U.S. patent application number 13/600661 was filed with the patent office on 2012-12-27 for methods and compositions for regenerating connective tissue.
This patent application is currently assigned to Encelle, Inc.. Invention is credited to Ronald Stewart Hill, Richard Chris Klann, Francis V. Lamberti.
Application Number | 20120328700 13/600661 |
Document ID | / |
Family ID | 34549273 |
Filed Date | 2012-12-27 |
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United States Patent
Application |
20120328700 |
Kind Code |
A1 |
Hill; Ronald Stewart ; et
al. |
December 27, 2012 |
METHODS AND COMPOSITIONS FOR REGENERATING CONNECTIVE TISSUE
Abstract
Connective tissue regenerative compositions and methods of
repairing and regenerating connective tissue using such
compositions are provided. The compositions generally comprise a
bioactive hydrogel matrix comprising a polypeptide, such as
gelatin, and a long chain carbohydrate, such as dextran. The
hydrogel matrix may further include polar amino acids, as well as
additional beneficial additives. Advantageously, the compositions
include further components, such as osteoinductive or
osteoconductive materials, medicaments, stem or progenitor cells,
and three-dimensional structural frameworks. The compositions are
useful for regenerating connective tissue, and can be administered
to an area having injury to, or a loss of, connective tissue, such
as bone, cartilage, tendon, and ligament.
Inventors: |
Hill; Ronald Stewart;
(Greenville, NC) ; Klann; Richard Chris;
(Washington, NC) ; Lamberti; Francis V.;
(Greenville, NC) |
Assignee: |
Encelle, Inc.
|
Family ID: |
34549273 |
Appl. No.: |
13/600661 |
Filed: |
August 31, 2012 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12039214 |
Feb 28, 2008 |
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13600661 |
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10971544 |
Oct 22, 2004 |
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12039214 |
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60513392 |
Oct 22, 2003 |
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Current U.S.
Class: |
424/488 ;
424/549; 424/602; 424/85.1; 424/93.7 |
Current CPC
Class: |
A61L 26/0057 20130101;
A61P 31/12 20180101; A61K 38/014 20130101; A61L 27/3645 20130101;
A61L 27/26 20130101; A61P 19/04 20180101; A61L 27/3608 20130101;
A61P 29/00 20180101; A61K 38/1875 20130101; A61L 2430/10 20130101;
A61P 37/06 20180101; A61P 7/02 20180101; A61L 27/3834 20130101;
A61K 35/32 20130101; A61P 19/02 20180101; A61L 27/52 20130101; A61P
31/04 20180101; A61L 27/48 20130101; A61L 26/0052 20130101; A61L
26/008 20130101; A61L 26/0052 20130101; C08L 5/02 20130101; A61L
27/26 20130101; C08L 5/02 20130101; A61K 35/32 20130101; A61K
2300/00 20130101; A61K 38/014 20130101; A61K 2300/00 20130101; A61K
38/1875 20130101; A61K 2300/00 20130101; A61L 27/26 20130101; C08L
89/06 20130101; A61L 27/48 20130101; C08L 5/02 20130101; A61L
26/0052 20130101; C08L 89/06 20130101 |
Class at
Publication: |
424/488 ;
424/602; 424/85.1; 424/549; 424/93.7 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 35/12 20060101 A61K035/12; A61K 35/32 20060101
A61K035/32; A61P 19/04 20060101 A61P019/04; A61K 33/42 20060101
A61K033/42; A61K 38/19 20060101 A61K038/19 |
Claims
1. A connective tissue regenerative composition comprising: a
bioactive hydrogel matrix comprising a polypeptide and a long chain
carbohydrate, the bioactive hydrogel matrix being in a dehydrated
form; and an osteoinductive or osteoconductive material comprising
hydroxyapatite.
2. The connective tissue regenerative composition of claim 1,
wherein the osteoinductive or osteoconductive material is dispersed
within the bioactive hydrogel matrix.
3. The connective tissue regenerative composition of claim 1,
wherein the bioactive hydrogel matrix is in particulate form.
4. The connective tissue regenerative composition of claim 3,
wherein the composition comprises a mixture of particles of the
bioactive hydrogel matrix and particles of the osteoinductive or
osteoconductive material.
5. The connective tissue regenerative composition of claim 1,
wherein the osteoinductive or osteoconductive material is present
at a concentration of about 0.01 volume percent to about 90 volume
percent, based upon the total volume of the composition.
6. The connective tissue regenerative composition of claim 1,
wherein at least a portion of the bioactive hydrogel matrix is in
crosslinked form.
7. The connective tissue regenerative composition of claim 6,
wherein the bioactive hydrogel matrix comprises gelatin crosslinked
to oxidized dextran.
8. The connective tissue regenerative composition of claim 6,
wherein the dehydrated form is a freeze-dried form.
9. The connective tissue regenerative composition of claim 6,
wherein the bioactive hydrogel matrix has been dehydrated using a
method that maintains the connective tissue regenerative properties
of the bioactive hydrogel matrix, said method comprising a first
step wherein the hydrogel matrix is frozen at a temperature below
the eutectic point of the hydrogel.
10. The connective tissue regenerative composition of claim 9,
wherein the first step in the dehydration method is carried out at
a temperature of less than or equal to -30.degree. C.
11. The connective tissue regenerative composition of claim 10,
wherein the dehydrating method further comprises a second, primary
drying step that is carried out at a temperature greater than used
in the first step
12. The connective tissue regenerative composition of claim 1,
wherein the bioactive hydrogel matrix comprises at least one
additional osteoinductive or osteoconductive material selected from
the group consisting of demineralized bone matrix (DBM), bone
morphogenetic proteins (BMPs), transforming growth factors (TGFs),
fibroblast growth factors (FGFs), insulin-like growth factors
(IGFs), platelet-derived growth factors (PDGFs), epidermal growth
factors (EGFs), vascular endothelial growth factors (VEGFs), and
vascular permeability factors (VPFs).
13. The connective tissue regenerative composition of claim 1,
wherein the polypeptide derived from tissue selected from the group
consisting of collagens, gelatins, keratin, decorin, aggrecan, and
glycoproteins.
14. The connective tissue regenerative composition of claim 1,
wherein the long chain carbohydrate is a polysaccharide or a
sulfated polysaccharide.
15. The connective tissue regenerative composition of claim 14,
wherein the long chain carbohydrate is a glycosaminoglycans or
glucosaminoglycans.
16. The connective tissue regenerative composition of claim 14,
wherein the long chain carbohydrate is selected from the group
consisting of dextran, dextrin, heparan, heparin, hyaluronic acid,
chondroitin, alginate, agarose, carageenan, amylopectin, amylose,
glycogen, starch, cellulose, chitin, chitosan, heparan sulfate,
chondroitin sulfate, dextran sulfate, dermatan sulfate, keratan
sulfate, and combinations thereof.
17. The connective tissue regenerative composition of claim 1,
wherein the bioactive hydrogel matrix further comprises one or more
components selected from the group consisting of polar amino acids,
polar amino acid analogs or derivatives, divalent cation chelators,
and combination thereof.
18. The connective tissue regenerative composition of claim 17,
wherein the bioactive hydrogel matrix comprises one or more polar
amino acids selected from the group consisting of tyrosine,
cysteine, serine, threonine, asparagine, glutamine, aspartic acid,
glutamic acid, arginine, lysine, histidine, and mixtures
thereof.
19. The connective tissue regenerative composition of claim 17,
wherein the bioactive hydrogel matrix comprises
ethylenediaminetetraacetic acid or a salt thereof.
20. The connective tissue regenerative composition of claim 1,
wherein the polypeptide is gelatin and the long chain carbohydrate
is dextran.
21. The connective tissue regenerative composition of claim 20,
wherein the gelatin has a molecular mass of about 80,000 to about
200,000 Da and the polydispersity of the molecular mass of the
gelatin is 1 to about 3.
22. The connective tissue regenerative composition of claim 20,
wherein the dextran has a molecular mass of about 200,000 to about
800,000 Da and the polydispersity of the molecular mass of the
dextran is about 1 to about 3.
23. A connective tissue regenerative composition comprising: a
bioactive hydrogel matrix comprising a polypeptide and a long chain
carbohydrate; and an osteoinductive or osteoconductive material
comprising hydroxyapatite; wherein the composition is in a cast
form adapted for integration into a bone defect.
24. A method of treating a bone defect, comprising: (i) hydrating a
dehydrated connective tissue regenerative composition, the
dehydrated composition comprising: a dehydrated bioactive hydrogel
matrix comprising a polypeptide and a long chain carbohydrate; and
an osteoinductive or osteoconductive material comprising
hydroxyapatite; and (ii) administering the hydrated connective
tissue regenerative composition to the site of the bone defect.
25. The method of claim 24, wherein the bioactive hydrogel matrix
is in particulate form and the osteoinductive or osteoconductive
material is dispersed within the bioactive hydrogel matrix.
26. The method of claim 24, wherein at least a portion of the
bioactive hydrogel matrix is in crosslinked form.
27. The method of claim 24, wherein the bioactive hydrogel matrix
comprises gelatin crosslinked to oxidized dextran.
28. The method of claim 24, wherein the dehydrated form is a
freeze-dried form.
29. The method of claim 24, wherein the bioactive hydrogel matrix
has been dehydrated using a method that maintains the connective
tissue regenerative properties of the bioactive hydrogel matrix,
said method comprising a first step wherein the hydrogel matrix is
frozen at a temperature below the eutectic point of the
hydrogel.
30. The method of claim 29, wherein the first step in the
dehydration method is carried out at a temperature of less than or
equal to -30.degree. C.
31. The method of claim 30, wherein the dehydrating method further
comprises a second, primary drying step that is carried out at a
temperature greater than used in the first step
32. The method of claim 24, wherein the polypeptide derived from
tissue selected from the group consisting of collagens, gelatins,
keratin, decorin, aggrecan, and glycoproteins.
33. The method of claim 24, wherein the long chain carbohydrate is
a polysaccharide or a sulfated polysaccharide.
34. The method of claim 33, wherein the polysaccharide is selected
from the group consisting of dextran, dextrin, heparan, heparin,
hyaluronic acid, chondroitin, alginate, agarose, carageenan,
amylopectin, amylose, glycogen, starch, cellulose, chitin, and
chitosan.
35. The method of claim 24, wherein the bioactive hydrogel matrix
further comprises one or more components selected from the group
consisting of polar amino acids, polar amino acid analogs or
derivatives, divalent cation chelators, and combination
thereof.
36. The method of claim 35, wherein the bioactive hydrogel matrix
comprises one or more polar amino acids selected from the group
consisting of tyrosine, cysteine, serine, threonine, asparagine,
glutamine, aspartic acid, glutamic acid, arginine, lysine,
histidine, and mixtures thereof.
37. The method of claim 35, wherein the bioactive hydrogel matrix
comprises ethylenediaminetetraacetic acid or a salt thereof.
38. The method of claim 24, wherein the composition further
comprises cells selected from the group consisting of stem cells,
progenitor cells, and mixtures thereof.
39. The method of claim 24, wherein the polypeptide is gelatin and
the long chain carbohydrate is dextran.
40. The method of claim 24, wherein said hydrating comprises
contacting the dehydrated connective tissue regenerative
composition with water.
41. The method of claim 24, wherein said hydrating comprises
contacting the dehydrated connective tissue regenerative
composition with bone marrow aspirate.
42. The method of claim 24, wherein, prior to said administering,
the method further comprises molding the hydrated connective tissue
regenerative composition to a putty form.
43. A method of preparing a connective tissue regenerative
composition, comprising: dehydrating a bioactive hydrogel matrix
comprising a polypeptide and a long chain carbohydrate; and
dispersing an osteoinductive or osteoconductive material comprising
hydroxyapatite within the dehydrated bioactive hydrogel matrix.
44. The method of claim 43, wherein said dehydrating step comprises
a method that maintains connective tissue regenerative properties
of the bioactive hydrogel matrix, said method comprising a first
step wherein the hydrogel matrix is frozen at a temperature below
the eutectic point of the hydrogel.
45. The method of claim 44, wherein the first step in the
dehydration method is carried out at a temperature of less than or
equal to -30.degree. C.
46. The method of claim 45, wherein the dehydrating method further
comprises a second, primary drying step that is carried out at a
temperature greater than used in the first step.
47. The method of claim 43, comprising particularizing the
dehydrated bioactive hydrogel matrix.
48. A method of preparing a connective tissue regenerative
composition, comprising: dispersing an osteoinductive or
osteoconductive material comprising hydroxyapatite within a
bioactive hydrogel matrix comprising a polypeptide and a long chain
carbohydrate; and dehydrating the bioactive hydrogel matrix with
the osteoinductive or osteoconductive material dispersed therein.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S. patent
application Ser. No. 12/039,214, filed Feb. 28, 2008, which is a
continuation of U.S. patent application Ser. No. 10/971,544, filed
Oct. 22, 2004, which claims the benefit of U.S. Provisional Patent
Application Ser. No. 60/513,392, filed Oct. 22, 2003, all of which
are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention is related to methods and compositions
for regenerating connective tissue, such as bone, cartilage,
ligament, tendon, and the like. In particular, the invention is
related to methods for regenerating connective tissue through
application of a hydrogel matrix, wherein the matrix is comprised
of a polypeptide, such as gelatin, and a long chain carbohydrate,
such as dextran.
