U.S. patent application number 11/825894 was filed with the patent office on 2007-11-15 for compositions comprising bone marrow cells together with demineralized and/or mineralized bone matrix and uses thereof in the induction of bone and cartilage formation.
This patent application is currently assigned to Hadasit Medical Research Services & Development, Ltd.. Invention is credited to Olga Gurevitch, Basan Gowda S. Kurkalli, Tatyana Prigozhina, Shimon Slavin.
Application Number | 20070264240 11/825894 |
Document ID | / |
Family ID | 11075198 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264240 |
Kind Code |
A1 |
Slavin; Shimon ; et
al. |
November 15, 2007 |
Compositions comprising bone marrow cells together with
demineralized and/or mineralized bone matrix and uses thereof in
the induction of bone and cartilage formation
Abstract
A composition comprising bone marrow cells (BMC) and
demineralized bone matrix (DBM) and/or mineralized bone matrix
(MBM) and optionally comprising bone morphogenetic protein/s (BMP)
and/or other active agents, particularly for use in the
transplantation of mesenchymal progenitor cells into a joint and/or
a cranio-facial maxillary bone, for restoring and/or enhancing the
formation of a new hyaline cartilage and subchondral bone
structure. The composition of the invention and method of treatment
employing the same may be used for the treatment of hereditary or
acquired bone disorders, hereditary or acquired cartilage
disorders, malignant bone or cartilage disorders, metabolic bone
diseases, bone infections, conditions involving bone or cartilage
deformities and Paget's disease. The composition and method may
further be used for the correction of complex fractures, bone
replacement and formation of new bone in plastic or sexual surgery,
for support of implants of joints, cranio-facial-maxillary bones,
or other musculoskeletal implants, including artificial implants.
The method of the invention may further be used for treating
damaged joints or degenerative arthropathy associated with
malformation and/or dysfunction of cartilage and/or subchondral
bone. A kit is provided for performing transplantation into a joint
or a cranio-facial-maxillary bone of a mammal of the composition of
the invention.
Inventors: |
Slavin; Shimon; (Jerusalem,
IL) ; Gurevitch; Olga; (Jerusalem, IL) ;
Kurkalli; Basan Gowda S.; (Jerusalem, IL) ;
Prigozhina; Tatyana; (Rehovot, IL) |
Correspondence
Address: |
John P. White;Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
Hadasit Medical Research Services
& Development, Ltd.
|
Family ID: |
11075198 |
Appl. No.: |
11/825894 |
Filed: |
July 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10471031 |
Apr 19, 2004 |
|
|
|
PCT/IL02/00172 |
Mar 5, 2002 |
|
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11825894 |
Jul 9, 2007 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61K 38/1875 20130101;
A61K 2300/00 20130101; A61K 38/1875 20130101; A61P 35/00 20180101;
A61P 19/02 20180101; A61K 35/28 20130101; A61P 19/08 20180101; A61K
35/32 20130101; A61K 2300/00 20130101; A61P 7/06 20180101; A61K
35/32 20130101 |
Class at
Publication: |
424/093.7 |
International
Class: |
C12N 5/06 20060101
C12N005/06; A61K 39/00 20060101 A61K039/00; A61P 19/02 20060101
A61P019/02 |
Foreign Application Data
Date |
Code |
Application Number |
May 5, 2001 |
IL |
141813 |
Claims
1-45. (canceled)
46. A mixture comprising bone marrow cells (BMC) and demineralized
bone matrix (DBM) and/or mineralized bone matrix (MBM) for use as a
graft of mesenchymal progenitor cells for transplantation into a
joint and/or a cranio-facial-maxillary bone of a subject in
need.
47. The mixture according to claim 46, wherein said BMC are
allogeneic or said subject's own.
48. The mixture according to claim 46, further comprising active
agents, preferably selected from bone morphogenetic proteins
(BMPs), immunosuppressants, immunomodulators, antibiotics and
anti-inflammatory agents.
49. A mixture according to claim 46, wherein the DBM and/or MBM are
of vertebrate origin.
50. The mixture according to claim 46, wherein the DBM and/or MBM
are of human origin.
51. The mixture according to claim 46, wherein the DBM or MBM are
in powder or slice form.
52. The mixture according to claim 46, wherein the particle size of
the DBM is about 50 to 2500u.
53. The mixture according to claim 52, wherein the particle size of
the DBM is about 250 to 500u.
54. The mixture according to claim 46, wherein the ratio between
BMC and DBM is between 1:1 and 20:1 (volume:volume).
55. The mixture according to claim 54, wherein the ratio between
BMC and DBM is between 2:1 and 9:1 (volume:volume).
56. The mixture according to claim 55, wherein the ratio between
BMC and DBM is 4:1 (volume:volume).
57. The mixture according to claim 46, wherein the subject is a
human.
58. The mixture according to claim 55, wherein the number of bone
marrow cells in the mixture is from about 10.sup.6 to 10.sup.8
cells/ml.
59. The mixture according to claim 46, further comprising a
pharmaceutically acceptable carrier or diluent.
60. A method for treating a patient suffering from a damaged joint,
degenerative arthropathy, a hereditary or acquired bone disorder,
hereditary or acquired cartilage disorder, a malignant bone or
cartilage disorder, metabolic bone diseases, bone infections,
conditions involving bone or cartilage deformities, or Paget's
disease, comprising administering to the patient the mixture of
claim 46.
61. A method for transplantation of a mixture comprising BMC with
DBM and/or MBM and optionally further comprising pharmaceutically
acceptable carrier or diluent, into a joint or a
cranio-facial-maxillary bone of a subject in need, wherein said
method comprises introducing into said joint or bone a mixture as
defined in claim 46.
62. The method according to claim 61, wherein said mixture is
administered non-invasively by a syringe, an arthroscopic procedure
or by open surgery into the site of implantation.
63. The method according to claim 62, for support of implants of
joints, cranio-facial-maxillary bones, or other musculoskeletal
implants.
64. A method of treating a damaged joint or degenerative
arthropathy associated with malformation and/or dysfunction of
cartilage and/or subchondral bone in a mammal in need of such
treatment, comprising administering into an affected joint or bone
of said mammal a mixture comprising BMC with DBM and/or MBM, said
mixture optionally further comprising a pharmaceutically acceptable
carrier or diluent and/or additional active agents.
65. The method according to claim 64, wherein the BMC are either
allogeneic or said mammal's own.
66. The method according to claim 64, wherein said DBM or MBM are
in a slice, powder, gel, semi-solid or solid form embedded in or
encapsulated in polymeric or biodegradable materials.
67. The method according to claim 66, wherein the particle size of
said DBM is about 50 to 2500u.
68. The method according to claim 67, wherein the particle size of
said DBM is about 250 to 500u.
69. The method according to claim 64, wherein the ratio between the
transplanted BMC and DBM is between 1:1 and 20:1
(volume:volume).
70. The method according to claim 69, wherein the ratio between the
transplanted BMC and DBM is between 2:1 and 9:1
(volume:volume).
71. The method according to claim 70, wherein the ratio between the
transplanted BMC and DBM is 4:1 (volume:volume).
72. A non-invasive implantation method for support of implants of
joints or other musculoskeletal implants, comprising introducing a
graft into a joint or a cranio-facial-maxillary bone of a subject
in need, wherein said graft comprises the mixture of claim 46.
73. A kit for performing transplantation into a joint or a
cranio-facial-maxillary bone of a mammal of a mixture as defined in
claim 46, wherein said kit comprises: (a) the mixture-as defined in
claim 46; (b) a BM aspiration needle; (c) an intra-osseous bone
drilling burr; (d) a needle with a thick lumen for infusion of
viscous bone marrow-DBM mixture; (e) a 2-way lumen connector for
simultaneous mixing of BMC-DBM and diluent; (f) a medium for
maintaining BMC; and optionally (g) cryogenic means for handling
and maintaining BMC or BMC together with DBM.
74. The kit according to claim 73, optionally further comprising a
carrier and/or diluent for the mixture.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions comprising
bone marrow cells (BMC) and demineralized and/or mineralized bone
matrix (DBM and MBM, respectively) and to their novel uses in
induction of new bone and cartilage formation in mammals.
BACKGROUND OF THE INVENTION
[0002] New bone formation, such as in the case of damage repair or
substitution of a removed part of the bone in postnatal mammals,
can only occur in the presence of the following three essential
components, (i) mesenchymal progenitor cells; (ii) a conductive
scaffold for these cells to infiltrate and populate; and (iii) Bone
Morphogenetic Proteins. Unfortunately, local conditions usually do
not satisfy the requirements of osteogenesis, and thus substitution
of removed, damaged or destroyed bones does not occur
spontaneously.
