinduction,
including osteoblasts/osteocytes, and new bone, was found in two of
the implants. Bone marrow also was found in one of the implants.
Thus, the test article from Donor 1 was considered to be
osteoinductive.
[0115] Test Article from Donor 2--Evidence of osteoinduction,
including osteoblasts/osteocytes and new bone, was found in all
five of the implants. Chondroblasts/chondrocytes, cartilage/osteoid
tissue, and bone marrow were found in three of the implants. Thus,
the test article from Donor 2 was considered to be
osteoinductive.
[0116] Test Article from Donor 3--Evidence of osteoinduction,
including chondroblasts/chondrocytes, osteoblasts/osteocytes,
cartilage/osteoid tissue, new bone, and bone marrow, was found in
two of the implants. Thus, the test article from Donor 3 was
considered to be osteoinductive.
[0117] Test Article from Donor 4--Evidence of osteoinduction,
including chondroblasts/ chondrocytes, was found in three of the
implants. Osteoblasts/osteocytes, cartilage/osteoid tissue, and new
bone were found in two of the implants. Bone marrow was found in
one of the implants. Thus, the test article from Donor 4 was
considered to be osteoinductive.
[0118] Test Article from Donor 5--Evidence of osteoinduction,
including osteoblasts/ osteocytes, was found in three of the
implants. Chondroblasts/chondrocytes, cartilage/ osteoid tissue,
and bone marrow were found in two of the implants. Thus, the test
article from Donor 5 was considered to be osteoinductive.
[0119] With respect to the control articles, no evidence of
osteoinduction was found in any of the implant sites from the
control articles from Donor 1 and Donor 3. Thus, the control
articles from Donor 1 and Donor 3 were considered to be
non-osteoinductive.
[0120] The results with respect to the remaining control articles
were as follows:
[0121] Control Article from Donor 2--Evidence of osteoinduction,
including osteoblasts/osteocytes and new bone, was found in four of
the implants. Bone marrow was found in three of the implants, and
chondroblasts/chondrocytes and cartilage/osteoid tissue were found
in one of the implant sites. Thus, the control article from Donor 2
was considered to be osteoinductive.
[0122] Control Article from Donor 4--Evidence of osteoinduction,
including osteoblasts/osteocytes, new bone, and bone marrow, was
found in two of the implant sites. Thus, the control article from
Donor 4 was considered to be osteoinductive.
[0123] Control Article from Donor 5--Evidence of osteoinduction,
including osteoblasts/osteocytes and new bone, was found in one of
the implant sites. Thus, the control article from Donor 5 was
considered to be osteoinductive.
EXAMPLE 7
[0124] The test and control samples from Donors 1 through 5 as
described in Example 6 hereinabove were tested for cell density and
cell viability as described in Example 2 hereinabove. The cell
density results are given in Table 4 below, and the cell viability
results are given in Table 5 below.
TABLE-US-00004 TABLE 4 Cell Density (cells/cc) Donor Control Sample
Test Sample Difference 1 7.07 .times. 10.sup.6 4.38 .times.
10.sup.6 -2.69 .times. 10.sup.6 2 1.43 .times. 10.sup.6 2.48
.times. 10.sup.6 1.05 .times. 10.sup.6 3 4.07 .times. 10.sup.6 3.16
.times. 10.sup.6 -0.93 .times. 10.sup.6 4 2.47 .times. 10.sup.6
2.24 .times. 10.sup.6 -0.23 .times. 10.sup.6 5 1.23 .times.
10.sup.6 4.05 .times. 10.sup.6 2.82 .times. 10.sup.6
TABLE-US-00005 TABLE 5 Cell Viability Donor Control Sample Test
Sample Difference 1 90.2% 82.0% -8.2% 2 80.6% 68.6% -12.0% 3 86.1%
80.5% -5.6% 4 88.3% 79.6% -8.7% 5 82.4% 79.9% -2.5%
EXAMPLE 8
[0125] The control and test samples from each of Donors 1 through 5
were tested for osteodifferentiation in a revision of the method
decribed in Example 4 hereinabove. In this example, two bone
samples from each of the control and test samples from Donors 1
through 5 of cells were obtained from cancellous bone samples
treated with collagenase are tested for their ability to
differentiate into osteogenic cells.
[0126] A stock of a standard culture medium was prepared by
pipetting 111 ml of FBS and 11 ml of antibiotic-antimycotic
(Invitrogen Cat. No. 15240-062) into 1,000 ml of DMEM-low glucose
(DMEM-lg) to provide a medium having a final concentration of 10%
FBS and 1% antibiotic-antimycotic.
[0127] A stock of a culture medium including osteogenic supplements
(OS medium) was prepared by mixing 246 ml of the standard medium
with 25 .mu.l of 1 mM dexamethasone solution, 2.5 ml of 1M
.beta.-glycerophosphate (PGP) solution, and 1.25 ml of 10 mM
ascorbic acid-2-phosphate (AsAP) solution in a 500 ml sterile
bottle. The materials are mixed by swirling the bottle gently for
30 seconds. The medium then is poured into the reservoir of a 500
ml 0.2.mu. filter with storage system. A vacuum line then is
attached to the 500 ml 0.2.mu. filter, and the medium is filter
sterilized. The reservoir then is removed and replaced with a cap.
The OS medium is stored at 2.degree.-8.degree. C.