BACKGROUND OF THE INVENTION
[0003] Injuries to bone, such as partial or complete fracture, can
be slow to heal, but such injuries generally heal on their own
accord with external immobilization as needed, such as by applying
a cast to the affected area. In more severe cases, more aggressive
internal immobilization, such as permanently reconnecting the
fractured bone with screws and/or metal plates, may be required.
Regeneration of bone tissue over the relatively short distances
generally present in bone fracture readily occurs in most healthy
patients. Bone injuries beyond simple fractures, however, present
greater challenges in treatment. Long segmental diaphyseal bone
loss, for example, can result from multiple causes including
high-energy trauma, such as blast injury, disease, such as
osteomyelitis or osteonecrosis, or wide excision of malignant
conditions, such as osteosarcoma. Such conditions often result in
cavitation of the bone or complete loss of bone tissue across an
extended length of the bone (i.e., a critical bone defect). Bone
regeneration in these cases becomes increasingly challenging and
sometimes impossible.
[0004] Many techniques have been used in an attempt to enhance bone
growth. Most commonly, an attempt is made to replace the lost bone.
Examples of such techniques include autologous vascularized bone
grafts, massive allograft (generally from cadaver), and use of
reabsorbable and non-reabsorbable artificial bone. Another method
for promoting bone regeneration is through the introduction of
osteoinductive bioactive factors, such as bone morphogenetic
proteins (BMPs), platelet rich plasma (PRP), synthetic peptides,
such as P-15 (Pepgen P-15.TM., Dentsply International, York, Pa.),
and bone marrow aspirates. Such bioactive factors can be introduced
into the area of bone loss through various vehicles. Mechanical
methods, such as distraction osteogenesis, are also employed for
promoting bone regeneration. Distraction osteogenesis is a process
involving gradual, controlled displacement of surgically created
fractures resulting in simultaneous expansion of soft tissue and
bone volume.
[0005] A somewhat less invasive technique that is used most
commonly for regenerating bone around teeth is known as "guided
bone regeneration." As the tissue surrounding a bone almost always
heals faster than the bone itself, the faster-healing tissue often
expands into and fills the space where the bone is missing,
hindering the bone regeneration. In guided bone regeneration, a
biocompatible membrane is placed between the tissue and the bone
acting as a barrier, which prevents growth of the tissue into the
bone. Often, a bone graft is inserted under the barrier. The
membranes are typically designed to dissolve away after several
weeks.
[0006] A variation on this procedure is known as "protected bone
regeneration" and is based on the theory that three prerequisites
for bone healing are required: 1) adequate blood supply, 2)
abundant bone forming cells, and 3) protected healing space. See,
Holmes, R. E., Lemperle, S. M., and Calhoun, C. J., "Protected Bone
Regeneration," Scientific Data Series in Resorbable Fixation,
distributed by Medtronic Sofamor Danek, available on-line at
http://www.macropore.com/pdf/Protected_Bone.pdf. Adequate blood
supply is a known requirement for bone regeneration as it supplies
the necessary oxygen and nutrients, as well as mesenchymal stem
cells (the bone forming cells). As described above, the healing
space of the bone must also be protected from the ingrowth of
surrounding tissue. According to the above-noted publication, all
of the stated prerequisites can be met through the use of a
reabsorbable polymer protective sheet offering a physiologically
balanced porosity for positive cellular exchange and the
opportunity for vascular infiltration, while preventing
interposition of adjacent soft tissues.
[0007] While there are several methods currently known, treatment
of injury resulting in major bone loss remains a difficult clinical
problem. Furthermore, approximately 10% of all long bone fractures
are non-union fractures that do not heal spontaneously. Thus, there
remains a need for methods for bone regeneration that are effective
at promoting bone tissue growth and that are as non-invasive as
possible.
SUMMARY OF THE INVENTION
[0008] It has been discovered that the matrix described herein is
capable of successfully promoting regeneration of connective
tissue. Surprisingly, the matrix is even useful for effecting bone
regeneration in bone with defects that will not normally
spontaneously heal. The present invention provides a method for
connective tissue regeneration comprising administration of a
bioactive hydrogel matrix into the site in need of connective
tissue regeneration. As used herein, "bioactive" is intended to
indicate the ability to facilitate a cellular or tissue response,
such as, induction of vasculogenesis, promotion of cellular
attachment to a scaffold material, and promotion of tissue
regeneration.
[0009] In one aspect of the invention, there is provided a method
for regenerating connective tissue. In one embodiment, the method
comprises administering to a site in need of connective tissue
regeneration a bioactive hydrogel matrix comprising a polypeptide
and a long chain carbohydrate. The polypeptide can be selected from
tissue-derived polypeptides or synthetic polypeptides. In one
embodiment, the polypeptide is skin-derived gelatin. In another
embodiment, the polypeptide is bone-derived gelatin. Exemplary long
chain carbohydrates include polysaccharides and sulfated
polysaccharides. In one embodiment, the long chain carbohydrate is
dextran. The bioactive hydrogel matrix further comprises one or
more components selected from the group consisting of polar amino
acids, polar amino acid analogues or derivatives and divalent
cation chelators, such as ethylenediaminetetraacetic acid (EDTA) or
salts thereof.
[0010] The bioactive hydrogel matrix, as used in the above method,
can further include one or more various structuring agents,
medicaments, or other agents useful for facilitating or mediating
connective tissue regeneration.
[0011] In one embodiment of the invention, the bioactive hydrogel
matrix can further comprise at least one osteoinductive or
osteoconductive material. In this embodiment of the invention, the
method is particularly useful for regenerating bone; moreover, the
use of osteoinductive or osteoconductive materials is not limited
to bone regeneration.
[0012] In yet another embodiment of the invention, the bioactive
hydrogel matrix further comprises at least one medicament. Any
medicament recognizable by one of skill in the art as useful in the
treatment of connective tissue injury, particularly in methods of
regenerating connective tissue, could be used. For example, the
medicaments can include antivirals, antibacterials,
anti-inflammatories, immunosuppresants, analgesics, anticoagulants,
or various wound healing promotion agents.
[0013] In one particular embodiment of the invention, the bioactive
hydrogel matrix further comprises stem or progenitor cells, such as
adipose-derived adult stem (ADAS) cells or mesenchymal stem cells.
Such cells are known in the art as useful in various therapies due
to their ability to differentiate into a number of cell types. ADAS
cells in particular are known to differentiate into cell types
including chondrocytes and osteoblasts.
[0014] In still another embodiment of the method of the invention,
the bioactive hydrogel matrix is at least partially contained
within a three-dimensional structural framework. Accordingly, the
structural framework can be included with the bioactive hydrogel
matrix prior to administration of the bioactive hydrogel matrix to
the site in need of connective tissue regeneration. Alternatively,
the structural framework can be formed around the site in need of
connective tissue regeneration at the time of administration of the
bioactive hydrogel matrix (i.e., formed shortly before or shortly
after administration of the bioactive hydrogel matrix). The
three-dimensional structural framework, therefore, includes any
material capable of providing load-bearing structural support or
anatomical space for cellular infiltration and includes, for
example, a metal cage, a sintered ceramic framework, a collagen
sponge, or allogenic or autologous bone. The structural framework
can further include three dimensional structures prepared from
polymeric materials, including biopolymers.
[0015] The bioactive hydrogel matrix can also be used in the method
of the invention in a dehydrated form. In such form, the bioactive
hydrogel matrix retains its beneficial properties yet can be stored
and transported in a solid form, being capable of re-hydration for
use in the method of the present invention. In one embodiment, the
bioactive hydrogel matrix is administered in a dehydrated form such
that body fluids re-hydrate the bioactive hydrogel matrix. In
another embodiment, the bioactive hydrogel matrix is in dehydrated
form and the method further comprises re-hydrating the bioactive
hydrogel matrix with a re-hydrating fluid prior to administering
the bioactive hydrogel matrix to the site in need of connective
tissue regeneration. In dehydrated form, the bioactive hydrogel
matrix can be shaped or processed into a variety of shapes and
forms. For example, the dehydrated bioactive hydrogel matrix can be
in a unitary piece capable of being shaped to precisely fit the
site in need of connective tissue regeneration. Alternately, the
dehydrated bioactive hydrogel matrix can be in particulate form.
The particulate dehydrated bioactive hydrogel matrix could be mixed
into a solution containing other beneficial ingredients, such as
stem or progenitor cells or medicaments, combined with
osteoinductive or osteoconductive materials to form a putty or
paste-like material for placement into the site in need of
connective tissue regeneration, or used in other preparations that
would be useful in the method of the invention.
[0016] In other embodiments of the invention, it may be useful for
the bioactive hydrogel matrix to have additional structure or
strength in the absence of additives. Accordingly, the present
invention further encompasses embodiments wherein the bioactive
hydrogel matrix is in crosslinked form, the long chain carbohydrate
being covalently crosslinked to the polypeptide. In such
embodiments, the bioactive hydrogel matrix can be used alone in the
method of the invention or may be used in conjunction with other
components as described herein.
[0017] In one embodiment of the invention, the bioactive hydrogel
matrix is inserted into an area of a bone in need of repair or
regeneration (i.e., a bone defect). The amount of the bioactive
hydrogel matrix used in the bone can vary depending upon the size
of the bone defect, the form of the bioactive hydrogel matrix, and
the presence or absence of additives as described herein.
Typically, the total amount of the bioactive hydrogel matrix used
is the amount required to fill the area of bone loss.
[0018] According to another embodiment of the present invention,
the hydrogel matrix can be used for repair of soft tissue either
separately or in conjunction with regeneration of nearby hard
tissue, such as bone. According to this embodiment, the bioactive
hydrogel matrix is administered around and/or injected into the
soft tissue.
[0019] According to another embodiment of the present invention,
the hydrogel matrix can be used for repair and/or regeneration of
non-bone connective tissue. According to this embodiment, the
bioactive hydrogel matrix is administered to an area having loss
of, or damage to, connective tissue, which includes tissue arising
from fibroblasts, such as tendon and ligament, or chondrocytes,
such as cartilage.
[0020] According to another aspect of the present invention, there
are provided various connective tissue regenerative compositions.
The compositions are particularly useful in the regeneration of
connective tissue or for treatment of patients having various
connective tissue degenerative diseases. Accordingly, the
compositions described herein are particularly useful in the
methods of the invention also described herein.
[0021] In one embodiment of this aspect of the invention, the
connective tissue regenerative composition comprises a
three-dimensional structural framework and a bioactive hydrogel
matrix at least partially contained within the three-dimensional
structural framework, wherein the bioactive hydrogel matrix
comprises a polypeptide and a long chain carbohydrate. The
bioactive hydrogel matrix preferably further comprises one or more
components selected from the group consisting of polar amino acids,
polar amino acid analogues or derivatives, and divalent cation
chelators, such as EDTA or salts thereof. In one particular
embodiment, the three-dimensional structural framework includes a
crosslinked hydrogel matrix. In another preferred embodiment, the
three-dimensional structural framework includes a collage
sponge.
[0022] In another embodiment, the connective tissue regenerative
composition comprises at least one osteoinductive or
osteoconductive material and a bioactive hydrogel matrix comprising
a polypeptide and a long chain carbohydrate. The osteoinductive or
osteoconductive material can be dispersed within the bioactive
hydrogel matrix. In one preferred embodiment, the osteoinductive or
osteoconductive material and the bioactive hydrogel matrix can be
in admixture. The bioactive hydrogel matrix can be in a hydrated
form or can be in a dehydrated form.
[0023] In still another embodiment of the invention, the connective
tissue regenerative composition comprises stem or progenitor cells
and a bioactive hydrogel matrix comprising a polypeptide and a long
chain carbohydrate. Again, the bioactive hydrogel matrix can be in
a hydrated form or can be in a dehydrated form.
[0024] According to another aspect of the present invention, the
bioactive hydrogel matrix can be used for attaching or reattaching
two or more connective tissues. In one embodiment of this aspect of
the invention, the method comprises: coating at least a portion of
at least one of a first and second connective tissue with a
bioactive hydrogel matrix comprising a polypeptide and a long chain
carbohydrate; contacting the first connective tissue to the second
connective tissue at a point of attachment; and attaching the first
connective tissue to the second connective tissue using sutures,
staples or other appropriate means. Such a method is particularly
useful for attaching connective tissue, such as tendon or ligament,
to bone. The method is further useful for attaching soft connective
tissue to other soft connective tissue, such as tendon to tendon or
ligament to ligament.
[0025] According to another aspect of the invention, the bioactive
hydrogel matrix is used in a method for treating degenerative
diseases of the natural joint of a patient in need of treatment
thereof. In one embodiment, the method comprises: applying to a
joint affected by a degenerative disease, a bioactive hydrogel
matrix comprising a polypeptide and a long chain carbohydrate.