[0003] Previous research has already uncovered somewhat about these
three components.
[0004] It was shown that multipotent mesenchymal stem cells, which
are capable of extensive proliferation and differentiation into
cartilage, bone, tendon, muscle, fat and etc. are present in the
bone marrow [Caplan, A. I. (1991) J Orthop Res 9:641-650; Prockop
J. D. (1997) Science 276:71-74; Pittehger, M. F. et al. (1999)
Science 284:143-147; Wakitani, S. W. et al. (1995) Muscle &
Nerve 18:1417-1426].
[0005] DBM (and/or MBM) has been shown to play the role of
supportive material or structure that is essential for promoting
engraftment of mesenchymal progenitor cells and their proliferation
and differentiation in the course of bone and cartilage
development, whenever mesenchymal cells are introduced as a cell
suspension (Inventor's unpublished results). It serves as a
conductive scaffold for cartilage and bone regeneration, while
providing a natural source for inducing both chondro- and
osteogenesis, thus combining all the essential inductive and
conductive features. DBM also has additional advantageous, that can
be summarized as follows: (i) it is mechanically flexible and
slowly biodegradable, with the degradation time compatible with the
period of de novo chondro- and osteogenesis; (ii) it is strong
enough to provide at least partially biomechanical properties of
the flat bone and joint surface during the period of new bone and
cartilage formation; (iii) it can be provided as an amorphous
powder that can be inserted locally, without major surgical
intervention, while avoiding iatrogenic damage; (iv) it is a low
immunogenic material even when used as a xenograft, and when used
in an allogeneic combination, it is practically non-immunogenic
[Block, J. E. and Poser, J. (1995) Med Hypotheses 45(1):27-32;
Torricelli, P. et al. (1999) Int Orthop 23(3):178-81; Hallfeldt, K.
K. et al. (1995) J Surg Res 59(5):614-20].
[0006] BMPs are growth factors that play an important role in the
formation of bone and cartilage [Ducy, P. and Karsenty, G. (2000)
Kidney Int 57(6):2207-14; Schmitt, J. M. et al. (1999) J Orthop Res
17(2):269-78]. Most importantly, DBM is a natural source of BMPs.
Moreover, induction of cartilage and bone may be enhanced by
additional exogenous supply of BMPs that are not even
species-specific [Sampath, T. K. and Reddi, A. H. (1983) Proc Natl
Acad Sci USA 80(21): 6591-5; Bessho, K. et al. (1992) J Oral
Maxillofac Surg 50(5):496-501], together with DBM [Niedewanger, M.
and Urist, M. R. (1996) J Oral Implantol 22(3-4):210-5].
[0007] Arthropathies are a group of chronic progressive joint
diseases that can result from degenerative changes in the cartilage
and hypertrophy of bone at the articular margins. Arthropathies can
be secondary to trauma, inflammatory (autoimmune or infectious),
metabolic or neurogenic diseases. Hereditary and mechanical factors
may be an additional factor involved in the pathogenesis of
arthropathies.
[0008] Restoration of a healthy joint surface in a damaged or
degenerative arthropathy requires addressing the treatment both
towards the cartilage and the subchondral bone.
[0009] Various attempts have been made to replace damaged
cartilage, including: [0010] 1. Stimulation of bone marrow from
subchondral bone to form a fibrotic repair tissue; [0011] 2.
Osteochondral transplantation (allogeneic and autologous); [0012]
3. Transplantation of autologous cultured chondrocyte or
mesenchymal cells; [0013] 4. Combined transplantation of
chondrocytes with different kinds of matrices; and [0014] 5.
Artificial implantation of mechanical joints.
[0015] Each of these methods has limitations and disadvantages and
most of them are expensive, cumbersome, innefective and rather
impractical. Autologous osteochondral graft is restricted to a
small area of damaged cartilage, up to 2 cm.sup.2, and could cause
discomfort, infection and morbidity in the donor site. Allogeneic
osteochondral graft is immunogenic, hence requires life-long use of
undesired, hazardous immunosuppressive agents, which would be an
impractical approach for routine orthopedic practice.
Transplantation of cultured chondrocytes is cumbersome and very
expensive, involving a two-stage procedure. The hyaline-like tissue
which is produced after transplantation has sub-optimal
biomechanical properties [Gilbert, J. E. (1998) Am J Knee Surg
11(1):42-6; Temeno, J. S. and Mikos, A. G. (2000) Biomaterials:
issue Engineering for Regeneration of Articular Cartilage,
21:431-440; Buckwalter, J. A. and Mankin, H. J. (1998) Instr Course
Lect 47:487-504; Stocum, D. L. (1998) Wound Repair Regen.
6(4):276-90]. Hence, adequate restoration of cartilage remains an
unsolved problem.
[0016] Currently, autologous grafts are the most commonly used bone
and cartilage graft material. However, the use of autografts has
limitations, such as donor site discomfort, infection and morbidity
and limited sizes and shapes of available grafts. Even if enough
tissue is transplanted there is an acute limitation in the number
of mesenchymal stem cells with high proliferative potential present
in the differentiated bone tissue implanted.
[0017] In theory, the most promising approach should involve the
combined transplantation of cells capable of hyaline cartilage
formation and a matrix, providing means for induction/conduction
and support of cartilage development and maintenance.
[0018] It is widely accepted that, for successful application of
combined cell-matrix graft, the basic requirements are the
following: [0019] 1. Rich source of progenitor cells capable of
differentiation into chondrocytes, for continuous repair of "wear
and tear" of weight bearing joints. [0020] 2. Conductive scaffold
for cell attachment should be maintained, leading to development of
hyaline cartilage. [0021] 3. Conductive scaffold should be
non-immunogenic, non-toxic and susceptible to biodegradation
simultaneously with the development of new cartilage. [0022] 4.
Conditions for stimulating development of chondrocytes from
mesenchymal precursor cells.
[0023] So far, most of the matrices that were tried in combined
cell-matrix grafts were either immunogenic or non-biodegradable,
and the remaining others did not possess conductive or inductive
properties needed to support formation of biomechanical strong
cartilage. Cells used in combined cell-matrix grafts were in most
of the cases chondrocytes, which were already fully differentiated
cells, with relatively low metabolic activity and limited
self-renewal capacity. Whereas the proliferative capacity of such
cells may be sufficient to maintain healthy cartilage, it is
certainly insufficient for the development de novo of large areas
of hyaline cartilage. In addition to being immunogenic, mesenchymal
progenitor cell allografts were not combined with optimal
supportive matrix. Thus, unfortunately, none of the available
options fulfill all basic requirements, and all options are far
from being satisfactory for reliable routine clinical
application.
[0024] The composition of the invention comprising BMC and DBM
and/or MBM overcomes the above shortcomings and provides, upon
administration into a damaged joint, replacement and/or restoration
of hyaline cartilage together with subchondral bone, in a one-step
transplantation procedure, without any preliminary cultivation of
mesenchymal progenitor cells.
[0025] Thus, it is the major object of the present invention to
provide a mixture of bone marrow cells and demineralized or
mineralized bone matrix, for use as a graft in patients in need of
restoration of damaged joints and cranio-facial-maxillary bones.
This and other objects of the invention will be elaborated on as
the description proceeds.
SUMMARY OF THE INVENTION
[0026] The present invention relates to compositions comprising a
mixture of bone marrow cells (BMC) and demineralized and/or
mineralized bone matrix DBM and MBM, respectively) and to their
novel uses in the transplantation of mesenchymal progenitor cells
into joints and cranio-facial-maxillary bones.
[0027] Thus, in a first aspect, the present invention relates to a
composition comprising bone marrow cells (BMC) and demineralized
bone matrix (DBM) and/or mineralized bone matrix (MBM).
[0028] In a second aspect, said composition comprising BMC and DBM
and/or MBM is for use in transplantation of mesenchymal progenitor
cells present in the bone marrow into a joint and/or a
cranio-facial-maxillary bone of a subject in need, wherein said
subject is a mammal, preferably a human.
[0029] In a first embodiment, the DBM and MBM comprised within the
composition of the invention are of vertebrate origin, and they may
be of human origin.
[0030] In a second embodiment, the DBM or MBM comprised within the
composition of the invention are in powder or slice form. The
particle size of the DBM may be about 50 to 2500.mu.. Preferably,
said particle size is about 250 to 500.mu.. The most preferable
particle size will depend on the specific needs of each case.