[0128] A BCIP/NBT working solution was prepared by placing 1 drop
each of Reagent 1, 2, and 3 from the BCIP/NBT Alkaline Phosphatase
Substrate Kit IV into 5 mL of Tris-HCL Buffer 100 mM. The resulting
solution then was aliquoted into a 50 ml conical tube and stored at
4.degree. C.
[0129] One of the two collagenase-treated cancellous bone samples
(Bone Sample 1) was prepared as described in Example 1, and
collagenase released (CR) cells from such samples were obtained as
described in Example 2. The cryopreservation solution from Bone
Sample 1 was placed into a conical tube and set aside. The other
bone sample (Bone Sample 2) was thawed in a water bath at
37.degree. C. and then the cryopreservation solution was aspirated
off and added to the conical tube containing the cryopreservation
solution from Bone Sample 1. 10 mL of standard medium was then
added to the Bone Sample 2 container, the sample was shaken, and
the medium was transferred to the conical tube containing the
cryopreservation solution from Bone Sample 1 and 2. Then the tube
was placed in a centrifuge, and spun at 1,960 rpm for 8 minutes
(about 878 g). The media was then aspirated from the tube and the
cells were resuspended in 4 mL of standard medium and placed into a
well in a 6 well plate labaled Cells from Cryopreservation Solution
(Samples 1 & 2). The remaining bone chips of Bone Sample 2 were
then transferred to another well in the 6 well plate labeled
Osteogenic Sample 2 Bone Chips.
[0130] CR cells were plated into one well of the six well plate in
4 mL of standard medium hereinabove described. The well was labeled
as CR Cells Sample 1. Bone samples were placed into 3 wells of the
same 6 well plate at 1.5-2 cc per well with 4 mL of standard media
per well and all three wells were labeled as Osteogenic Sample 1.
The plate was covered with a lid, and transferred to a 37.degree.
C. incubator set at 5% CO.sub.2 and 90%.+-.5% humidity. Medium
changes were performed every 2-4 days with a medium volume of 4 ml
per well.
[0131] Plates were examined daily until confluence was greater than
60%. Once confluence was greater than 60%, plates were trypsinized
for cells.
[0132] An appropriate volume of trypsin was warmed to 37.degree. C.
in a water bath. The plate that is to be trypsinized was removed
from the incubator. The medium was aspirated off, and each well
containing bone chips was washed with 3.5 mL of PBS and the well
containing cells with 3 mL. The PBS was then aspirated and 2 mL of
trypsin was added to each well containing bone chips and 1.5 mL to
each well containing cells. The plate was placed back in the
37.degree. C. incubator for 5 minutes. The plate then was removed
from the incubator, and the sides and bottom of the plate were
tapped to dislodge attached cells. 3 ml of standard medium then
were added to the wells, the cell suspensions then were mixed, and
the entire volumes from the wells were transferred into a 50 ml
conical tube. The wells then were rinsed with 3 ml of standard
medium, and the rinse medium was added to the tube. The total
volume of the tube then was brought to 45 ml. The tube then was
placed in a centrifuge, and spun at 1,960 rpm for 8 minutes (about
878 g). The supernatant then was aspirated off, and the pellet was
loosened gently by dragging the tube across a tube rack. The pellet
then was re-suspended in 9 mL of standard medium. 3 mL of the cell
suspension was plated in each of 3 wells of a 6 well plate and the
plate was covered and transferred to a 37 C incubator. The plate
then was labeled "OS", covered with a lid, and transferred to a
37.degree. C. incubator for 24 to 48 hours.
[0133] The OS medium was warmed to 37.degree. C. in a water bath.
The standard medium then was aspirated off from each well of the
plate, and 3 ml of OS medium then was added to each well. The OS
medium was changed every third or fourth day, and the cultures were
assayed for alkaline phosphatase expression between days 7 and
14.
[0134] Alkaline phosphatase staining then was conducted. Each well
was rinsed twice with 1 ml of PBS. 1 ml of BCIP/NBT Working
solution was added to each well and plates were incubated at room
temperature in the dark for 6-30 hours. All wells were then
evaluated for the presence of the blue/purple color of the positive
Alkaine Phosphatase reaction. Each of the test and control samples
from each of Donors 1 through 5 was positive for
osteodifferentiation.
EXAMPLE 9
[0135] Evaluation of cortical chip size was performed through a
series grinding and sifting procedures in which cortical bone was
sorted into multiple size ranges. The various sized cortical chip
fragments were then compared. Blinded evaluation of mixtures of
cancellous chips and cortical chips of various chip sizes with
varied cortical to cancellous ratios was performed by seven people
with previous experience in osteoimplantation techniques. Six of
the seven persons evaluating the trial mixtures selected the same
chip size and formulation when asked to select the sample having
optimal physical characteristics, such optimal mixture was found to
comprise 70% cancellous bone chips, and 30% cortical bone chips of
780 microns to 1500 microns in size, compared to a mixture of 100%
cancellous bone chips. Blinded evaluation of this optimal mixture
was then performed by orthopedic surgeons, operating under test
conditions similar to typical surgical conditions, also found
products comprising this optimal mixture to exhibit superior
handling and physical characteristics.
[0136] The disclosures of all patents, publications (including
published patent applications), depository accession numbers, and
database accession numbers were incorporated herein by reference to
the same extent that each patent, publication, depository accession
number and database accession number were specifically and
individually incorporated by reference.
[0137] It is to be understood, however, that the scope of the
present invention is not to be limited to the specific embodiments
described above. The invention may be practiced other than as
particularly described and still be within the scope of the
accompanying claims.
* * * * *