Further, optionally, the bioactive hydrogel matrix can include stem
or progenitor cells. Preferentially, the administering step
comprises injecting the bioactive hydrogel matrix into the affected
joint. The method is particularly useful for halting progression of
or reversing degenerative joint diseases, such as
osteoarthritis.
[0026] The compositions and methods of the present invention are
particularly useful for repairing connective tissue of the knee,
such as the anterior cruciate ligament, the posterior cruciate
ligament, the patellar tendon, the quadriceps tendon, and the
anterior meniscofemoral ligament.
[0027] The compositions and methods of the invention are further
useful for treating a patient having an artificial joint. In
particular, the connective tissue regenerative compositions can be
administered around the site of the artificial joint, either during
placement of the artificial joint or post-surgery, to facilitate
integration of the artificial joint into the surrounding
tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Having thus described the invention in general terms,
reference will now be made to the accompanying drawings,
wherein:
[0029] FIG. 1 illustrates formation of open alpha chains derived
from collagen monomers;
[0030] FIGS. 2A and 2B illustrate the effect of the association of
the collagen-derived alpha chains with dextran;
[0031] FIG. 3 illustrates the effect of other additives used in the
bioactive hydrogel matrix of the invention;
[0032] FIG. 4 graphically illustrates cellular aggregation across
various cell types in the presence of the bioactive hydrogel matrix
of the present invention;
[0033] FIG. 5 illustrates the effect of the bioactive hydrogel
matrix of the present invention on the expression of the BMP-2 gene
as compared to expression in cells in serum free medium (SFM);
[0034] FIG. 6 illustrates the increased expression of connective
tissue growth factor (CTGF) messenger RNA in chondrosarcoma cells
treated with the bioactive hydrogel matrix of the invention as
compared to cells in SFM;
[0035] FIG. 7 illustrates the expression of aggrecan messenger RNA
in chondrosarcoma cells treated with the bioactive hydrogel matrix
of the invention compared to cells in SFM;
[0036] FIG. 8 illustrates a crosslinked bioactive hydrogel matrix
of the invention comprising dextran and gelatin;
[0037] FIG. 9 illustrates the effect of the bioactive hydrogel
matrix of the present invention on the production of BMP-2 protein
as compared to production in cells in serum containing medium
(SCM); and
[0038] FIG. 10 illustrates the effect of the crosslinked bioactive
hydrogel matrix of the present invention on the expression of the
BMP-2 gene.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention now will be described more fully
hereinafter with reference to the accompanying drawings, in which
preferred embodiments of the invention are shown. This invention
may, however, be embodied in many different forms and should not be
construed as limited to the embodiments set forth herein; rather,
these embodiments are provided so that this disclosure will be
thorough and complete, and will fully convey the scope of the
invention to those skilled in the art.
[0040] The formulation of a thermoreversible hydrogel matrix
providing a cell culture medium and composition for preserving cell
viability is taught by U.S. Pat. No. 6,231,881, herein incorporated
by reference in its entirety. Additionally, a hydrogel matrix
useful in promoting vascularization is provided in U.S. Pat. No.
6,261,587, herein incorporated by reference in its entirety. The
thermoreversible hydro gel matrix taught by these references is a
gel at storage temperatures and molten at physiologic temperatures,
and comprises a combination of a collagen-derived component, such
as gelatin, a long chain carbohydrate, such as dextran, and
effective amounts of other components, such as polar amino
acids.
[0041] The present invention provides connective tissue
regenerative compositions and methods of regenerating connective
tissue at a site in need of connective tissue regeneration. The
compositions and method of the invention include a bioactive
hydrogel matrix generally comprising a polypeptide and a long chain
carbohydrate.
[0042] A polypeptide, as used herein, is intended to encompass any
tissue-derived or synthetically produced polypeptide, such as
collagens or collagen-derived gelatins. Although collagen-derived
gelatin is the preferred polypeptide component, other gelatin-like
components characterized by a backbone comprised of sequences of
amino acids having polar groups that are capable of interacting
with other molecules can be used. For example, keratin, decorin,
aggrecan, glycoproteins (including proteoglycans), and the like
could be used to provide the polypeptide component. In one
embodiment, the polypeptide component is porcine gelatin from
partially hydrolyzed collagen derived from skin tissue.
Polypeptides derived from other types of tissue could also be used.
Examples include, but are not limited to, tissue extracts from
arteries, vocal chords, pleura, trachea, bronchi, pulmonary
alveolar septa, ligaments, auricular cartilage or abdominal fascia;
the reticular network of the liver; the basement membrane of the
kidney; or the neurilemma, arachnoid, dura mater or pia mater of
the nervous system. Purified polypeptides including, but not
limited to, laminin, nidogen, fibulin, and fibrillin or protein
mixtures such as those described by U.S. Pat. No. 6,264,992 and
U.S. Pat. No. 4,829,000, extracts from cell culture broth as
described by U.S. Pat. No. 6,284,284, submucosal tissues such as
those described in U.S. Pat. No. 6,264,992, or gene products such
as described by U.S. Pat. No. 6,303,765 may also be used. Another
example of a suitable polypeptide is a fusion protein formed by
genetically engineering a known reactive species onto a
protein.
[0043] The polypeptide component preferably has a molecular mass
range of about 3,000 to about 3,000,000 Da, more preferably about
30,000 to about 300,000 Da, most preferably about 50,000 to about
250,000 Da. Molecular mass can be expressed as a weight average
molecular mass (M.sub.w) or a number average molecular mass
(M.sub.n). Both expressions are based upon the characterization of
macromolecular solute containing solution as having an average
number of molecules (n.sub.i) and a molar mass for each molecule
(M.sub.i). Accordingly, number average molecular mass is defined by
formula 1 below.
M n = n i M i n i ( 1 ) ##EQU00001##
Weight average molecular mass (also known as molecular mass
average) is directly measurable using light scattering methods and
is defined by formula 2 below.
M w = n i M i 2 n i M i ( 2 ) ##EQU00002##
Molecular mass can also be expressed as a Z-average molar mass
(M.sub.z), wherein the calculation places greater emphasis on
molecules with large molar masses. Z-average molar mass is defined
by formula 3 below.
M z = n i M i 3 n i M i 2 ( 3 ) ##EQU00003##
Unless otherwise noted, molecular mass is expressed herein as
weight average molecular mass.
[0044] In addition to molecular mass, polymer solutions can also be
physically described in terms of polydispersity, which represents
the broadness of the molecular mass distribution within the
solution, such distribution being the range of different molecular
masses of the individual polymer molecules in the solution.
Polydispersity is the ratio of the number average molecular mass to
the weight average molecular mass, which is defined by formula 4
below.
Polydispersity = M w M n ( 4 ) ##EQU00004##
[0045] If polydispersity is equal to 1 (i.e., M.sub.n equals
M.sub.w), the polymer is said to be monodisperse. A truly
monodisperse polymer is one where all polymer molecules within the
solution are of a single, identical molecular mass. As M.sub.n
changes with M.sub.w, the polydispersity changes, always being
greater than 1. The polydispersity of a given polymer solution can
affect the physical characteristics of the polymer, and, therefore,
the interaction of the polymer with another polymer. Research has
shown that in aqueous mixtures of biopolymers (including gelatin
and dextran), an increase in molecular weight results in a less
compatible system with a higher phase separation temperature,
whereas a decrease in concentration results in a more compatible
system with a lower phase separation temperature (see E. H. A. de
Hoog and R. H. Tromp, On the phase separation kinetics of an
aqueous biopolymer mixture in the presence of gelation: the effect
of the quench depth and the effect of the molar mass, Colloids and
Surfaces A: Physicochemical and Engineering Aspects, 213 (2-3),
Pages 221-234). Preferably, the polypeptide used according to the
present invention has a polydispersity close to 1. In one preferred
embodiment, the polypeptide has a polydispersity of 1 to about 4,
more preferably, about 1 to about 3, most preferably about 1.1 to
about 2.4.
[0046] The polypeptide used in the bioactive hydrogel matrix of the
invention is preferably a gelatin, such as collagen derived
gelatin.
[0047] Collagen is a major protein component of the extracellular
matrix of animals. Early in fetal development, a more open form of
collagen (compared to tightly bound mature collagen) is associated
with large carbohydrate molecules, and serves as the predominant
tissue scaffolding. It is believed that attachment of
differentiated or incompletely differentiated cells of mesenchymal
origin to this polar, proteoglycan-like, collagen scaffolding
results in a specific host tissue response. This response is to
guide the differentiation of mesenchymal tissue.
[0048] Collagen is assembled into a complex fibrillar organization.
The fibrils are assembled into bundles that form the fibers. The
fibrils are made of five microfibrils placed in a staggered
arrangement. Each microfibril is a collection of collagen rods.
Each collagen rod is a right-handed triple-helix, each strand being
itself a left-handed helix. Collagen fibrils are strengthened by
covalent intra- and intermolecular cross-links which make the
tissues of mature animals insoluble in cold water. When suitable
treatments are used, collagen rods are extracted and solubilized
where they keep their conformation as triple-helices. This is
denatured collagen and differs from the native form of collagen,
but has not undergone sufficient thermal or chemical treatment to
break the intramolecular stabilizing covalent bonds found in
collagen. When collagen solutions are extensively heated, or when
the native collagen containing tissues are subjected to chemical
and thermal treatments, the hydrogen and covalent bonds that
stabilize the collagen helices are broken, and the molecules adopt
a disordered conformation. By breaking these hydrogen bonds, the
polar amine and carboxylic acid groups are now available for
binding to polar groups from other sources or themselves. This
material is gelatin and is water-soluble at 40-45.degree. C.
[0049] As noted above, gelatin is a form of denatured collagen, and
is obtained by the partial hydrolysis of collagen derived from the
skin, white connective tissue, or bones of animals. Gelatin may be
derived from an acid-treated precursor or an alkali-treated
precursor. Gelatin derived from an acid-treated precursor is known
as Type A, and gelatin derived from an alkali-treated precursor is
known as Type B. The macromolecular structural changes associated
with collagen degradation are basically the same for chemical and
partial thermal hydrolysis. In the case of thermal and
acid-catalyzed degradation, hydrolytic cleavage predominates within
individual collagen chains. In alkaline hydrolysis, cleavage of
inter- and intramolecular cross-links predominates.
[0050] Preferably, the gelatin used in the present invention is
skin-derived gelatin or bone derived gelatin. In one preferred
embodiment, the gelatin has a molecular mass of about 80,000 Da to
about 200,000 Da. Further, it is preferred that the gelatin have a
polydispersity of 1 to about 3. In one preferred embodiment, the
gelatin has a polydispersity of about 1.1 to about 2.4.
[0051] The polypeptide, such as gelatin, is preferentially present
at a concentration of about 0.01 to about 40 mM, preferably about
0.05 to about 30 mM, most preferably about 0.25 to about 5 mM.
Advantageously, the gelatin concentration is approximately 0.75 mM.
The above concentrations provide a non-flowable phase at storage
temperature (below about 33.degree. C.) and a flowable phase at
treatment temperature (about 35 to about 40.degree. C.).
[0052] The bioactive hydrogel matrix of the present invention also
comprises a long chain carbohydrate. The phrase long chain
carbohydrate is generally intended to encompass any polysaccharide
or sulfated polysaccharide consisting of more than about 10
monosaccharide residues joined to each other by glycosidic
linkages. The phrase is also intended to encompass other long chain
carbohydrates, including heterosaccharides, and specific classes of
carbohydrates, such as starches, sugars, celluloses, and gums. The
long chain carbohydrate may consist of the same monosaccharide
residues, or various monosaccharide residues or derivatives of
monosaccharide residues. Dextran, a preferred polysaccharide,
solely comprises glucose residues.
[0053] Any polysaccharide, including glycosaminoglycans (GAGs) or
glucosaminoglycans, with suitable viscosity, molecular mass and
other desirable properties may be utilized in the present
invention. By glycosaminoglycan is intended any glycan (i.e.,
polysaccharide) comprising an unbranched polysaccharide chain with
a repeating disaccharide unit, one of which is always an amino
sugar. These compounds as a class carry a high negative charge, are
strongly hydrophilic, and are commonly called mucopolysaccharides.
This group of polysaccharides includes heparin, heparan sulfate,
chondroitin sulfate, dermatan sulfate, keratan sulfate, and
hyaluronic acid. These GAGs are predominantly found on cell
surfaces and in the extracellular matrix. By glucosaminoglycan is
intended any glycan (i.e. polysaccharide) containing predominantly
monosaccharide derivatives in which an alcoholic hydroxyl group has
been replaced by an amino group or other functional group such as
sulfate or phosphate. An example of a glucosaminoglycan is
poly-N-acetyl glucosaminoglycan, commonly referred to as chitosan.
Exemplary polysaccharides that may be useful in the present
invention include dextran, heparan, heparin, hyaluronic acid,
alginate, agarose, carageenan, amylopectin, amylose, glycogen,
starch, cellulose, chitin, chitosan and various sulfated
polysaccharides such as heparan sulfate, chondroitin sulfate,
dextran sulfate, dermatan sulfate, or keratan sulfate.