[0031] In another embodiment, the composition of the invention is
for restoring and/or enhancing the formation of a new hyaline
cartilage and subchondral bone structure.
[0032] In a further embodiment, the composition of the invention is
intended for the treatment of a patient suffering from any one of a
hereditary or acquired bone disorder, a hereditary or acquired
cartilage disorder, a malignant bone or cartilage disorder,
conditions involving bone or cartilage deformities and Paget's
disease. Additionally, the invention is also intended for the
treatment of a patient in need of any one of correction of complex
fractures, bone replacement and formation of new bone in plastic or
sexual surgery.
[0033] In a yet further embodiment, the composition of the
invention may further optionally comprise a pharmaceutically
acceptable carrier or diluent, as well as additional active
agents.
[0034] In another aspect, the present invention relates to a method
for transplantation of a mixture comprising BMC with DBM and/or MBM
and optionally further comprising pharmaceutically acceptable
carrier or diluent, into a joint and/or a cranio-facial-maxillary
bone of a subject in need, wherein said method comprises
introducing into said joint or bone the composition of the
invention.
[0035] In a first embodiment of the method of the invention, the
mixture is administered by any one of the following procedures
injection, minimally invasive arthroscopic procedure, or by
surgical arthroplasty into the site of implantation, wherein said
method is for support or correction of congenital or acquired
abnormalities of the joints, cranio-facial-maxillary bones,
orthodontic procedures, bone or articular bone replacement
following surgery, trauma or other congenital or acquired
abnormalities, and for supporting other musculoskeletal implants,
particularly artificial and synthetic implants.
[0036] Thus, in a further aspect, the invention relates to a method
of treating a damaged or degenerative arthropathy associated with
malformation and/or dysfunction of cartilage and/or subchondral
bone in a mammal in need of such treatment, comprising
administering into an affected joint or bone of said mammal a
mixture comprising BMC with DBM and/or MBM, said mixture optionally
further comprising a pharmaceutically acceptable carrier or diluent
and/or additional active agents.
[0037] In one embodiment, the BMC which are present in the
administered mixture are either allogeneic or said mammal's
own.
[0038] In another embodiment, the DBM or MBM which is present in
the administered mixture is in a slice, powder, gel, semi-solid or
solid form embedded in or encapsulated in polymeric or
biodegradable materials.
[0039] In a yet further aspect, the present invention relates to a
non-invasive (through injection), minimally invasive (through
arthroscopy) or surgical transplantation method for support of
implants of joints or other musculoskeletal implants, comprising
introducing a graft into a joint or a cranio-facial-maxillary bone
of a subject in need, wherein said graft comprises a mixture of BMC
and DBM or MBM.
[0040] In an even further aspect, the present invention relates to
the use of a composition comprising BMC and DBM and/or MBM as a
graft of mesenchymal and/or mesenchymal progenitor cells for
transplantation/implantation into a mammal, wherein said mammal is
preferably a human. The transplantation is to be performed into a
joint or into a cranio-facial-maxillary bone, for the development
of new bone and/or cartilage.
[0041] Furthermore, the composition used in said transplantation is
intended for the treatment of a patient suffering from any one of a
hereditary or acquired bone disorder, a hereditary or acquired
cartilage disorder, a malignant bone or cartilage disorder,
conditions involving bone or cartilage deformities and Paget's
disease. In addition, said composition is intended for the
treatment of a patient in need of any one of correction of complex
fractures, bone replacement and formation of new bone in plastic or
sexual surgery.
[0042] In one embodiment, the composition used in the invention
further comprises an active agent.
[0043] In another embodiment, the DBM and MBM comprised within the
composition used in the invention are of vertebrate origin, and
they may be of human origin. Moreover, said DBM and MBM may be in
powder, strips, thin layers, or slice form.
[0044] In an additional aspect, the present invention concerns the
use of a mixture of BMC with DBM and/or MBM in the preparation of a
graft for the treatment of a bone or cartilage disorder.
[0045] Lastly, the present invention provides a kit for performing
transplantation into a joint or a cranio-facial-maxillary bone of a
mammal of BMC in admixture with DBM and/or MBM, wherein said kit
comprises: [0046] (a) DBM and/or MBM in a compacted form; [0047]
(b) a BM aspiration needle; [0048] (c) an intra-osseous bone
drilling burr; [0049] (d) a needle with a thick lumen for infusion
of viscous bone marrow-DBM mixture; [0050] (e) a 2-way lumen
connector for simultaneous mixing of BMC with DBM and diluent;
[0051] (f) a medium for maintaining BMC; and optionally [0052] (g)
cryogenic means for handling and maintaining BMC or BMC together
with DBM.
[0053] The kit of the invention may optionally further comprise a
carrier and/or a diluent for the BMC and DBM and/or MDM
mixture.
BRIEF DESCRIPTION OF THE FIGURES
[0054] FIGS. 1A-L: Photomacrographs and micrographs of sagital knee
joint sections 2 to 24 weeks after the experimentally created
microfracture drilling defect (Picroindigocarmin, PIC,
staining).
[0055] FIG. 1A: Photomacrograph of a normal rat knee joint
section.
[0056] FIG. 1B: Photomicrograph of the entire normal osteo-chondral
complex in the interchondylar region of the femur.
[0057] FIG. 1C: Photomicrograph of the articular cartilage in the
normal osteo-chondral complex shown in FIG. 1B.
[0058] FIG. 1D: Microfracture drilling (full thickness defect),
immediately after damage.
[0059] FIG. 1E: Micro-fracture left without the implant, two weeks
after damage. The drilled hole, filled with connective tissue, can
be seen.
[0060] FIG. 1F: Micro-fracture left without the implant, 24 weeks
after damage. Regenerated subchondral bone and damaged joint
surface constituted of fibro-cartilaginous tissue can be seen.
[0061] FIG. 1G: DBM particles alone were transplanted into defect
area, two weeks after transplantation. DBM particles are clearly
seen in the site of transplantation surrounded mostly with
connective tissue.
[0062] FIG. 1H: DBM particles alone were transplanted into defect
area, 24 weeks after transplantation. Regenerated sub-chondral bone
and damaged joint surface covered with connective tissue together
with fibro-cartilage could be observed.
[0063] FIG. 1I: DBM particles together with BMC were transplanted
into defect area, 2 weeks after transplantation. Extensively
developing hyaline cartilage surrounding the implanted DBM
particles could be seen.
[0064] FIG. 1J: DBM particles together with BMC were transplanted
into defect area, 4 weeks after transplantation. Extensively
developing hyaline cartilage, as well as considerably degraded DBM
particles can be seen.
[0065] FIG. 1K: DBM particles together with BMC were transplanted
into defect area, 8 weeks after transplantation. Almost complete
regeneration of subchondral bone; surface of the damaged area is
built of a continues layer of extensively developing young hyaline
cartilage.
[0066] FIG. 1L: DBM particles together with BMC were transplanted
into defect area, 24 weeks after transplantation. The histological
structure of the regenerated osteo-chondral complex is
indistinguishable from normal.
[0067] Abbreviations: Typ. Kn. J., typical knee joint; Osteoch.
Comp., osteochondral complex; Norm. Cart., normal cartilage; Def.,
defect; D., day(s); Al., alone; We., week(s); NROC, newly
reconstituted osteochondral complex.
[0068] FIGS. 2A-G: Laser Capture Microdissection and PCR analysis
of cells captured from the newly reconstituted osteochondral
complex of the knee joint (6 months after transplantation of DBM
with donor male BMC into female recipient).
[0069] FIG. 2A: Laser shot general area, new cartilage
formation.
[0070] FIG. 2B: Laser shot cap, new cartilage formation.
[0071] FIG. 2C: Magnification (.times.20) of laser shot cap, new
cartilage formation.
[0072] FIG. 2D: Laser shot general area, new subchondral bone
formation.
[0073] FIG. 2E: Laser shot cap, new subchondral bone formation.
[0074] FIG. 2F: Magnification (.times.20) of laser shot cap, new
subchondral bone formation.
[0075] FIG. 2G: Detection of donor-derived cells by PCR analysis.
Lanes: 1, DNA size markers (+X714 cut with HaeIII), arrows point to
the 194 bp-long and 118 bp-long bands, respectively; 2,
Amplification of male rat DNA derived from cartilage area of female
rat knee joint; 3, Amplification of male rat DNA derived from
subchondral bone area of female rat knee joint; 4, Male rat DNA
derived from hematopoietic marrow area of female rat knee joint; 5,
Internal positive control DNA from male blood. The PCR results
confirm the expression of donor derived cells in all the three
tissues composing the newly reconstituted osteochondral
complex.
[0076] Abbreviations: Targ. Ar. LCM Kn. J., target area for LCM in
the knee joint; Las. Sh. Ar., laser shot area; Las. Sh. Ca., laser
shot cap; Ca. Marn., caps magnified; Cart., cartilage; S. Bo.,
subchondral bone; Ost. Ch. Comp., osteo-chondral complex; Det. Don.
Der. Cel. PCR Anal., detection of donor-derived cells by PCR
analysis; Lan., lanes.
[0077] FIGS. 3A-F: Correction of the calvarial defect by
transplantation of demineralized bone matrix (DBM) and bone marrow
cells (BMC) in rats, shown by sagital sections stained with
Picroindigocarmin (PIC).
[0078] FIG. 3A: Photomacrographs of a normal rat cranium. Region
marked by a square (D) is shown in FIG. 3B in higher
magnification.
[0079] FIG. 3B: Site of the artificial defect (D) in the parietal
region of the cranium.
[0080] FIG. 3C: Photomacrograph of the defect area (DA) between the
two cut edges.
[0081] FIG. 3D: Photomicrograph of cranial section 8 days after the
experimentally created calvarial defect (PIC staining). Defect left
untreated. Cut edge (CE) and the fibrous connective tissue can be
seen.
[0082] FIG. 3E: Photomicrograph of cranial section 8 days after the
experimentally created calvarial defect (PIC staining). DBM
particles alone were transplanted into defect area. Actively
proliferating fibroblastic cells surrounding the cut edge and DBM
particles could be seen.
[0083] FIG. 3F: Photomicrograph of cranial section 8 days after the
experimentally created calvarial defect (PIC staining). DBM
particles together with BMC were transplanted into defect area.
Active remodeling of the transplanted DBM particles, areas of new
bone formation are clearly visible.
[0084] Abbreviations: Norm. Ra. Cran., normal rat cranium; Def.,
defect; Def. Ar., defect area; Def. Al., defect alone; D., day(s);
Po. Transpl., post-transplantation.
[0085] FIGS. 4A-L: Photomacro- and micrographs of cranial sections
15 and 30 days after the experimentally created calvarial defect
(Sagital sections, Picroindigocarmin, PIC, staining).
[0086] FIG. 4A: 15 days post-operation, control (no
transplant).
[0087] FIG. 4B: 15 days post-transplantation, transplantation of
DBM alone.
[0088] FIG. 4C: 15 days post-transplantation, transplantation of
DBM and BMC.
[0089] FIG. 4D: 15 days post-operation, control (no transplant),
10.times. magnification. There is no new bone formation in the area
of defect.
[0090] FIG. 4E: 15 days post-transplantation of DBM alone,
10.times. magnification. Remodeling of DBM particles results in
bridging the area of defect with the newly formed bone tissue.
[0091] FIG. 4F: 15 days post-transplantation of DBM and BMC,
10.times. magnification. The cut edge of the parietal bone could
hardly be distinguished in the continuous uniform layer of actively
remodeling bony tissue.
[0092] FIG. 4G: 30 days post-operation, control (no
transplant).
[0093] FIG. 4H: 30 days post-transplantation of DBM alone.
[0094] FIG. 4I: 30 days post-transplantation of DBM and BMC.
[0095] FIG. 4J: 30 days post-operation, control (no transplant),
10.times. magnification. There is no new bone formation in the area
of defect.
[0096] FIG. 4K: 30 days post-transplantation of DBM alone,
10.times. magnification. Remodeling of DBM particles results in
bridging the area of defect with the newly formed bone tissue.
[0097] FIG. 4L: 30 days post-transplantation of DBM and BMC,
10.times. magnification. The cut edge of the parietal bone could
hardly be distinguished in the continuous uniform layer of actively
remodeling bony tissue.
[0098] Abbreviations: Def. Al., defect alone; Def., defect; D.,
day(s); DA, area of defect; CE, cut edge.
[0099] FIGS. 5A-F: Laser Capture Microdissection (LCM) and PCR
analysis of cells captured from the newly developing bony tissue in
the area of the experimentally created calvarial defect after
transplantation of DBM together with donor male BMC to female
recipient.
[0100] FIG. 5A: General view of the normal rat cranium with the
place where the defect was inflicted highlighted (D).
[0101] FIG. 5B: Regenerating bony tissue, area target for LCM.
[0102] FIG. 5C: Higher magnification, area target for LCM.
[0103] FIG. 5D: Laser shot caps; cells captured from this area were
used for PCR analysis.
[0104] FIG. 5E: Laser shot caps, 10.times. magnification, cells
captured for PCR analysis.
[0105] FIG. 5F: Detection of donor-derived cells by PCR analysis.
Lanes: 1, DNA size markers (+X714 cut with HaeIII), arrows point to
the 194 bp-long and 118 bp-long bands, respectively; 2, male rat
DNA derived from bone area of female knee cranium; 3, internal
positive control, DNA from male blood. The results of the PCR
analysis confirm the expression of donor derived cells in the newly
forming bony tissue.
[0106] Abbreviations: Norm. Ra. Cran., normal rat cranium; Bo.,
bone; Las. Sh. Ar., laser shot area; Las. Sh. Ca., laser shot caps;
Ca. Magn., caps magnified; Targ. Ar. LCM, target area for LCM; CE,
cut edge; D, area of defect; Det. Don. Der. Cel. PCR Anal.,
detection of donor-derived cells by PCR analysis; bp,
base-pair(s).
DETAILED DESCRIPTION OF THE INVENTION
[0107] The following abbreviations are utilized throughout this
specification: [0108] BM: bone marrow [0109] BMC: bone marrow
cell(s) [0110] BMP: bone morphogenetic protein [0111] DBM:
demineralized bone matrix [0112] LCM: Laser Capture Microdissection
[0113] MBM: mineralized bone matrix [0114] PCR: polymerase chain
reaction [0115] PIC: Picroindigocarmin, a dye used in histological
staining.
[0116] In search for improving regeneration of damaged
osteochondral complex in joint and cranio-facial-maxillary areas,
the inventors have found that using a composition comprising BMC
and DBM and/or MBM as a graft results in the development of bone
and cartilage according to the local conditions of the site of
transplantation. New tissue formation follows a differentiation
pathway producing different types of bone and cartilage, depending
on the local conditions. Thus, the newly formed tissue meets
precisely the local demands.
[0117] The present invention relates to compositions comprising a
mixture of bone marrow cells (BMC) and demineralized and/or
mineralized bone matrix (DBM and MBM, respectively) and to their
novel uses in the transplantation of mesenchymal progenitor cells
into joints and cranio-facial-maxillary bones.
[0118] Thus, in a first aspect, the present invention relates to a
composition comprising bone marrow cells (BMC) and demineralized
bone matrix (DBM) and/or mineralized bone matrix (MBM).
[0119] DBM is a preferable essential ingredient in the composition
of the invention in view of its advantageous ability to combine all
the features needed for making it an excellent carrier for
mesenchymal progenitor cells. The properties of DBM can be
summarized as follows: [0120] 1. DBM can be a conductive scaffold
essential for the engraftment, proliferation and differentiation of
mesenchymal progenitor cells, in the course of bone and cartilage
formation. [0121] 2. DBM is the natural source of BMPs, which are
active in stimulating osteo- and chondrogenesis, thus also
fulfilling the inductive function. [0122] 3. DBM is slowly
biodegradable, the degradation time being compatible with the
period of de novo chondro- and osteogenesis. [0123] 4. DBM has very
low immunogenicity when used as a xenograft, and it is practically
non-immunogenic when used in allogeneic combinations. [0124] 5. DBM
is sufficiently flexible and strong to provide biomechanical
properties to the joint surface during the period of new cartilage
formation. [0125] 6. DBM can be provided as an amorphous powder
that can be injected locally, without major surgical intervention,
thus avoiding iatrogenic damage to complex joints.
[0126] In a second aspect, said composition comprising BMC and DBM
and/or MBM is for use in transplantation of mesenchymal cells
and/or mesenchymal progenitor cells into a joint and/or a
cranio-facial-maxillary area of a subject in need, wherein said
subject is a mammal, preferably a human.
[0127] It is an object of the present invention to provide the said
composition for transplantation of BMC into damaged joints for the
replacement and/or restoration of hyaline cartilage as well as of
subchondral bone, originating from the mesenchymal precursor cells
existing in the transplanted BMC.