[0054] The long chain carbohydrate preferably has a molecular mass
of about 2,000 to about 8,000,000 Da, more preferably about 20,000
to about 1,000,000 Da, most preferably about 200,000 to about
800,000 Da. In one embodiment, the long chain carbohydrate has a
molecular mass of approximately 500,000 Da.
[0055] Preferably, the long chain carbohydrate used according to
the present invention has a polydispersity close to 1. In one
preferred embodiment, the polypeptide has a polydispersity of 1 to
about 3, more preferably, about 1.1 to about 2.4.
[0056] As previously noted, one preferred long chain carbohydrate
for use in the present invention is dextran. Dextran typically
comprises linear chains of .alpha.(1.fwdarw.6)-linked D-glucose
residues, often with .alpha.(1.fwdarw.2)- or
.alpha.(1.fwdarw.3)-branches. Native dextran, produced by a number
of species of bacteria of the family Lactobacilliaceae, is a
polydisperse mixture of components. Dextrans have been widely used
as plasma substitutes and blood extenders, are considered fully
biocompatible, and are metabolizable. Dextrans are available in a
wide range of average molecular masses, varying from about 4,000 to
about 40,000,000 Da. Preferably, the dextran used in the invention
has a molecular mass of about 200,000 to about 800,000 Da, most
preferably about 300,000 to about 600,000 Da. In one preferred
embodiment, the dextran has a molecular mass of approximately
500,000 Da. Dextrans have varying rates of resorption in vivo from
about two to about 20 days depending on their molecular mass.
[0057] The long chain carbohydrate, such as dextran, is
preferentially present at a concentration of about 0.01 to about 10
mM, preferably about 0.01 to about 1 mM, most preferably about 0.01
to about 0.5 mM. In one embodiment, dextran is present at a
concentration of about 0.1 mM.
[0058] While native dextran is generally used in the present
invention, the use of dextran derivatives, such as dextran sulfate
and dextran phosphate is also within the scope of the invention. In
one embodiment, the derivatives are free radical polymerizable,
preferably photopolymerizable derivatives, such as acrylates.
According to this embodiment, the composition can be injected as a
viscous liquid and polymerized in situ to form a solid material.
The dextran can also be selected to degrade at a rate which
approximates ingrowth of new bone or tissue. Those compositions
that include free radical polymerizable groups may also include
polymerization initiators, such as photoinitiators, such as benzoin
ethers, and thermally activatable initiators, such as
azobisisobutyronitrile (AIBN) and di-t-butyl ether. Free radical
polymerization initiators, and conditions for carrying out free
radical polymerizations, are well known to those of skill in the
art, and any of such methods are encompassed by the present
invention.
[0059] In a preferred embodiment, gelatin and dextran are
components of the bioactive hydrogel matrix of the present
invention. For ease of describing the invention, the terms
"gelatin" and "dextran" are used throughout with the understanding
that various alternatives as described above, such as other
polypeptides and other long chain carbohydrates readily envisioned
by those skilled in the art, are contemplated by the present
invention.
[0060] Although not bound by any particular theory, the present
invention is intended to provide a matrix scaffolding designed to
maximize the polar amino acid hydrogen bonding sites found in alpha
chains derived from collagen. These alpha chains, or gelatin, are
preferably derived from pig gelatin, and stabilized by 500,000 Da
molecular mass dextran, or other long chain carbohydrates, added
while the alpha chains are heated. The positively charged polar
groups of the collagen-derived alpha chains are then able to
associate with the negatively charged --OH groups of the repeating
glucose units found in the dextran. The gelatin and the dextran
form a proteoglycan-type structure. FIGS. 1-3 illustrate the
interaction between the various components of the preferred
embodiment of the matrix of the invention and interaction between
the matrix and the tissue of a patient.
[0061] FIG. 1 illustrates the creation of polar alpha chains 15
from tropocollagen 10 derived from mature collagen. Heating
tropocollagen 10 disrupts the hydrogen bonds that tightly contain
the triple stranded monomers in mature collagen. By breaking these
hydrogen bonds, the polar amine and carboxylic acid groups are now
available for binding to polar groups from other sources or
themselves.
[0062] FIGS. 2A-2B illustrate stabilization of the matrix monomeric
scaffolding by the introduction of a long chain carbohydrate 20,
such as dextran. As shown in FIG. 2B, without the long chain
carbohydrate 20, the alpha chain 15 will form hydrogen bonds
between the amino and carboxylic acid groups within the linear
portion of the monomer and fold upon itself, thus limiting
available sites for cellular attachment. As depicted in FIG. 2A,
the long chain carbohydrate 20 serves to hold the alpha chain 15
open by interfering with this folding process.
[0063] In addition to the polypeptide and long chain carbohydrate,
the bioactive hydrogel matrix can further comprise one or more
components useful for enhancing the bioadhesiveness of the hydrogel
matrix. Examples of such components include polar amino acids,
polar amino acid analogues or derivatives, divalent cation
chelators, and combinations thereof. In one preferred embodiment,
all of the bioactive hydrogel matrix ingredients are provided in
admixture.
[0064] The bioactive hydrogel matrix preferably includes one or
more polar amino acids in an effective amount to increase the
rigidity of the hydrogel matrix and allow direct administration of
the hydrogel matrix, such as through injection, to a site in need
of connective tissue regeneration. As used herein, polar amino
acids are commonly defined and intended to include tyrosine,
cysteine, serine, threonine, asparagine, glutamine, asparatic acid,
glutamic acid, arginine, lysine, and histidine. Preferentially, the
amino acids are selected from the group consisting of cysteine,
arginine, lysine, histidine, glutamic acid, aspartic acid. When
polar amino acids are present in the bioactive hydrogel matrix, the
polar amino acids are preferentially present in a concentration of
about 3 to about 150 mM, preferably about 10 to about 65 mM, and
more preferably about 15 to about 40 mM.
[0065] Advantageously, the added polar amino acids comprise
L-glutamic acid, L-lysine, and L-arginine. The final concentration
of L-glutamic acid is generally about 2 to about 60 mM, preferably
about 5 to about 40 mM, most preferably about 10 to about 30 mM. In
one embodiment, the concentration of L-glutamic acid is about 20
mM. The final concentration of L-lysine is generally about 0.5 to
about 30 mM, preferably about 1 to about 15 mM, most preferably
about 1 to about 10 mM. In one embodiment, the concentration of
L-lysine is about 5.0 mM. The final concentration of L-arginine is
generally about 1 to about 40 mM, preferably about 1 to about 30
mM, most preferably about 5 to about 20 mM. In one embodiment, the
final concentration of arginine is about 15 mM.
[0066] By amino acid is intended all naturally occurring alpha
amino acids in both their D and L stereoisomeric forms, and their
analogues and derivatives. An analog is defined as a substitution
of an atom or functional group in the amino acid with a different
atom or functional group that usually has similar properties. A
derivative is defined as an amino acid that has another molecule or
atom attached to it. Derivatives would include, for example,
acetylation of an amino group, amination of a carboxyl group, or
oxidation of the sulfur residues of two cysteine molecules to form
cystine. As previously noted, the bioactive hydrogel matrix of the
invention can include one or more polar amino acid analogues or
derivatives.
[0067] Amino acids used in the bioactive hydrogel matrix of the
present invention can also be present as dipeptides, which are
particular beneficial for delivery of amino acids having decreased
water solubility, such as L-glutamine. Accordingly, amino acids
added to the hydrogel matrix can include dipeptides, such as
L-alanyl-L-glutamine. When present in the hydrogel matrix, the
concentration range for L-alanyl-L-glutamine is preferably about
0.001 to about 1 mM, more preferably about 0.005 to about 0.5 mM,
most preferably about 0.008 to about 0.1 mM. In one particular
embodiment, the final concentration of L-alanyl-L-glutamine is
about 0.01 mM.
[0068] The added amino acids can also include L-cysteine, which is
advantageous in many regards. Cysteine is useful for providing
disulfide bridges, further adding support and structure to the
bioactive hydrogel matrix and increasing its resistance to force.
The final concentration of L-cysteine is generally about 5 to about
5000 .mu.M, preferably about 10 to about 1000 .mu.M, most
preferably about 100 to about 1000 .mu.M. In one embodiment, the
final concentration of cysteine is about 700 .mu.M. L-cysteine also
acts as a nitric oxide scavenger or inhibitor. Nitric oxide
inhibitors include any composition or agent that inhibits the
production of nitric oxide or scavenges or removes existing nitric
oxide. Nitric oxide, a pleiotropic mediator of inflammation, is a
soluble gas produced by endothelial cells, macrophages, and
specific neurons in the brain, and is active in inducing an
inflammatory response. Nitric oxide and its metabolites are known
to cause cellular death from nuclear destruction and related
injuries.
[0069] Accordingly, the bioactive hydrogel matrix can optionally
include one or more additional nitric oxide inhibitors, such as
aminoguanidine, N-monomethyl-L-arginine, N-nitro-L-arginine,
cysteine, heparin, and mixtures thereof. When present in the
hydrogel matrix, the final concentration of nitric oxide inhibitors
is generally about 5 to about 500 .mu.M, preferably about 10 to
about 100 .mu.M, most preferably about 15 to about 25 .mu.M. In one
embodiment, the final concentration is about 20 .mu.M.
[0070] Advantageously, intact collagen can be optionally added to
the bioactive hydrogel matrix to provide an additional binding
network and provide additional support to the matrix. The final
concentration of the intact collagen present in the hydrogel matrix
is from about 0 to about 5 mM, preferably about 0 to about 2 mM,
most preferably about 0.05 to about 0.5 mM.
[0071] Additionally, the bioactive hydrogel matrix may optionally
include one or more divalent cation chelators, which increase the
rigidity of the matrix by forming coordinated complexes with any
divalent metal ions present. The formation of such complexes leads
to the increased rigidity of the matrix by removing the inhibition
of hydrogen bonding between --NH.sub.2 and --COOH caused by the
presence of the divalent metal ions. A preferred example of a
divalent cation chelator that is useful in the present invention is
ethylenediaminetetraacetic acid (EDTA) or a salt thereof. The
concentration range for the divalent cation chelator, such as EDTA,
is generally about 0.01 to about 10 mM, preferably 1 to about 8 mM,
most preferably about 2 to about 6 mM. In a one embodiment, EDTA is
present at a concentration of about 4 mM.
[0072] EDTA is also an example of another group of compounds useful
as additives for the bioactive hydrogel matrix, superoxide
inhibitors. Superoxide is a highly toxic reactive oxygen species,
whose formation is catalyzed by divalent transition metals, such as
iron, manganese, cobalt, and sometimes calcium. Highly reactive
oxygen species such as superoxide (O.sub.2.sup.-) can be further
converted to the highly toxic hydroxyl radical (OH.sup.-) in the
presence of iron. By chelating these metal catalysts, EDTA serves
as an antioxidant. Accordingly, the bioactive hydrogel matrix can
include one or more superoxide inhibitor.
[0073] Optionally, trace mineral nutrients and salts thereof, such
as zinc sulfate, can be added to the bioactive hydrogel matrix.
Zinc has beneficial wound healing effects that are particularly
useful in the present invention. When present in the hydrogel
matrix, the concentration range for zinc is generally about 0.005
mM to about 3 mM, preferably about 0.01 to about 2 mM, most
preferably about 0.02 to about 1 mM. In one particular embodiment,
the final concentration of zinc is about 0.03 mM.
[0074] The bioactive hydrogel matrix is preferably based upon a
physiologically compatible buffer, one embodiment being Medium 199,
a common nutrient solution used for in vitro culture of various
mammalian cell types (available commercially from Sigma Chemical
Company, St. Louis, Mo.). The buffer can be further supplemented
with additives and additional amounts of some medium components,
such as supplemental amounts of polar amino acids as described
above.
[0075] The bioactive hydrogel matrix can also be formulated in
other buffered solutions, including buffered solutions regarded as
simplified in relation to Medium 199. For example, a phosphate
buffer formulated to yield physiological osmotic pressures after
hydrogel matrix compounding can be prepared using 1.80 mM
KH.sub.2PO.sub.4 and 63 mM Na.sub.2HPO.sub.4.
[0076] The bioactive hydrogel matrix of the present invention is
particularly useful for repairing and regenerating connective
tissue because of the open structure of the hydrogel matrix and the
inherent ability of the hydrogel matrix to interact with
physiological material. FIG. 3 illustrates the effect of polar
amino acids and/or L-cysteine added to stabilize the
monomer/carbohydrate units 25 by linking the exposed monomer polar
sites to, for example, arginine's amine groups or glutamic acid's
carboxylic acid groups. Furthermore, disulfide linkages can be
formed between L-cysteine molecules (thereby forming cystine),
which in turn forms hydrogen bonds to the monomeric alpha chains
15. The stability imparted by the polar amino acids, polar amino
acid analogues and derivatives, and intact collagen is particularly
advantageous for maintaining the open structure of the gelatin and
keeping the active sites available for therapeutic benefit.