[0128] In a first embodiment, the DBM and MBM comprised within the
composition of the invention are of vertebrate origin, and they may
be of human origin.
[0129] In a second embodiment, the DBM and MBM comprised within the
composition of the invention are in powder or slice form. The
particle size of the DBM may be about 50 to 2500.mu.. Preferably,
said particle size is about 250 to 500.mu.. The most preferable
particle size will depend on the specific needs of each case.
[0130] In another embodiment, the composition of the invention is
for restoring and/or enhancing the formation of a new hyaline
cartilage and subchondral bone structure.
[0131] The idea underlying the present invention is that bone
marrow cells (BMC) may provide a source for mesenchymal stem cells,
which are capable of inducing osteo- and chondrogenesis. Thus, as
described in the following examples, when a BMC suspension in
admixture with DBM and/or MBM powder was administered directly into
either a joint bearing a damage in the osteo-chondral complex, or
in the cranium of an animal with a partial bone defect in the
parietal bone, significant restoration occurred. Treated recipients
were mobile with no need for fixation of the joints, and full
restoration of the anatomic structure of the treated joint was
accomplished. Likewise, newly reconstituted parietal bone replacing
surgically removed parietal bone in the skull showed normal
remodeling. In the damaged joint, there was formation of
subchondral bone structure and hyaline cartilage, and in the
cranial defect, new flat bone was formed, both originating from the
mesenchymal cells present in the transplanted BMC, as confirmed by
the LCM-PCR analysis.
[0132] In a further embodiment, the composition of the invention is
intended for the treatment of a patient suffering from any one of a
hereditary or acquired bone disorder, a hereditary or acquired
cartilage disorder, a malignant bone or cartilage disorder,
metabolic bone diseases, bone infections, conditions involving bone
or cartilage deformities and Paget's disease. Said disorders are
listed in detail in Table 1. Additionally, the invention is also
intended for the treatment of a patient in need of any one of
correction of complex fractures, bone replacement, treatment of
damaged or degenerative arthropathy and formation of new bone in
plastic or sexual surgery. TABLE-US-00001 TABLE 1 Congenital and
Hereditary Metabolic Non-neoplastic Bone Disorders Bone Infections
Bone Diseases Disorders of the Bone Achondroplasia Hematogenous
Osteoporosis Fibrous Dysplasia of (Pyogenic) the Bone Osteomyelitis
Osteogenesis Osteomyelitis Rickets and Fibrous Cortical Imperfecta
from a Osteomalacia Defect and Non- (Brittle Bones, Contiguous
ossifying Fibroma Fragilitas Infection Ossium) Osteopetrosis
Osteomyelitis Bone Changes Solitary Bone Cyst (Marble Bone from an
in (Unicameral Bone Disease, Introduced Hyperparathyroidism Cyst)
Osteosclerosis) Infection (Generalized Osteitis, Cystic Fibrosis,
Von Recklinghausen's Bone Disease) Hereditary Bone Renal Aneurysmal
Bone Multiple Tuberculosis Osteodystrophy Cyst Exotosis
(Osteochondromatosis) Enchondromatosis Bone Syphilis Paget's
Eosinophilic (Ollier's Disease of Granuloma of Bone Disease) Bone
(Osteitis Deformans) Bone Fungus Bone Lesions of Infections
Gaucher's Disease
[0133] In a yet further embodiment, the composition of the
invention may further optionally comprise a pharmaceutically
acceptable carrier or diluent, as well as additional active
agents.
[0134] A pharmaceutically acceptable (or physiologically
acceptable) additive, carrier and/or diluent mean any additive,
carrier or diluent that is non-therapeutic and non-toxic to
recipients at the dosages and concentrations employed, and that
does not affect the pharmacological or physiological activity of
the active agent.
[0135] The preparation of pharmaceutical compositions is well known
in the art and has been described in many articles and textbooks,
see e.g., Remington's Pharmaceutical Sciences, Gennaro A. R. ed.,
Mack Publishing Company, Easton, Pa., 1990, and especially pages
1521-1712 therein.
[0136] Active agents of particular interest are those agents that
promote tissue growth or infiltration, such as growth factors. One
example is BMPs, which may enhance the activity of the composition
of the invention. Other exemplary growth factors for this purpose
include epidermal growth factor (EGF), osteogenic growth peptide
(OGP), fibroblast growth factor (FGF), platelet-derived growth
factor (PDGF), transforming growth factors (TGFs), parathyroid
hormone (PTH), leukemia inhibitory factor (LIF), insulin-like
growth factors (IGFs), and growth hormone. Other agents that can
promote bone growth, such as the above-mentioned BMPs, osteogenin
(Sampath et al. (1987) Proc. Natl. Acad. Sci. USA 84:7109-[3] and
NaF [Tencer et al. (1989) J. Biomed. Mat. Res. 23: 571-89] are also
preferred.
[0137] Other active agents may be anti-rejection or tolerance
inducing agents, as for example immunosupressive or
immunomodulatory drugs, which can be important for the success of
bone marrow allografts or xenografts transplantion.
[0138] Alternatively, said active agents may be for example
antibiotics, provided to treat and/or prevent infections at the
site of the graft. On the same token, anti-inflammatory drugs can
also be added to the composition of the invention, to treat and/or
prevent inflammations at the site of the graft. Said inflammations
could be the result of for example rheumatoid arthritis, or other
conditions.
[0139] Polymeric or biodegradable materials are pharmaceutically
acceptable carriers and diluents. Biodegradable films or matrices,
semi-solid gels or scaffolds include calcium sulfate, tricalcium
phosphate, hydroxyapatite, polylactic acid, polyanhydrides, bone or
dermal collagen, fibrin clots and other biologic glues, pure
proteins, extracellular matrix components and combinations thereof.
Such biodegradable materials may be used in combination with
non-biodegradable materials, to provide desired mechanical,
cosmetic or tissue or matrix interface properties.
[0140] In preferred embodiments, the composition of the invention
contains BMC suspensions at cell concentrations ranging from
1.times.10.sup.6/ml to 1.times.10.sup.8/ml and DBM at a ratio of
from 1:1 to 20:1, preferably between 2:1 to 9:1, most preferably
the composition of the invention is at a ratio of 4 parts BMC
concentrate to 1 part of DBM in powder form (volume:volume). The
absolute number of BMC and DBM is dependent on the size of the
joint that needs to be corrected or the size (surface, shape and
thickness) of the bone that needs to be replaced.
[0141] In another aspect, the present invention relates to a method
for transplantation of a mixture comprising BMC with DBM and/or MBM
and optionally further comprising pharmaceutically acceptable
carrier or diluent, into a joint and/or a cranio-facial-maxillary
bone of a subject in need, wherein said method comprises
introducing into said joint or bone the composition of the
invention.
[0142] The composition of the invention, which possesses all the
essential features for accomplishing local bone formation wherever
it is implanted, could be efficiently applied for all kinds of bone
repair or substitution, especially in places lacking or deprived of
mesenchymal stem cells. Amongst the most problematic places in this
sense are joints, cranio-facial-maxillary areas and different kinds
of segmental bony defects. Thus, the present invention may be
explained as a complex graft, comprising all necessary components,
and which its implantation into a damaged joint or bone is
sufficient for regeneration or substitution of removed, damaged or
destroyed cartilage and/or bone.
[0143] In a first embodiment of the method of the invention, the
mixture is administered by any one of the following procedures,
injection, minimally invasive arthroscopic procedure, or by
surgical arthroplasty into the site of implantation, wherein said
method is for support or correction of congenital or acquired
abnormalities of the joints, cranio-facial-maxillary bones,
orthodontic procedures, bone or articular bone replacement
following surgery, trauma or other congenital or acquired
abnormalities, and for supporting other musculoskeletal implants,
particularly artificial and synthetic implants.
[0144] Thus, in a further aspect, the invention relates to a method
of treating a damaged or degenerative arthropathy associated with
malformation and/or dysfunction of cartilage and/or subchondral
bone in a mammal in need of such treatment, comprising
administering into an affected joint or bone of said mammal a
mixture comprising BMC with DBM and/or MBM, said mixture optionally
further comprising a pharmaceutically acceptable carrier or diluent
and/or additional active agents.