[0077] The hydrogen bonds formed between these additional amino
acids and monomer/carbohydrate units 25 are broken when the matrix
is liquefied upon heating, and the polar groups are freed to attach
the monomer/dextran units to exposed patient tissue surfaces. In
preferred embodiments, EDTA or a salt thereof is also present to
chelate divalent cations and thereby prevent divalent cations from
being preferentially attracted to the exposed polar groups of the
monomer/carbohydrate units 25 to the exclusion of the polar amino
acids.
[0078] Normally, the tearing of tissue secondary to trauma
stimulates production and release of nitric oxide, initiating
recruitment of immune and inflammatory cells that phagocytise or
release chemicals to destroy foreign substances. By providing local
and temporal inhibition of nitric oxide and superoxide release and
production, nitric oxide inhibitors, such as aminoguanidine and
cysteine, and superoxide inhibitors, such as EDTA, allow the
collagen derived alpha chain/dextran units 25 to bind and become
integrated on the exposed tissue surface. The alpha chain/dextran
units 25 then serve as the scaffolding on which formerly
differentiated host cells de-differentiate into "mesenchymoid"
morphology. This de-differentiation process is followed by
integration of these incompletely differentiated cells into host
tissue. These mesenchymoid cells are then able to promote areas of
their genome that leads to differentiation into cell types required
for tissue healing and regeneration.
[0079] By providing a proteoglycan-like scaffolding similar to that
found in the early stages of fetal development, and using
structural stabilizers that serve a secondary purpose in enhancing
host response to the scaffolding upon exposure to host tissues, the
matrix serves as a biocompatible device capable of increasing
vascularization and promoting wound healing and local tissue
regeneration, even in the case of large areas of bone loss. Because
the matrix promotes tissue-specific regeneration, as occurs during
embryogenesis and fetogenesis where similar types of scaffolding
are present, it has now been discovered that the matrix of the
invention can be used to successfully treat bone injuries that are
typically non-responsive to conventional treatments, such as long
segmental diaphyseal bone loss, cavitation, and simple fractures in
patients having abnormally low ability to regenerate bone tissue.
Furthermore, it has been discovered that the bioactive hydrogel
matrix of the present invention can be used to successfully treat
additional types of injuries often known to be difficult to treat
or slow to heal, such as injuries to non-bone connective tissues,
such as tendon, ligament, and cartilage.
[0080] In vitro testing has shown that the bioactive hydro gel
matrix of the invention exhibits a remarkable ability to bind to
and hence promote cell aggregation across multiple cell types.
Treatment of cultured osteoblasts (human osteosarcoma cell line
SAOS-2) with the bioactive hydrogel matrix resulted in
approximately 80% cellular aggregation. In one comparative study,
cells were treated with the bioactive hydro gel matrix of the
invention, and cells (control) were treated with gelatin alone.
Cell types tested were fibroblasts, osteoblasts, chondrocytes, and
adipocytes. The cells were stained with trypan blue and visually
inspected. The cells treated with the bioactive hydrogel matrix
were evident as large clumps (i.e., aggregates), while the control
cells (those treated with gelatin alone) were evident as single
cells and not aggregated. This illustrates how the intact bioactive
hydrogel matrix binds to and aggregates cells important in wound
healing, bone repair, and non-bone connective tissue repair. This
binding and subsequent interaction does not occur when only gelatin
is present. Furthermore, previous similar studies with fibroblasts
indicated the binding and aggregation also did not occur after
treatment with dextran alone.
[0081] FIG. 4 provides quantification of the aggregation of the
cells in the study described above. As shown in FIG. 4, after
treatment with the bioactive hydrogel matrix of the invention, all
four cell types demonstrated approximately 80% aggregation.
Comparatively, the cells treated with gelatin alone demonstrated
less than 30% aggregation. The binding of the bioactive hydrogel
matrix to cells as evidenced by the aggregation is believed to be
the first key step in the action of the bioactive hydrogel matrix
on cellular activity. The aggregation is a result of the cells
interacting with the open polar co-polymer structure of the
bioactive hydrogel matrix.
[0082] The bioactive hydrogel matrix of the invention also exhibits
additional action necessary for bone regeneration. In one study,
treatment of cultured osteoblasts with the bioactive hydrogel
matrix of the invention resulted in a greater than 20-fold increase
in bone morphogenetic protein-2 (BMP-2) messenger RNA. BMP-2 is a
member of the transforming growth factor (TGF) beta superfamily of
proteins and a key regulator of osteoblast differentiation. BMP is
known to stimulate wound healing and includes various bone
morphogenetic proteins in addition to BMP-2. This alteration and
increase of gene activity is indicative of the ability of the
matrix to produce healing of bone fractures. This activity of the
bioactive hydrogel matrix in stimulating BMP-2 production is
illustrated in FIG. 5, which demonstrates an acute and dramatic
increase in BMP-2 gene expression after a 40 minute treatment with
the bioactive hydrogel matrix as compared to a control.
[0083] The useful activity of the bioactive hydrogel matrix is
further demonstrated in FIGS. 6 and 7, which illustrate the effects
of treatment of cultured chondrocytes (cells leading to the
production of tendon, ligament, and cartilage) with the bioactive
hydrogel matrix of the invention in causing a greater than 3-fold
increase in Connective Tissue Growth Factor (CTGF) and aggrecan
gene expression. CTGF is a profibrotic protein induced by TGF beta
and is a key regulator of chondrocyte proliferation and
differentiation. It is an early marker of chondrogenesis expressed
at the highest levels in vivo during chondrocyte growth. Aggrecan
is a major cartilage extracellular matrix (ECM) component and a
marker for the chondrocyte phenotype. FIG. 6 again illustrates an
acute and marked increase in CTGF gene expression in the presence
of the bioactive hydrogel matrix. FIG. 7 illustrates a similar
increase in aggrecan gene expression and also illustrates a more
prolonged effect of such increase.
[0084] In addition to being in its usual, hydrated form (as
generally described above), the bioactive hydrogel matrix of the
present invention can further be in a dehydrated form. This is a
particularly advantageous form of the bioactive hydrogel matrix
increasing the practical usefulness of the hydrogel matrix,
providing for ease of storage and transportation, and preserving
the shelf-life of the hydrogel matrix and compositions made using
the hydrogel matrix. Any method generally known in the art for
dehydrating materials normally in a hydrated state would be useful
according to the present invention, so long as it is not
detrimental to the connective tissue regenerative properties of the
hydrogel matrix as described herein. For example, one preferred
method of dehydrating the bioactive hydrogel matrix is freeze
drying. Other methods of preparing dehydrated biopolymers, such as
spray-drying or speed-vac, can also be used and are known to those
skilled in the art.
[0085] Freeze drying generally comprises the removal of water or
other solvent from a frozen product through sublimation, which is
the direct transition of a material (e.g., water) from a solid
state to a gaseous state without passing through the liquid phase.
Freeze drying allows for the preparation of a stable product being
readily re-hydratable, easy to use, and aesthetic in appearance.
The freeze drying process consists of three stages: 1)
pre-freezing, 2) primary drying, and 3) secondary drying.
[0086] Since freeze drying involves a phase change from solid to
gaseous, material for freeze drying must first be adequately
pre-frozen. The pre-freezing method and the final frozen product
temperature can both affect the ability to successfully freeze dry
the material. Rapid cooling forms small ice crystals. While small
crystals are useful in preserving structure, they result in a
product that is more difficult to freeze dry. Slower cooling
results in larger ice crystals and produces less restrictive
channels in the matrix during the drying process. Pre-freezing to
temperatures below the eutectic temperature, or glass transition
temperature, is necessary for complete drying of hydrogels.
Inadequate freezing may produce small pockets of unfrozen material
remaining in the product which may expand and compromise the
structural stability of the freeze dried product.
[0087] After pre-freezing the product, conditions must be
established in which ice (i.e., frozen solvent) can be removed from
the frozen product via sublimation, resulting in a dry,
structurally intact product. This requires careful control of the
two parameters, temperature and pressure, involved in the freeze
drying system. It is important that the temperature at which a
product is freeze dried is balanced between the temperature that
maintains the frozen integrity of the product and the temperature
that maximizes the vapor pressure of the solvent.
[0088] After primary freeze drying is complete, and all ice has
sublimed, bound moisture is still present in the product. The
product appears dry, but the residual moisture content may be as
high as 7-8%. Continued drying is necessary at a warmer temperature
to reduce the residual moisture content to optimum values. This
process is called isothermal desorption, as the bound water is
desorbed from the product. Secondary drying is normally continued
at a product temperature higher than ambient but compatible with
the sensitivity of the product. All other conditions, such as
pressure and collector temperature, remain the same. Because the
process is desorptive, the vacuum should be as low as possible (no
elevated pressure) and the collector temperature as cold as can be
attained. Secondary drying is usually carried out for approximately
1/3 to 1/2 the time required for primary drying.
[0089] One example of equipment useful in preparing freeze dried
hydrogels is the FreeZone 12 Liter Freeze Dry System with
Stoppering Tray Dryer (Labconco Kansas City, Mo.). With such
system, tubes with porous caps containing hydrogels are frozen to
-30.degree. C. at a cooling rate of 0.05.degree. C./min using the
cooling shelf unit of the freeze dryer and are held at -30.degree.
C. for 12 hours. A vacuum is applied to the frozen hydrogel at
-30.degree. C. for 24 hours before the temperature is incrementally
increased to -10.degree. C. at a rate of 0.25.degree. C./minute.
The hydrogel is held under vacuum at -10.degree. C. for at least 12
hours before the temperature is further increased to 20.degree. C.
at a rate of 0.05.degree. C./minute.
[0090] The dehydrated bioactive hydrogel matrix can comprise the
bioactive hydrogel matrix in any of the embodiments described
herein. Furthermore, the bioactive hydrogel matrix can be used in
preparing any of the connective tissue regenerative compositions
described herein prior to being dehydrated. Therefore, the present
invention also encompasses dehydrated connective tissue
regenerative compositions.
[0091] In one embodiment of the invention, the bioactive hydrogel
matrix can be prepared as described herein and then dehydrated to
form a single mass. The single mass can then be customized for
specific uses. For example, the dehydrated hydrogel matrix could be
sliced into wafer-like slices of varying dimensions. The dehydrated
hydrogel matrix could also be ground to a particulate form. The
dehydrated hydrogel matrix could also be cut to various shapes and
dimensions for specified uses, such as pre-formed plugs for use in
bone cavitation. Also, advantageously, the dehydrated bioactive
hydrogel matrix could be formed to a standardized shape and size
and packaged for various uses. The pre-packaged dehydrated
bioactive hydrogel matrix could then be customized to a desired
shape and size at the time of use. In a further embodiment, the
dehydrated hydrogel matrix can be shaped around a central mandrel
to form porous tubes useful for tissue regenerative guidance
conduits. These can be wrapped around specific sites which may
require or benefit from guided tissue regeneration. Dehydrated
hydrogels can also be partially rehydrated to form putties and
pastes appropriate for filling bony voids caused by surgery or
trauma
[0092] The dehydrated hydrogel matrix, when re-hydrated, retains is
connective tissue regenerative properties as described herein and
can be used according to the methods of the invention as
effectively as a freshly prepared bioactive hydrogel matrix of the
invention. The re-hydration of the hydrogel matrix can be performed
according to various methods, all of which are encompassed by the
invention. In one embodiment, the dehydrated bioactive hydrogel
matrix is re-hydrated immediately prior to use, such as by
contacting with water or a physiologically compatible buffer
solution, such as Medium 199. In another embodiment, the dehydrated
bioactive hydrogel matrix could be placed in the site in need of
connective tissue regeneration and then contacted with re-hydrating
fluids, such as water or a physiologically compatible buffer
solution. In still another embodiment, the dehydrated hydrogel
matrix could be placed in the site in need of connective tissue
regeneration and then re-hydrated through contact with natural body
fluids.
[0093] It is, of course, understood that any of the above
embodiments described in relation to the dehydrated hydrogel matrix
are also intended to encompass similar or identical embodiments
using the connective tissue regenerative compositions of the
present invention comprising the bioactive hydrogel matrix.
[0094] While the bioactive hydrogel matrix of the present invention
is useful in multiple types of tissue repair, it is particularly
advantageous in areas where tissue repair or regeneration is
especially difficult. As described previously, such is often the
case with bone regeneration and repair of non-bone connective
tissue. Connective tissue is a generalized term for mesodermally
derived tissue that may be more or less specialized. Many types of
tissue can fall under the term, such as bone, cartilage, dura
mater, tendon, and ligament. The term can also be used for less
specialized tissue that is rich in components such as collagen and
proteoglycans, and that surrounds other more highly ordered tissues
and organs.
[0095] The bioactive hydrogel matrix is especially useful in the
regeneration of bone, particularly in situations where bone repair
does not occur or where more rapid healing of a bone defect would
be beneficial to a patient. In situations where there is bone loss
over of relatively large area of the bone, the bioactive hydrogel
matrix can be inserted into the area of the bone loss and allowed
to remain in place to facilitate healing of the wound and
regeneration of bone in the area of the loss. The matrix provides
multiple regenerative functions as described above. The matrix
interacts with osteocytes leading to more rapid formation of bone
tissue. The matrix also promotes osteoblast gene expression as
demonstrated by the increased production of BMP-2. The presence of
the matrix in the wound site also inhibits ingrowth of non-bone
tissue into the wound inhibiting the formation of new bone tissue.