[0145] As demonstrated in the following examples, the process of
induced development (i.e. proliferation and differentiation) of
mesenchymal progenitor cells present within the BMC/DBM mixture can
accomplish bone and cartilage formation wherever the mixture is
transferred to. The findings presented by the inventors indicate
that administration of the composition of the invention into a
damaged area of the joint, results in generation of new
osteochondral complex consisting of articular cartilage and
subchondral bone. When administered into an experimentally created
calvarial defect, the composition of the invention results in
generation of full intramembranous bone development at the site of
transplantation. New tissue formation follows a differentiation
pathway, producing different types of bone and cartilage, depending
on the local conditions. Thus, the newly formed tissue meets
precisely the local demands.
[0146] In one embodiment, the BMC which is present in the
administered mixture are either allogeneic or said mammal's
own.
[0147] In another embodiment, the DBM or MBM which is present in
the administered mixture is in a slice, powder, gel, semi-solid or
solid form embedded in or encapsulated in polymeric or
biodegradable materials.
[0148] The procedure of applying the composition of the invention
into a damaged joint or cranial area comprises the following steps:
[0149] 1. Selecting the source for BMC. The donor may be allogeneic
or the BMC may be obtained from the same treated subject
(autologous transplantation). [0150] 2. Selecting the source of DMB
and/or MBM. The DBM may be supplied commercially and since it is
not immunogenic, there are no limitations for a specific donor. DMB
and/or MBM may be in powder, granules or in slice form. The
particle size of the DBM may be about 50 to 2500.mu.. Preferably,
said particle size is about 250 to 500.mu.. The most preferable
particle size will depend on the specific needs of each case.
[0151] 3. Preparing a composition comprising a suspension of BMC,
at a cell concentration ranging from 1.times.10.sup.6/ml to
1.times.10.sup.8/ml and mixing it with DBM at a ratio of from 1:1
to 20:1, preferably between 2:1 to 9:1, most preferably the
composition of the invention is at a ratio of 4 parts BMC
concentrate to 1 part of DBM in powder form (volume:volume). MDM
may be used instead of DBM. If so desired, BMP may optionally be
included in the composition. [0152] 4. Administering said
composition into a subject in need either through a syringe
(non-invasive injection), closed arthroscopy or open surgical
procedure. Alternatively, the composition may be administered so
that it is encapsulated within normal tissue membranes. Still
alternatively, the composition may be contained within a membranous
device, made of a selective biocompatible membrane that allows
cells, nutrients, cytokines and the like to penetrate the device,
and at the same time retains the DBM and/or MBM particles within
the device. Such a membranous device, bone strips or additional
scaffolds are preferably surgically introduced. Or, still
alternatively, the composition may be administered within a
biocompatible and biodegradable polymeric device retaining the DBM
and/or MBM particles within the device and suitable to create the
needed shape of the transplanted complex. [0153] 5. Providing glue,
preferably consisting of fibrinogen and thrombin, this may be used
for fixation of the implant composition at the site of
implantation, if necessary.
[0154] In a yet further aspect, the present invention relates to a
non-invasive transplantation method comprising introducing a graft
into a joint or a cranio-facial-maxillary bone of a subject in
need, wherein said graft comprises a mixture of BMC and DBM or
MBM.
[0155] In the examples presented herein (see Examples), the
inventors show that administration of the composition of the
present invention (e.g. BMC in admixture with DBM, as in Example 1)
into a damaged area of the joint is essential and sufficient for
the generation of new osteochondral complex, consisting of
articular cartilage and subchondral bone, at the site of
transplantation. The newly formed donor-derived osteochondral
complex was capable of long-term maintenance, remodeling and
self-renewal, as well as carrying out specific functions of joint
surface, such as motion and weight bearing.
[0156] In an even further aspect, the present invention relates to
the use of a composition comprising BMC and DBM and/or MBM as a
graft of mesenchymal and/or mesenchymal progenitor cells for
transplantation into a mammal, wherein said mammal is preferably a
human. The transplantation is to be performed into a joint or into
a cranio-facial-maxillary bone, for the development of new bone
and/or cartilage. The graft of said transplantation may also be for
supporting orthodontical procedures for bone augmentation caused by
aging, or by congenital, acquired or degenerative processes.
[0157] Furthermore, the composition used in said transplantation is
intended for the treatment of a patient suffering from any one of a
hereditary or acquired bone disorder, a hereditary or acquired
cartilage disorder, a malignant bone or cartilage disorder,
conditions involving bone or cartilage deformities and Paget's
disease. In addition, said composition is intended for the
treatment of a patient in need of any one of correction of complex
fractures, bone replacement, treatment of damaged or degenerative
arthropathy and formation of new bone in plastic or sexual
surgery.
[0158] The method of the invention may also be used to induce or
improve the efficiency of bone regeneration in damaged
cranio-facial-maxillary areas, for therapeutic and cosmetic
purposes.
[0159] In one embodiment, the composition used in the invention
further comprises an additional active agent.
[0160] In another embodiment, the DBM and MBM comprised within the
composition used in the invention are of vertebrate origin, and
they may be of human origin. Moreover, said DBM and MBM is in
powder or slice form.
[0161] In an additional aspect, the present invention concerns the
use of a mixture of BMC with DBM and/or MBM in the preparation of a
graft for the treatment of a bone or cartilage disorder, and/or for
support of musculoskeletal implants, as a `glue` to enforce metal
implants, joints, etc. that may become lose with time, or to
provide a constantly adapting "biological glue" to support such
non-biological implants. Alternatively, the invention could be for
the support of limb transplants, especially in the articular/bone
junction.
[0162] Lastly, the present invention provides a kit for performing
transplantation into a joint or a cranio-facial-maxillary bone of a
mammal of BMC in admixture with DBM and/or MBM, wherein said kit
comprises: [0163] (a) DBM and/or MBM in a compacted form; [0164]
(b) a BM aspiration needle; [0165] (c) an intra-osseous bone
drilling burr; [0166] (d) a needle with a thick lumen for infusion
of viscous bone marrow-DBM mixture; [0167] (e) a 2-way lumen
connector for simultaneous mixing of BMC-DBM and diluent; [0168]
(f) a medium for maintaining BMC; and optionally [0169] (g)
cryogenic means for handling and maintaining BMC or BMC together
with DBM.
[0170] The kit of the invention may optionally further comprise a
carrier and/or a diluent for the BMC and DBM and/or MDM
mixture.
[0171] The present inventors have concluded that transplantation of
multipotent mesenchymal stem cells, and not of differentiated bone
or chondrocytes, for remodeling and restoration of a healthy joint
or cranio-facial-maxillary structure in arthropathy, is especially
important for the following reasons: [0172] (1) Chondrocytes, as
well as the cells transferred within a bone transplant are already
fully differentiated cells, with relatively low metabolic activity
and limited self-renewal capacity that may be sufficient to
maintain healthy cartilage or bone, but is certainly insufficient
for the development of large areas of bone or of hyaline cartilage
de novo. [0173] (2) Most frequently in joints, both cartilage and
subchondral bone are damaged. Thus, even a successfully developed
new hyaline cartilage is unlikely to be maintained for long if the
subchondral bone is left damaged. Based on these findings, it was
observed in the following examples that mesenchymal stem cells
present in bone marrow, if transplanted under the appropriate
conditions, will create a self-supporting osteochondral complex
providing healthy joint surface.
[0174] It is not yet clear what makes multipotential mesenchymal
stem cells, under the influence of DBM, to choose between an
osteogenic and a chondrogenic differentiation pathway. It has
however been reported that the ratio of cartilage to bone
production depends in particular on the site of DBM implantation,
which is naturally influenced by the local conditions [Inoue, T. et
al. (1986) J Dent Res 65(1):12-22], such as the local source of
mesenchymal cells and blood supply [Reddi, A. H. and Huggins, C. H.
(1973) P.S.E.B.M. 143:634-637]. Low oxygen tension favors
chondrogenesis [Bassett, C. A. L. (1962) J Bone Joint Surg
44A:1217], most likely due to the low O.sub.2 tension in poorly
vascularized cartilage [Sledge, C. B. and Dingle, J. T. (1965)
Nature (London) 205: 140]. Interestingly, a successful substitution
of anterior cruciate ligament (ACL) by demineralized cortical bone
matrix has been reported in a goat model [Jackson, D. W. et al.
(1996) Amer J Sports Medicine 24(4):405-414]. The remodeling
process included new bone formation within the matrix in the
osseous tunnels and a ligament-like transition zone developing at
the extra-articular tunnel interface [Jackson, D. W. et al. (1996)
id ibid.]. Taking into consideration that hyaline cartilage is
naturally developed and maintained only in the joints, where
contact with synovial membranes and lubrication with synovial fluid
is available and probably essential, it seems reasonable to assume
that the environmental conditions in the joint play a major role in
enhancing chondrogenesis.