The presence of the matrix also promotes vascularization, which is
necessary for the rapid growth of new bone tissue in providing
nutrients, growth factors, oxygen, and other components necessary
to bone regeneration.
[0096] Closely related to the ability of the matrix to promote
regeneration of bone is the function of the matrix in relation to
stem or progenitor cells. This is an important aspect of the
ability of the matrix to support tissue regeneration for multiple
reasons. First, stem cells are found in bone marrow, and these
adult stem cells can be induced to differentiate into bone tissue
or other types of connective tissue, including cartilage, and
important adjacent tissues, such as neurons and skeletal muscle.
Further, progenitor cells, which are precursors giving rise to
cells of a particular cell type, are also useful for inducing bone
tissue growth, or other connective tissue growth, where applicable.
Thus, interacting with those cells in the areas surrounding bone
injury, for example, could stimulate stem or progenitor cells in
the injured area to differentiate into bone cells, further
hastening the regeneration of the bone. This is also significant in
that often times, repair of hard tissue, such as bone, is
accompanied by the need to repair soft tissue as well. One example
is in the periodontal field where the presence of a material that
would promote healing of the gums as well as the underlying bone
would be advantageous. A patient having severe periodontal disease
with significant bone loss could be treated using the bioactive
hydrogel matrix of the present invention. The bioactive hydrogel
matrix could be inserted into the area of bone loss and the gum
tissue replaced over the area. The bioactive hydrogel matrix,
through its interaction with stem or progenitor cells and
subsequent changes in gene expression, as well as the other
activities described above, would not only facilitate the
regeneration of the bone, but also hasten the repair of the gum
tissue overlying the injured bone. The same type of action would be
expected to take place in other types of injury resulting in damage
to bone as well as the surrounding tissue.
[0097] The present invention, in one aspect, is a method for
regenerating connective tissue comprising administering a bioactive
hydrogel matrix comprising a polypeptide and a long chain
carbohydrate, as described herein, to a site in need of connective
tissue regeneration. Preferentially, the polypeptide is a gelatin,
and the long chain carbohydrate is dextran.
[0098] The bioactive hydrogel matrix as used in the method of the
invention can include one or more of the additional components
previously noted herein. Additionally, the bioactive hydrogel
matrix can incorporate further components facilitating the
regeneration of connective tissue according to the method of the
invention.
[0099] According to another aspect, the present invention provides
various connective tissue regenerative compositions. Generally, the
compositions comprise a bioactive hydrogel matrix as described
herein and at least one additional component useful for
accomplishing the methods of the invention. Accordingly, any of the
compositions described herein can be used in the various methods of
the invention.
[0100] In one embodiment of the invention, the bioactive hydrogel
matrix further comprises at least one osteoinductive or
osteoconductive material. By "osteoinductive" is meant materials
that lead to a mitogenesis of undifferentiated perivascular
mesenchymal cells leading to the formation of osteoprogenitor cells
(i.e., cells with the capacity to form new bone). By
"osteoconductive" is meant materials that facilitate blood vessel
incursion and new bone formation into a defined passive trellis
structure. Various compounds, minerals, proteins, and the like are
known to exhibit osteoinductive or osteoconductive activity.
Accordingly, any of such materials would be useful according to the
present invention.
[0101] In particular, any of the following could be used for their
osteoinductive or osteoconductive ability according to the present
invention: demineralized bone matrix (DBM), bone morphogenetic
proteins (BMPs), transforming growth factors (TGFs), fibroblast
growth factors (FGFs), insulin-like growth factors (IGFs),
platelet-derived growth factors (PDGFs), epidermal growth factors
(EGFs), vascular endothelial growth factors (VEGFs), vascular
permeability factors (VPFs), cell adhesion molecules (CAMs),
calcium aluminate, hydroxyapatite, coralline hydroxyapatite,
alumina, zirconia, aluminum silicates, calcium phosphate,
tricalcium phosphate, calcium sulfate, polypropylene fumarate,
bioactive glass, porous titanium, porous nickel-titanium alloy,
porous tantalum, sintered cobalt-chrome beads, ceramics, collagen,
autologous bone, allogenic bone, xenogenic bone, coralline, and
derivates or combinations thereof, or other biologically produced
composite materials containing calcium or hydroxyapatite structural
elements.
[0102] By "alumina" is meant the commonly held definition of
materials comprised of the natural or synthetic oxide of aluminum,
which may be exemplified in various forms, such as corundum
Bioactive glasses generally contain silicon dioxide (SiO.sub.2) as
a network former and are characterized by their ability to firmly
attach to living tissue. Examples of bioactive glasses available
commercially and their manufacturers include Bioglass.RTM.
(American Biomaterials Corp., USA, 45% silica, 24% calcium oxide
(CaO), 24.5% disodium oxide (Na.sub.2O), and 6% pyrophosphate
(P.sub.2O.sub.5)), Consil.RTM. (Xeipon Ltd., UK), NovaBone.RTM.
(American Biomaterials Corp.), Biogran.RTM. (Orthovita, USA),
PerioGlass.RTM.(Block Drug Co., USA), and Ceravital.RTM. (E.Pfeil
& H. Bromer, Germany). Corglaes.RTM. (Giltech Ltd., Ayr, UK)
represents another family of bioactive glasses containing
pyrophosphate rather than silicon dioxide as a network former.
These glasses contain 42-49 mole % of P.sub.2O.sub.5, the remainder
as 10-40 mole % as CaO and Na.sub.2O.
[0103] When present in the bioactive hydrogel matrix of the present
invention, the osteoinductive or osteoconductive material is
preferably present at a volume concentration of about 0.01 percent
to about 90 percent based upon the total volume of the connective
tissue regenerative composition. Such concentration is further
dependent upon the ability to form compositions having suitable
putty or paste-like properties. Preferably, the osteoinductive or
osteoconductive material is present at a volume concentration of
about 50 percent to about 80 percent, based upon the total volume
of the connective tissue regenerative composition. In one
particular embodiment, a composition according to the invention
comprises a 75% volume/volume mixture of osteoinductive or
osteoconductive material, such as calcium sulfate, and bioactive
hydrogel matrix (e.g., 12 mL calcium sulfate to 4 mL hydrogel
matrix).
[0104] As a connective tissue regenerative composition, the
bioactive hydrogel matrix and the osteoinductive or osteoconductive
materials can be variously combined. Preferably, the osteoinductive
or osteoconductive materials and the hydrogel matrix are in
admixture, which can be according to any means generally known to
one of skill in the art. For example, the bioactive hydrogel matrix
could be prepared, and the osteoinductive or osteoconductive
material (e.g., powdered calcium phosphate) could be poured into
and mixed into the hydrogel matrix by mechanical mixing means. The
mixture could be flowable or could be substantially thickened to a
putty or paste-like consistency. According to another embodiment,
the bioactive hydrogel matrix could be dehydrated and,
preferentially, in particulate or pelletized form. The particulate
dehydrated bioactive hydrogel matrix could be mixed with an
osteoinductive or osteoconductive material to form a substantially
uniform mixture. In particular, the osteoinductive or
osteoconductive material could be in the form of a putty or paste,
and the particulate dehydrated bioactive hydrogel matrix kneaded or
otherwise mixed therein.
[0105] In yet another embodiment of the invention, the bioactive
hydrogel matrix of the invention can further comprise at least one
medicament useful for treating patients having connective tissue
damage or in need of connective tissue regeneration. The medicament
can be any medicament useful in facilitating the healing and
regenerative process. Such medicaments useful according to the
invention include, but are not limited to, antivirals,
antibacterials, anti-inflammatories, immunosuppressants,
analgesics, anticoagulants, and wound healing promotion agents.
[0106] According to another embodiment of the invention, the
bioactive hydro gel matrix can further comprise stem or progenitor
cells, such as ADAS cells, which are known to be capable of
differentiating into adipogenic, osteogenic, chondrogenic, and
myogenic lineages. Accordingly, the presence of stem or progenitor
cells can be beneficial for stimulating and increasing connective
tissue regrowth, particularly bone and cartilage. Further, the
presence of the stem or progenitor cells can be beneficial for
stimulating and increasing growth of surrounding tissue, providing
support for the damaged connective tissue. Preferably, stem or
progenitor cells are present at a concentration of about 10,000 to
about 1,000,000 cells per ml of hydrogel matrix, more preferably
about 50,000 to about 750,000 cells per ml of hydrogel matrix, most
preferably about 100,000 to about 500,000 cells per ml of hydrogel
matrix. In one particular embodiment, the final concentration is
about 250,000 cells per ml of hydrogel matrix. In a further
embodiment of the invention, the bioactive hydrogel matrix includes
stem cells and progenitor cells. In a particularly preferred
embodiment, the progenitor cells are osteoprogenitor cells.
[0107] In one particular embodiment, the bioactive hydrogel matrix
could be in a particulate, dehydrated form and the particles mixed
into a solution containing stem or progenitor cells, such as ADAS
or mesenchymal stem cells.
[0108] In another embodiment of the invention, the bioactive
hydrogel matrix includes a three-dimensional structural framework.
As previously noted, the bioactive hydro gel matrix of the present
invention becomes flowable at physiological temperatures. As such,
it is beneficial, in certain embodiments, for the bioactive
hydrogel matrix to include structural components. Preferentially,
the bioactive hydrogel matrix is at least partially contained
within the three-dimensional structural framework. Accordingly, the
structural framework can take on various embodiments.
[0109] In one particular embodiment, the three-dimensional
structural framework includes a scaffold or cage-like structure at
least partially containing the bioactive hydrogel matrix. Such an
embodiment is particularly useful in areas of long segmental
diaphyseal bone loss or bone cavitation, or in the spinal column.
The scaffold or cage-like structure spans the area of bone loss and
encloses the bioactive hydrogel matrix within the area of bone
loss. For example, the three-dimensional structural framework could
be a cylindrical metal mesh, such as titanium mesh. Accordingly,
the three-dimensional structural framework can include materials
that are non-bioreabsorbable (i.e., persist in the body in a
virtually unchanged state or must be later removed).
Advantageously, the three-dimensional structural framework includes
a bioreabsorbable material that persists in the body long enough to
perform its structure-providing function, later being broken down
through natural body processes or being incorporated into the newly
formed bone. In one particular embodiment, the three-dimensional
structural framework includes calcium-containing or calcified
materials easily incorporated into newly formed bone.
[0110] In another embodiment, the three-dimensional structural
framework is at least partially internal to the bioactive hydrogel
matrix. In such embodiments, the three-dimensional structural
framework preferably comprises a material capable of physically or
chemically interacting with the hydrogel matrix. Preferably, the
three-dimensional structural framework provides an array of
structural formations for providing support and structure to the
bioactive hydrogel matrix.
[0111] It is particularly advantageous that the three-dimensional
structural framework be a structure that provides support and
simultaneously provides a space, or network of spaces, for cellular
infiltration. It is particularly beneficial for the
three-dimensional structural framework to include a porous
structure, such as a collagen or gelatin sponge. Any commercially
available collagen sponge would be useful generally in the present
invention. Examples of commercially available collagen sponges
include the Avitene Ultrafoam.TM. collagen sponge (available from
Davol, Inc., a subsidiary of C.R. Bard, Inc., Murray Hill, N.J.
--available online at http://www.davol.com), DuraGen.RTM. collagen
sponge (available from Integra LifeSciences Corp., Plainsboro,
N.J.), and Gelfoam.RTM., a gelatin based sponge (available from
Pharmacia & Upjohn, Kalamazoo, Mich.). Ceramic foams such as
those produced by Hi-Por Ceramics (Sheffield, UK), could also be
used.
[0112] The three-dimensional structural framework can be a single
unit having an inherent three-dimensional structure. As such, the
structural framework can be shaped as desired to precisely fit into
the site in need of connective tissue regeneration. This is
particularly beneficial in cases of long segmental diaphyseal bone
loss or bone cavitation. In such cases, the structural framework
can be precisely shaped and sized to the bone loss segment or
cavitation to fill the space. The bioactive hydrogel matrix is
retained in the bone loss segment or cavitation for an extended
time period to facilitate bone regeneration by being at least
partially contained within the structural framework. To further
placement of the bioactive hydrogel matrix, the structural
framework, with the hydrogel matrix contained therein, can
optionally be sutured into place.
[0113] The three-dimensional structural framework can comprise
various materials useful for providing structure and support and
having an inherent three dimensional structure. The
three-dimensional structural framework can be a structure
substantially in the form as found in nature, such as coralline or
natural sponge. Further, the three-dimensional structural framework
can be a fabricated structure made from materials not naturally
exhibiting a three-dimensional structure but being formed into such
a structure, such as, for example, sintered calcium phosphate.
Similarly, the three-dimensional structural framework can comprise
one or more polymeric materials that have been made, through
processing (such as casting, molding, or sintering), into a
three-dimensional structure, particularly having a series or
network of pores or cavities throughout the structure for allowing
cellular infiltration. Examples of various materials useful as, or
in the preparation of, a three-dimensional structural framework
include, but are not limited to, metals, calcium salts, coralline,
bioactive glass, sponges, ceramics, collagen, keratin, fibrinogen,
alginate, chitosan, hyaluronan, and other biologically-derived
polymers. The three-dimensional structural framework can also
comprise degradable and non-degradable polymers, such as those
commonly used in tissue engineering applications. Exemplary
non-degradable polymers include polyethylene, poly(vinylidene
fluoride), poly(tetrafluoroethylene), poly(vinyl alcohol),
poly(hydroxyalkanoate), poly(ethylene terephthalate), poly(butylene
terephthalate), poly(methyl methacrylate), poly(hydroxyethyl
methacrylate), poly(N-isopropylacrylamide), poly(dimethyl
siloxane), polydioxanone, and polypyrrole. Exemplary degradable
polymers include poly(glycolic acid), poly(lactic acids),
poly(ethylene oxides), poly(lactide-co-glycolides),
poly(s-caprolactone), polyanhydrides, polyphosphazenes,
poly(ortho-esters), and polyimides.
[0114] In one particularly preferred embodiment, the
three-dimensional structural framework comprises a crosslinked
hydrogel matrix. Particularly preferred is a crosslinked bioactive
hydrogel matrix comprising a polypeptide, such as gelatin, and a
long-chain carbohydrate, such as dextran. Published U.S. Patent
Application No. 2003/0232746, which is incorporated herein by
reference in its entirety, describes a crosslinked bioactive
hydrogel matrix, wherein the hydrogel matrix of the present
invention is further stabilized and imparted a three-dimensional
type structure through crosslinking of the matrix components. Such
crosslinked bioactive hydrogel matrix is also described in PCT
Publication No. WO 03/072155, which is also incorporated herein by
reference in its entirety. Additionally, Published U.S. Patent
Application No. 2003/0232198 and PCT Publication No. WO 03/072157,
both of which are also incorporated herein by reference in their
entirety, describe a stabilized bioactive hydrogel matrix as a
surface coating. Such crosslinked hydrogel matrices are also useful
in the various additional embodiments of the present invention as
described herein.
[0115] An example of a crosslinked bioactive hydrogel matrix
comprising dextran and gelatin is provided in FIG. 8 wherein
dextran 20 is covalently crosslinked to gelatin 15 by linkages 70,
thereby forming a crosslinked network 50. The linkages 70 either
result from reaction of functional groups on the gelatin 15 with
functional groups on the dextran 20, or result from reaction of a
bifunctional crosslinker molecule with both the dextran 20 and
gelatin 15. As explained in greater detail below, one method of
crosslinking gelatin and dextran is to modify the dextran molecules
20, such as by oxidation, in order to form functional groups
suitable for covalent attachment to the gelatin 15. This stabilized
cross-linked bioactive network 50 yields therapeutically useful
gels and pastes that are insoluble in physiologic fluids at
physiological temperatures.
[0116] Crosslinked hydrogel matrices as useful according to the
present invention can be prepared by various methods. In one
particular embodiment, one of the polypeptides and long chain
carbohydrates is modified to form reactive groups suitable for
crosslinking. For instance, the dextran or other long chain
carbohydrate component can be modified, such as by oxidation, in
order to cross-link with the polypeptide component. One known
reaction for oxidizing polysaccharides is periodate oxidation. The
basic reaction process utilizing periodate chemistry is well known
and appreciated by those skilled in the art. Periodate oxidation is
described generally in Affinity Chromatography: A Practical
Approach, Dean, et al., IRL Press, 1985 ISBN0-904147-71-1, which is
incorporated by reference in its entirety. The oxidation of dextran
by the use of periodate-based chemistry is described in U.S. Pat.
No. 6,011,008, which is herein incorporated by reference in its
entirety.
[0117] In periodate oxidation, polysaccharides may be activated by
the oxidation of the vicinal diol groups. With long chain
carbohydrates, such as dextran, this is generally accomplished
through treatment with an aqueous solution of a salt of periodic
acid, such as sodium periodate (NaIO.sub.4), which oxidizes the
sugar diols to generate reactive aldehyde groups (e.g. dialdehyde
residues). This method is a rapid, convenient alternative to other
known oxidation methods, such as those using cyanogen bromide.
Dextran activated by periodate oxidation may be stored at 4.degree.
C. for several days without appreciable loss of activity.
[0118] Long chain carbohydrate materials, such as dextran,
activated in this manner readily react with materials containing
amino groups, such as polypeptides, particularly gelatin, producing
a crosslinked material through the formation of Schiff's base
links. A Schiff base is a name commonly used to refer to the imine
formed by the reaction of a primary amine with an aldehyde or
ketone. The aldehyde groups formed on the cellulosic surface react
with most primary amines between pH values from about 4 to about 6.
The Schiff's base links form between the dialdehyde residues of the
dextran and the free amino groups on the gelatin. The crosslinked
product may subsequently be stabilized (i.e. formation of stable
amine linkages) by reduction with a borohydride, such as sodium
borohydride (NaBH.sub.4) or sodium cyanoborohydride (NaBH.sub.3CN).
The residual aldehyde groups may be consumed with ethanolamine or
other amine containing species to further modify the crosslinked
matrix. Other methods known to those skilled in the art may be
utilized to provide reactive groups on either one or both of the
polypeptide and long-chain carbohydrate.
[0119] In preparing crosslinked bioactive hydrogel matrices for use
in the present invention, periodate chemistry is preferentially
used with dextran to form a multifunctional polymer that can then
react with gelatin and other components, such as polar amino acids,
present during the manufacturing process. The periodate reaction
leads to the formation of polyaldehyde polyglycans that are
reactive with primary amines. For example, polypeptides and long
chain carbohydrates may form covalent hydrogel complexes that are
colloidal or covalently crosslinked gels. Covalent bonding occurs
between reactive groups of the dextran and reactive groups of the
gelatin component. The reactive sites on the gelatin include amine
groups provided by arginine, asparagine, glutamine, and lysine.
These amine groups react with the aldehyde or ketone groups on the
dextran to form a covalent bond. These hydrogels can be readily
prepared at temperatures from about 34.degree. C. to about
90.degree. C. Additionally, the hydrogels can be prepared at a pH
range of from about 5 to about 9, preferably from about 6 to about
8, and most preferably from about 7 to about 7.6.
[0120] By controlling the extent of dextran activation and the
reaction time, one can produce stabilized biomimetic scaffolding
materials of varying viscosity and stiffness. By "biomimetic" is
intended compositions or methods imitating or simulating a
biological process or product. Some biomimetic processes have been
in use for several years, such as the artificial synthesis of
vitamins and antibiotics. More recently, additional biomimetic
applications have been proposed, including nanorobot antibodies
that seek and destroy disease-causing bacteria, artificial organs,
artificial arms, legs, hands, and feet, and various electronic
devices. The biomimetic scaffolding materials of the present
invention yield therapeutically useful gels and pastes that are
stable at about 37.degree. C., or body temperature. These gels are
capable of expansion and/or contraction, but will not dissolve in
aqueous solution. Accordingly, such biomimetic crosslinked hydrogel
matrices are particularly useful as a three-dimensional structural
framework as described herein. The crosslinked hydrogel matrix
provides the structure and support required, remains stable and
maintains its three-dimensional structure at physiological
temperatures, and can beneficially provide many of the same
connective tissue regenerative properties of the non-crosslinked
bioactive hydrogel matrix of the invention.
[0121] As an alternate method for forming the crosslinked
dextran/gelatin network, a multifunctional crosslinking agent may
be utilized as a reactive moiety that covalently links the gelatin
and dextran chains. Such bifunctional crosslinking agents may
include glutaraldehyde, epoxides (e.g., bis-oxiranes), oxidized
dextran, p-azidobenzoyl hydrazide,
N-[.alpha.-maleimidoacetoxy]succinimide ester, p-azidophenyl
glyoxal monohydrate,
bis-[.beta.-(4-azidosalicylamido)ethyl]disulfide,
bis[sulfosuccinimidyl]suberate, dithiobis[succinimidyl proprionate,
disuccinimidyl suberate,
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, and
other bifunctional crosslinking reagents known to those skilled in
the art.
[0122] In another embodiment utilizing a crosslinking agent,
polyacrylated materials, such as ethoxylated (20) trimethylpropane
triacrylate, may be used as a non-specific photo-activated
crosslinking agent. Components of an exemplary reaction mixture
would include a thermoreversible hydrogel held at 39.degree. C.,
polyacrylate monomers, such as ethoxylated (20) trimethylpropane
triacrylate, a photo-initiator, such as eosin Y, catalytic agents,
such as 1-vinyl-2-pyrrolidinone, and triethanolamine Continuous
exposure of this reactive mixture to long-wavelength light (>498
nm) would produce a crosslinked hydrogel network.
[0123] As with the non-crosslinked bioactive hydrogel matrix of the
invention, the crosslinked hydrogel matrix can further comprise
various additional components (in addition to the polypeptide and
long chain carbohydrate) to enhance the crosslinked matrix by
providing further stability or functional advantages. Such
additional components can include any compound, especially polar
compounds, that, when incorporated into the crosslinked hydrogel
matrix, enhance the hydrogel matrix by providing further stability
or functional advantages.
[0124] Preferred additional components for use with the stabilized
crosslinked hydrogel matrix include polar amino acids, polar amino
acid analogues and derivatives, intact collagen, and divalent
cation chelators. Suitable concentrations of each particular
preferred additional component are the same as noted above in
connection with the bioactive hydrogel matrix of the present
invention. Polar amino acids, EDTA, and mixtures thereof, are
particularly preferred. The additional components can be added to
the hydrogel matrix composition before or during the crosslinking
of the polypeptide and long chain carbohydrate.
[0125] In one embodiment of the invention, the bioactive hydrogel
matrix of the invention is in a crosslinked form, the
three-dimensional structural framework thus being inherent to the
hydrogel matrix itself. In another embodiment of the invention, the
bioactive hydrogel matrix of the invention is first prepared, and
the three-dimensional structural framework, in the form of a
separate, crosslinked hydrogel matrix, as described above, is added
to the non-crosslinked hydro gel matrix. Such addition can include
physical admixture of the two hydrogels to form a composition
comprising a bioactive hydrogel matrix of the invention and a
three-dimensional structural framework, in the form of a
crosslinked hydrogel matrix. In one particular embodiment,
dehydrated cross-linked bioactive hydrogel matrix formulations can
be used with the non-crosslinked hydrogel matrix or for providing
structural support generally. In this embodiment, the
non-crosslinked hydrogel matrix can be mixed with dehydrated
crosslinked hydro gel matrix in the form of disks, rods, cylinders,
granules, or other suitable geometric forms. Such compositions
provide additional support to the surrounding tissue, and increase
the localized residence time of the non-crosslinked hydrogel
matrix
[0126] Various embodiments of the invention can also be combined,
particularly in preparing the various connective tissue
regenerative compositions of the invention. For example, in one
particular embodiment, the bioactive hydrogel
matrix/three-dimensional structural framework composition could
also include osteoinductive or osteoconductive materials, stem or
progenitor cells (such as ADAS or mesenchymal stem cells), or
medicaments as described herein. Furthermore, any of these
combinations could be used according to the methods of the
invention.
[0127] The methods of the invention wherein the bioactive hydrogel
matrix includes a three-dimensional structural framework are
intended to encompass situations wherein the three-dimensional
structural framework is included with the bioactive hydrogel matrix
prior to administration of the composition. Further, the methods
encompass situations wherein the three-dimensional structural
framework is included with the bioactive hydrogel matrix after
administration of the bioactive hydrogel matrix.
[0128] The methods and compositions of the invention as described
herein are useful in the repair and regeneration of connective
tissue, particularly bone, cartilage, ligament, and the like. As
such, the methods and compositions of the invention are
particularly useful in various treatments involving portions of the
human body particularly susceptible to connective tissue damage or
degeneration.
[0129] According to one embodiment of the invention, the bioactive
hydro gel matrix can be used in the surgical attachment or
reattachment of one or more connective tissues. Because of the
generally decreased vascularization of connective tissue types,
healing times related to injury repair are typically long in
duration. Administration of the bioactive hydrogel matrix to the
injured site during repair of the connective tissue damage can
improve integration of the tissues resulting in a much stronger
repair and decreased healing time. This is particularly true in the
reattachment of tendon or ligament to bone. Similar effects would
be expected in the repair of torn ligaments or torn tendons.
[0130] Accordingly, the present invention provides a method for
reattaching connective tissues to one another. The method generally
comprises coating at least a portion of at least one of the
connective tissues with the bioactive hydrogel matrix according to
any of the various embodiments of the present invention, contacting
the connective tissues, and, optionally, suturing the connective
tissues together. Generally, suturing (or use of other attachment
aids, such as staples, glues, and adhesive strips) is advisable
when using the bioactive hydrogel matrix to maintain the connection
between the connective tissues during healing and reduce occurrence
of separation of the tissues prior to sufficient re-growth and
reattachment of the connective tissues. In one particular
embodiment, at least a portion of the bioactive hydrogel matrix can
be in crosslinked form.