[0175] In the following examples the inventors have shown, for the
first time, that a graft composed of DBM and/or MBM and bone marrow
cells transplanted into a damaged joint or cranial bone, led to
successful replacement of damaged cartilage and subchondral bone.
This was the result of osteogenesis on the side of contact with
bone and chondrogenesis on the free joint surface, thus the
physiological environmental conditions favored osteogenesis or
chondrogenesis, respectively. The same kind of a graft composed of
DBM and/or MBM and bone marrow cells transplanted into
experimentally created partial bone defect in the parietal bone of
the cranium led to successful replacement of the removed part of
the bone. Thus, the new tissue formation follows a differentiation
pathway, producing different types of bone and cartilage depending
on the local conditions, such that the newly formed tissue meets
precisely the local demands.
[0176] Many publications are referred to throughout this
application. The contents of all of these references are fully
incorporated herein by reference.
[0177] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0178] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
[0179] The following examples are representative of techniques
employed by the inventors in carrying out aspects of the present
invention. It should be appreciated that while these techniques are
exemplary of preferred embodiments for the practice of the
invention, those of skill in the art, in light of the present
disclosure, will recognize that numerous modifications can be made
without departing from the spirit and intended scope of the
invention.
EXAMPLES
Experimental Procedures
1. Animals
[0180] 8 weeks old C57BL/6 male mice and Lewis male rats with body
weight of 180-200 g were used as the donors of bones (for matrix
preparation) and BMC. Rats from the same batches were used as graft
recipients.
2. Preparation of Demineralized Bone Matrix (DBM)
[0181] Demineralized bone matrix (DBM) was prepared as described
[Reddi and Huggins (1973) id ibid.] with the inventors'
modification. Diaphyseal cortical bone cylinders from Lewis rats
were cleaned from bone marrow and surrounding soft tissues,
crumbled and placed in a jar with magnetic stirring. Bone chips
were rinsed in distilled water for 2-3 hrs; placed in absolute
ethanol for 1 hr and in diethyl ether for 0.5 hr, then dried in a
laminar flow, pulverized in a mortar with liquid nitrogen and
sieved to select particles between 400 and 1,000.mu.. The obtained
powder was demineralized in 0.6M HCl overnight, washed for several
times to remove the acid, dehydrated in absolute ethanol and
diethyl ether and dried.
[0182] Mineralized bone matrix (MBM) was prepared according to the
same procedure but the stage of demineralization with HCl was
omitted.
[0183] With the exception of the drying step, all steps of the
procedure were performed at 4.degree. C.; to prevent degradation of
Bone Morphogenetic Proteins (BMP) by endogenous proteolytic
enzymes. The matrices were stored at -20.degree. C.
3. Preparation of the Implanted Material
Preparation of Donor BMC Suspensions for Transplantation:
[0184] The femurs of donor mice or rats were freed of muscle.
Marrow plugs were mechanically pressed out of the femoral canal by
a mandrin. Highly concentrated single cell suspensions of BMC were
prepared by dissolving 4-5 femoral plugs into 100 .mu.l of RPMI
1640 medium (Biological Industries, Beit Haemek, Israel), and
passing the cells through the needle several times to dissolve the
bone marrow tissue into a single-cell suspension. The number of
nucleated cells per femoral bone marrow plug is rather stable
(about 10.sup.7 cells/plug for a C57BL/6 male, 8 week old mouse).
Several reproducible verifications have shown that BMC prepared for
transplantation in a form of a single cell suspension contains an
approximate concentration of 3.times.10.sup.8 cells/ml.
Composition of the Grafts:
[0185] Grafts were composed of the following ingredients, in
different combinations: [0186] 1. 20 .mu.l of BMC suspension
(concentration 3.times.10.sup.8 cells/ml); [0187] 2. 4 mg of DBM
(or MBM); [0188] 3. 0.5 .mu.g BMP-2 (R & D systems, USA),
optionally.
[0189] The exogenous BMP that is optionally added to the
composition of the invention is not a mandatory ingredient. DBM
exhibits conductive properties essential for the engraftment,
proliferation and differentiation of mesenchymal progenitor cells
transplanted within BMC suspension, in the course of bone and
cartilage formation. At the same time, DBM is the natural source of
BMPs (bone morphogenetic proteins) active in stimulating osteo- and
chondrogenesis, thus fulfilling also the inductive function.
Addition of exogenous BMPs may enhance the efficiency of the
induction.
4. Implantation of a Mixture of BMC and Demineralized (or
Mineralized) Bone Matrix into the Area of Local Damage in the
Articular Cartilage of the Knee Joint
[0190] A standard artificial damage in the articular cartilage and
subchondral bone in the rat knee joint was induced as described.
Following anesthesia, the knee joint was accessed by a medial
parapatellar incision, and the patella was temporarily displaced
towards the side. A microfracture drilling (for a full thickness
defect) of 1.5 mm in diameter and 2.0 mm in depth was made in the
interchondylar region of the femur.
[0191] The defect was filled with DBM (or MBM) in the form of
powder (with particle size of 300-450 micron, or in the form of
slice), alone (control) or together with the BMC suspension,
prepared as described above. Another control consisted of
transferring only BMC into the damaged area. The transplanted
material was fixed in place with fibrinogen-thrombin tissue
adhesive glue, the patella was returned into its place and the
incision was sutured with bioresorbable thread. The skin was closed
with stainless clips. In another control group, the damaged area
was closed with fibrinogen-thrombin tissue adhesive glue only,
without the addition of any of DBM, MBM or BMC.
5. Implantation of a Mixture of BMC and Demineralized Bone Matrix
into the Experimentally Created Calvarial Defect.
[0192] Male Lewis rats were anesthetized by intraperitoneal
injection of Ketamine. An incision was performed in the frontal
region of the rat cranium. The muscular flap was removed from the
parietal bone area and a bony defect (0.4.times.0.5 mm.sup.2) was
made lateral to the saggital suture using a dental burr. The defect
was then filled with DBM in powder form (particle size of 300-450
micron) together with BMC suspension, prepared as described above.
In one control group, only DBM particles were transferred into the
damaged area. The transplanted material was fixed in place with
fibrinogen-thrombin tissue adhesive glue. In another control group
the damaged area was only closed with fibrinogen-thrombin tissue
adhesive glue, without addition of the transplanted material. The
skin was closed with stainless clips.
[0193] 6. Laser Capture Microdissection (LCM) and Polymerase Chain
Reaction (PCR) Analysis of the Reconstituted Bone Articular
Cartilage and Hematopoietic Tissue after Implantation of BMC and
Demineralized Bone Matrix into Damaged Intracondylar Region of
Femoral Bone or into the Cranial Defect
[0194] In the experiments in which BMC of male donors was
transplanted either into the damaged intracondylar region of
femoral bone, or into the cranial defect, both in female
recipients, the newly formed articular cartilage, cranial and
subchondral bone were checked by PCR analysis for the origin of the
donor. The new technology of Laser-Capture Microdissection (LCM)
(service provided by the Common Facility Unit of Hadassah
University Hospital, Jerusalem, Israel) allows the isolation of
individual cells from tissue sections under precise microscopic
control, and was used to harvest isolated cells from newly formed
articular cartilage, cranial and subchondral bone. PCR analysis of
the harvested cells (about 70-100 cells per test) was performed
using a set of primers specific to the Sry gene--the sex
determination region of the Y chromosome [An, J. et al. (1997) J
Androl. 18(3):289-93].
7. Histological Evaluation
[0195] The autopsied material was fixed in 4% neutral buffered
formaldehyde, decalcified, passed through a series of ethanol
grades and xylene, and then embedded in paraffin. Serial sections
(5-7 microns thick) were obtained. One set of representative serial
sections of each sample was stained with Hematoxylin & Eosin
(H&E), and another one with Picroindigocarmin (PIC).
Example 1
Transplantation of BMC into the Joint Together with Demineralized
(or Mineralized) Bone Matrix
[0196] FIG. 1 presents the results of experiments carried out to
test whether the mesenchymal stem cells present within the bone
marrow cells of the composition of the invention could be induced
to develop hyaline (articular) cartilage and subchondral bone, when
transplanted into the damaged areas of the knee joints.
[0197] Male Lewis rats were anesthetized by intraperitoneal
injection of Ketamine. Microfracture drilling (full thickness
defect) was inflicted in articular cartilage and subchondral bone
in the interchondylar region of the femur. The defects were then
filled with DBM (or MBM) together with BMC. In separate groups of
experimental animals with defects in articular cartilage and
subchondral bone, said defects were filled with DBM (or MBM) or BMC
alone. The optional addition of BMPs was also tested (data not
shown). The implanted material was fixed in place with
fibrinogen-thrombin tissue adhesive glue. In one group of control
animals, only glue (and no DBM, MBM or BMC) was grafted into the
defect area.
[0198] FIGS. 1A, 1B and 1C show healthy undamaged knee joint of the
rat with osteo-chondral complex in the interchondylar region of the
femur. In FIG. 1D, the microfracture drilling (full thickness
defect) can be seen immediately after damage. When DBM powder mixed
with BMC was transplanted into the drilled hole, areas of
extensively developing hyaline cartilage surrounding implanted DBM
particles were observed already two weeks after transplantation,
when slight degradation of DBM particles could be observed (FIG.
1I). One month after transplantation the DBM particles were already
considerably degraded, and areas of extensively developing hyaline
cartilage surrounding implanted DBM particles were still present
(FIG. 1J). After two months, regeneration of the subchondral bone
was almost complete, while the surface of the damaged area was
built of a thick and continuous layer of extensively developing
young hyaline (articular) cartilage (FIG. 1K). Six months after
transplantation of DBM powder, mixed with BMC, into the
microfracture drilling defect in the interchondylar region of the
femur, the histological structure of the regenerated osteo-chondral
complex was indistinguishable from normal (FIG. 1L).
[0199] PCR analysis of isolated cells from different tissues
composing newly developed osteo-chondral complex, captured by LCM
techniques after implantation of DBM together with BMC into the
micro-fracture drilling was performed (FIGS. 2A-F). The results of
the PCR showed the presence of donor derived cells within newly
formed articular cartilage and subchondral bone (FIG. 2G). This is
strong evidence that active mesenchymal progenitor cells,
transplanted within the donor BMC suspension, took an active part
in the development of a new osteo-chondral complex.
[0200] Most importantly, bone and cartilage regeneration, to the
same extent as that of the experimental group that received a DBM
together with BMC graft, were not observed in the control groups.
Thus, the inventors concluded that the DBM+BMC mixture included the
entire array of components essential for the successful
regeneration of the osteo-chondral complex in the damaged
joint.
[0201] The specificity of the artificial defect model used in the
present experiments resided in the penetration of the microfracture
drill into the subchondral bone, thus supplying the damaged area
with locally existing bone marrow containing mesenchymal progenitor
cells potentially capable of restoring both subchondral bone and
articular cartilage, when local conditions stimulating osteo- and
chondrogenesis were supplied.
[0202] However, without the implant, regeneration of micro-fracture
was incomplete, and two weeks after drilling the hole was filled
with connective tissue (FIG. 1E). During the following month, the
subchondral bone was repaired, although with no regeneration of the
articular cartilage on the surface of the damaged area (data not
shown). Six months after the micro-fracture had been inflicted, the
regenerating surface of the damaged joint constituted of
fibro-cartilaginous tissue (FIG. 1F). Implantation of exogenous BMC
does not bring any considerable changes in the pathway of
regeneration, i.e. the regenerating surface of the damaged joint
constituted of fibro-cartilaginous tissue usually deteriorating
over time. It is important to note that, unfortunately, despite the
restricted efficiency of this procedure, as also shown by these
results, this is the most used procedure for damaged joint surface
repair and for the treatment of osteoarthritis in the current
clinical practice.
[0203] When DBM particles alone were transplanted into the drilled
hole, it seems that the number of locally available mesenchymal
progenitor cells was not sufficient for effective regeneration of
osteo-chondral complex. No extensive developing hyaline cartilage
could be seen among the implanted DBM particles two weeks after
transplantation (FIG. 1G). In most of the cases the degradation and
remodeling of DBM particles as well as the process of new bone- and
cartilage formation was not efficient enough and the damaged area
was finally covered with connective tissue together with
fibro-cartilage (FIG. 1H).
Example 2
Transplantation of BMC Together with DBM into the Experimentally
Created Calvarial Defect.
[0204] Experiments were carried out to test whether the mesenchymal
stem cells within the BMC comprised in the composition of the
invention could initiate and accomplish the intramembranous
development of bone, when transplanted together with DBM into the
experimentally created calvarial defect. The results of these
experiments are shown in FIGS. 3 and 4. This method could then be
extended to treat facial-maxillary defects.
[0205] An incision was performed in the frontal cranium region of
anesthetized Lewis rats (8-12 weeks old) and the skin flap was
moved aside. The muscular flap was removed from the parietal bone
area and a bony defect (0.4.times.0.5 mm.sup.2) was created
laterally to the sagital suture using a dental burr. The defect
area was either left empty, filled with DBM alone, or filled with
DBM together with BMC, as described above. In all the groups
(experimental and control) the defect area was finally covered with
fibrin glue. Lastly, the skin flap was returned to place and fixed
with stainless clips.
[0206] The utilization of non-healing cranial defects allows for
the observation of both osteo-conductive and osteo-inductive
components of the healing process. Thus, the non-healing cranial
defect represents an appropriate model for evaluating the ability
of the composition of the present invention (in this Example, BMC
together with DBM) to accomplish intramembranous bone formation
when transplanted into a damaged area of the crania.
[0207] FIG. 3A shows the normal (undamaged) cranium of a rat. FIGS.
3B and 3C show the experimental defect in the parietal bone
area.
[0208] 15 and 30 days after the operation, absence of bony tissue
regeneration could be observed when the site of removed bone was
left empty (FIGS. 4A, 4D, 4G and 4J), suggesting that the size of
the defect sufficiently large, compatible with the definition of
non-healing cranial defect.
[0209] Filling of the experimental cranial defect with DBM alone
resulted in the gradual degeneration and remodeling of transplanted
DBM particles, and blood vessels and new bone formation on
different stages of maturity could be observed (FIG. 3E, FIGS. 4B,
4E, 4H and 4K). However, the process of intensive new bone
formation was not presented uniformly in the defect area. New bone
formation was considerably more active in the periphery, close to
the edges of the cut bone, suggesting that the number of
mesenchymal progenitor cells that could be induced and conducted to
osteogenesis was limited in the central area of the defect.
[0210] When DBM powder together with BMC was transplanted into the
site of the experimental cranial defect, extensive remodeling of
the transplanted DBM particles and developing areas of new bone
could be observed as early as 8 days after transplantation (FIG.
3F). 15 and mostly 30 days after transplantation, the cut edge of
the parietal bone could hardly be distinguished from the
surrounding new bony tissue (FIGS. 4F and 4L). The defect area was
reconstituted with a continuous layer of newly developing bone
(FIGS. 4C, 4F, 4I and 4L). It should be especially stressed that
extensive remodeling of transplanted DBM particles and active new
bone formation were presented uniformly throughout the defect area,
suggesting that the quantity of available mesenchymal progenitor
cells capable of being induced and conducted to osteogenesis was
sufficient when the implant consisted of DBM particles mixed with
BMC.
[0211] PCR analysis of the cells isolated by LCM from the newly
developed bone tissue, in the site of experimental calvarial
damage, 8 and 30 days after transplantation of DBM together with
BMC (from male donor to female recipient) showed the presence of
donor derived cells (FIG. 5). This is strong evidence that the
mesenchymal progenitor cells transplanted within the donor BMC
suspension play an active role in the development of this new
cranial bone, and are subject to the osteo-inductive and
osteo-conductive properties of DBM.
[0212] These findings indicate that administration of the
composition of the present invention (in this case, DBM together
with BMC) into an experimentally created calvarial defect was
sufficient for active and complete intramembranous bone formation
at the site of transplantation. This procedure could be extended to
treat facial-maxillary defects.
[0213] Pilot experiments utilizing BMC in combination with MBM
rather than DBM also showed positive results. Mainly, the
difference between employing DBM and MBM lies on delayed bone and
cartilage formation with MBM. Also, since MBM particles are much
more dense and hard, as compared to DBM particles, they are more
useful when weight bearing or shape preservation of the transplant
are needed. Transplantation of a mixture of both DBM and MBM
together with BMC should enable the best of the advantages of both:
(a) significantly prolonging the period of osteo- and chondrogenic
activity (with DBM acting fast and MBM after a delay); (b)
improving the shape preservation of the implant throughout the
whole period of new tissue formation.
[0214] Addition of BMP to the mixture of BMC and DBM has
considerably accelerated formation of new tissue both in the
osteochondral complex of the knee joint and in the flat bones of
the skull.
* * * * *