[0131] The improved repair/regeneration supplied by the bioactive
hydrogel matrix would be beneficial to a large number of patients.
While relatively moderate numbers of patients suffer from large
areas of bone loss, the matrix could also be used to treat injuries
that are very common in occurrence, such as a torn anterior
cruciate ligament (ACL), rotator cuff injuries, damaged cartilage
in knees and other joints, and other such injuries that would be
readily obvious to one of ordinary skill in the art.
[0132] The bioactive hydrogel matrix of the invention further would
be expected to be beneficial for improved performance of any
bioreabsorbable tissue anchors. Accordingly, in a further
embodiment of the invention, the bioactive hydrogel matrix,
according to any of the various compositions described herein,
could be used in a combination therapy with other known
reattachment devices. For example a torn connective tissue, such as
a tendon, could be reattached using sutures, staples, glues, or the
like, with at least one piece of the torn connective tissue being
coated at the area of the tear with a bioactive hydrogel matrix of
the present invention to facilitate re-growth of connective tissue
at the injury site and hasten reattachment of the connective
tissue.
[0133] According to another embodiment of the invention, the matrix
is useful in treating degenerative diseases, such as
osteoarthritis, of the natural joint of a patient. Osteoarthritis
is characterized by degeneration of the articular cartilage,
hypertrophy of bone at the margins, and changes in the synovial
membrane of the affected joint. Treatment of the affected joint
with the bioactive hydrogel matrix of the invention, especially
prior to complete loss of cartilage in the joint, can stabilize the
progression of the degeneration and even promote
repair/regeneration of cartilage within the joint and regeneration
of marginal bone structure. Further, the bioactive hydrogel matrix
of the invention provides an effective, minimally invasive
treatment, the hydrogel matrix being capable of injection directly
into the affected joint.
[0134] In this embodiment of the invention, the bioactive hydrogel
matrix can be in many of the various compositions as provided
herein. Advantageously, the bioactive hydrogel matrix further
comprises one or more medicaments, osteoinductive or
osteoconductive materials, or stem or progenitor cells.
[0135] In yet another embodiment of the invention, the bioactive
hydrogel matrix is useful in promoting the healing and
effectiveness of artificial joint replacements. According to this
embodiment, the matrix is inserted into the area surrounding the
artificial joint, particularly where the artificial joint is in
contact with the natural tissue of the patient. The hydrogel matrix
facilitates the integration of the artificial joint into the
surrounding tissue promoting faster healing of the surgical site
and greater duration of the effectiveness of the artificial
joint.
[0136] Preferably, a therapeutic amount of the matrix of the
invention is administered to a patient suffering from connective
tissue injury, particularly an injury to bone, cartilage, tendon,
or ligament. The patient can be any animal, including mammals such
as dogs, cats and humans. The term "therapeutic amount" refers to
the amount required to promote tissue repair/regeneration as
evidenced by, for example, the formation of new bone tissue across
an area previously lacking in bone tissue. The therapeutic amount
will be primarily determined by the size and type of injury being
treated. Typically, the volume of bioactive hydrogel matrix applied
is about 1 to about 60 mL, but could be greater, especially in
large injuries, such as, for example, an area of great bone loss in
a large bone, such as a femur. Preferably, the therapeutic amount
is sufficient to provide a uniform scaffolding for cellular
attachment and differentiation in the area where tissue
regeneration is needed. The non-crosslinked version of the hydrogel
matrix is typically warmed to a temperature of about 35 to about
40.degree. C. prior to administration in order to liquefy the
matrix.
[0137] The bioactive hydrogel matrix used according to the methods
of the present invention for regenerating connective tissue may be
comprised solely of the polypeptide and long chain carbohydrate as
described herein. Preferably, the hydrogel matrix may incorporate
additional components, such as the polar amino acids, polar amino
acid analogues and derivatives, and cation chelators, as described
above. Table 1 below lists particularly preferred components of the
matrix of the present invention along with suitable concentrations
as well as preferred concentrations for each component. Note that
the concentrations listed in Table 1 for gelatin and dextran would
also be suitable for alternative polypeptide and long chain
carbohydrate components. Bioactive hydrogel matrices prepared
having the preferred components and concentrations provided in
Table 1 would also be particularly suitable for use in the
preparation of any of the connective tissue regenerative
compositions as described herein.
TABLE-US-00001 TABLE 1 Concentration Preferred Component Range
Concentration L-glutamic acid 2 to 60 mM 20 mM L-lysine 0.5 to 30
mM 5.0 mM Arginine 1 to 40 mM 15 mM Gelatin 0.01 to 40 mM 0.75 mM
L-cysteine 5 to 5000 .mu.M 700 .mu.M L-alanyl-L-glutamine 0.001 to
1 mM 0.01 mM EDTA 0.01 to 10 mM 4 mM Dextran 0.01 to 10 mM 0.1 mM
Zinc 0.005 to 3 mM 0.03 mM
EXPERIMENTAL
[0138] The present invention is more fully illustrated by the
following examples, which are set forth to illustrate the present
invention and are not to be construed as limiting thereof.
Example 1
Matrix Preparation
[0139] In one embodiment, the bioactive hydrogel matrix was
compounded to yield a final formulation as described above in Table
1. Modified Medium 199 (2.282 L) was placed into a stirred beaker.
To the beaker were added L-cysteine, L-glutamic acid, L-lysine,
L-alanyl-L-glutamine, and EDTA. While stirring, the solution was
heated to 50.degree. C. Next, dextran was added, followed by the
addition of gelatin. NaOH (10%) was used to adjust the pH of the
matrix solution to a final pH of 7.50.+-.0.05. Finally, additional
L-glutamic acid, L-arginine, and L-cysteine were added followed by
the addition of zinc sulfate. The amounts of each component used
were the amounts necessary to bring the final concentration of each
component to the preferred concentration provided in Table 1.
Example 2
Effect of Bioactive Hydrogel Matrix on Critical Size Defect in
Bone
[0140] The in vivo effect of the matrix on bone repair was examined
using the critical size defect model in the rabbit ulna. A defect
in a rabbit ulna was created in which the length of the defect was
purposefully made to be three times the diameter of the bone, i.e.,
a 15 mm defect was created in each ulna (the diameter of the bone
was approximately 4 mm). It is well documented in the literature
that defects of this size will not spontaneously heal (i.e., a
critical size defect). Seven rabbits had 15 mm defects surgically
created in the ulna of each forelimb. Further, the periosteum of
the radius parallel to the defect was scraped off. In each rabbit,
the defect in one forelimb was treated with the bioactive hydrogel
matrix, and the other was treated with a collagen sponge soaked
with the bioactive hydrogel matrix. The muscle surrounding the bone
defect was sutured closed and the limb tightly wrapped.
[0141] The forelimbs of the rabbit were x-rayed to document the
size of the defect, with follow-up X-rays taken at two-week
intervals to 10 weeks of total testing. Micro CT scans and
histological examination were also performed at 10 weeks.
Radiographs of defect sites were scored for calcification on a 0 to
4 scale. Mineralization within the defect treated with the
bioactive hydrogel matrix alone in combination with the collagen
sponge was noted as early as two weeks after the procedure, and by
six weeks, clear and dramatic mineralization was evident within the
area of removed bone. The bioactive hydrogel matrix in the collagen
sponge tended to increase calcification compared to the hydrogel
matrix alone, but the differences were not statistically
significant. New bone formation within the defects was confirmed by
both micro CT scans and histopathology performed at 10 weeks.
Example 3
Effect of Bioactive Hydrogel Matrix on Tendon Re-Growth and
Strength
[0142] Four sheep had a 4 mm length of the central portion of the
patellar tendon removed from the point of attachment of the tibia
to the patella of one leg. A small block of the patella with the
attached patellar tendon was also removed. The contra-lateral leg
served as the unoperated control. Two defects were filled with a
collagen sponge (DuraGen.RTM., Integra LifeSciences) infiltrated
with the bioactive hydrogel matrix of the invention, and two
defects were filled with a DuraGen.RTM. collagen sponge infiltrated
with saline. The implants were sutured into the patellar tendon
defect and surgical site was sutured closed. After 12 weeks, the
patellar tendons were removed for gross observation and mechanical
testing for stiffness. The tendons treated with the bioactive
hydrogel matrix appeared thicker than the control tendons. Further,
the tendons treated with the bioactive hydrogel matrix had an
average increase in stiffness over the control tendons of about
17.2% compared to a decrease of 4.5% in stiffness of the 2 tendons
treated with the collagen sponge soaked with saline.
Example 4
BMP-2 Gene Expression in Presence of Bioactive Hydrogel Matrix
[0143] Human osteosarcoma cells were plated in T75 flasks, allowed
to grow to confluency, and then shifted to serum-free medium (SFM)
for three days. At this point cultures were treated for 40 minutes
at 37.degree. C. with either the bioactive hydrogel matrix of the
invention or serum-free medium as a control. Cultures were rinsed
and re-fed with serum-free medium and sampled over a subsequent 24
hour period for extraction of nucleic acids. Messenger RNA from
these preparations was used to create complementary DNA using
reverse transcription, and specific DNA sequences were amplified
and quantified using real-time polymerase chain reaction
methods.
[0144] In several replicate experiments, induction of messenger RNA
for bone morphogenetic protein-2 (BMP-2) was induced as much as
44-fold, with a peak response seen 2 hours after treatment with the
bioactive hydrogel matrix with a return to baseline levels in 9
hours. Controls retained basal expression of the BMP-2 message over
the entire 24 hour sampling period.
[0145] Expression of BMP-2 protein was also measured in these cell
cultures following identical culture and hydrogel matrix treatment
methods, with the exception that serum-containing medium (SCM) was
used to avoid loss of analyte by adsorption to culture surfaces.
For these studies cultures were sampled over a 3-day period
following either 40 or 120-minutes treatment by removal of medium
and snap freezing the culture to release cell-associated protein
for analysis. Resulting samples were analyzed by enzyme-linked
immunosorbent assay (ELISA) using a commercial BMP-2 detection kit
(R&D Systems, Minneapolis, Minn.). In replicate studies,
treatment with the bioactive hydrogel matrix increased BMP-2
protein levels over controls within 24 hours post-treatment and
provided as much as a 3-fold increase over controls by day 3. These
data are presented graphically in FIG. 9. These data demonstrate
that the rapid increase in gene expression described earlier (FIG.
5) leads to a sustained increase in BMP-2 protein production.
Example 5
BMP-2 Gene Expression in Presence of Crosslinked Bioactive Hydrogel
Matrix
[0146] Samples of crosslinked bioactive hydro gel matrix prepared
with 1% oxidized dextran were cast into disks to fit into 24-well
plates. Disks were sterilized in alcohol and equilibrated to
culture medium and SAOS-2 human osteosarcoma cells were seeded at
approximately 330,000 per disk. After allowing time for cell
attachment, disks were transferred to new wells and sampled on days
1, 2, 3 and 6 for extraction of nucleic acids and quantitative PCR
measurement of transcripts for BMP-2. Controls were seeded cells
that settled past the disks and attached and grew on the bottom of
the original wells used for disk seeding. These studies showed a
rise in BMP-2 mRNA that reached a peak nearly 3.5-fold greater than
initial levels by day 3 and remained elevated at day 6. The results
are displayed graphically in FIG. 10.
Example 6
Preparation of Compositions Including Bioactive Hydrogel Matrix and
Orthopedic Materials
[0147] A putty containing 2.4 g calcium sulfate and 1.5 g
demineralized bone matrix (DBM) was prepared and used to
incorporate particulate dehydrated bioactive hydrogel matrix. After
briefly hand-kneading the material, about 0.5 g of particulate
dehydrated bioactive hydrogel matrix (equivalent to about 3 mL of
fully hydrated hydrogel matrix) was incorporated into the putty.
The putty was mixed and kneaded to uniformity, and the putty
retained the ability to fill a bony defect created in a synthetic
bone
[0148] In a second composition, an injectable calcium sulfate
formulation was prepared using the bioactive hydrogel matrix of the
invention rather than the conventional water base (12 g calcium
sulfate powder and 4 mL of the bioactive hydrogel matrix). The
material had a prolonged set time (over 2 hours to solidify), and
during the early stages of setting was judged to be suitable for
filling a bony defect.
[0149] In a third composition, the bioactive hydrogel matrix was
mixed with a granulated tricalcium phosphate ceramic to form a
moldable putty easily packed into a bony defect.
[0150] Many modifications and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing descriptions and associated drawings. Therefore, it is to
be understood that the invention is not to be limited to the
specific embodiments disclosed and that modifications and other
embodiments are intended to be included within the scope of the
appended claims. Although specific terms are employed herein, they
are used in a generic and descriptive sense only and not for
purposes of limitation.
* * * * *
References