U.S. patent application number 10/374772 was filed with the patent office on 2004-04-22 for biocompatible scaffolds with tissue fragments.
Invention is credited to Binette, Francois, Dhanaraj, Sridevi, Gosiewska, Anna, Hwang, Julia.
Application Number | 20040078090 10/374772 |
Document ID | / |
Family ID | 32045864 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040078090 |
Kind Code |
A1 |
Binette, Francois ; et
al. |
April 22, 2004 |
Biocompatible scaffolds with tissue fragments
Abstract
A biocompatible tissue repair implant or scaffold device is
provided for use in repairing a variety of tissue injuries,
particularly injuries to cartilage, ligaments, tendons, and nerves.
The repair procedures may be conducted with implants that contain a
biological component that assists in healing or tissue repair. The
biocompatible tissue repair implants include a biocompatible
scaffold and particles of living tissue, such that the tissue and
the scaffold become associated. The particles of living tissue
contain one or more viable cells that can migrate from the tissue
and populate the scaffold.
Inventors: |
Binette, Francois;
(Weymouth, MA) ; Hwang, Julia; (Wayland, MA)
; Dhanaraj, Sridevi; (Raritan, NJ) ; Gosiewska,
Anna; (Skilman, NJ) |
Correspondence
Address: |
NUTTER MCCLENNEN & FISH LLP
WORLD TRADE CENTER WEST
155 SEAPORT BOULEVARD
BOSTON
MA
02210-2604
US
|
Family ID: |
32045864 |
Appl. No.: |
10/374772 |
Filed: |
February 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60420093 |
Oct 18, 2002 |
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60419539 |
Oct 18, 2002 |
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Current U.S.
Class: |
623/23.76 ;
435/395 |
Current CPC
Class: |
A61L 2400/18 20130101;
A61F 2310/0097 20130101; A61L 27/18 20130101; A61L 27/3817
20130101; A61F 2/02 20130101; C12N 5/0068 20130101; A61L 2430/10
20130101; A61L 27/36 20130101; A61L 27/3604 20130101; A61L 27/3616
20130101; A61L 27/58 20130101; A61L 2430/06 20130101; A61L 27/3895
20130101; A61L 2430/34 20130101; A61F 2/08 20130101; C12N 5/0655
20130101; A61L 27/3612 20130101 |
Class at
Publication: |
623/023.76 ;
435/395 |
International
Class: |
A61F 002/02 |
Claims
What is claimed is:
1. A biocompatible implant, comprising: a biocompatible scaffold;
and at least one tissue fragment that is associated with at least a
portion of the scaffold, wherein the tissue fragment includes an
effective amount of viable cells that can migrate out of the tissue
fragment and populate the scaffold.
2. The implant of claim 1, wherein the scaffold comprises a
synthetic polymer, a natural polymer, an injectable gel, a ceramic
material, autogeneic tissue, allogeneic tissue, xenogeneic tissue
and combinations thereof.
3. The implant of claim 1, wherein the at least one tissue fragment
includes a plurality of cells and, upon implantation at a surgical
site, at least a portion of the plurality of cells is able to
migrate out of the tissue fragment associated with the scaffold to
proliferate and integrate with surrounding tissue at a site of
implantation.
4. The implant of claim 1, wherein the at least one tissue fragment
includes a plurality of cells and, prior to implantation at a
surgical site, at least a portion of the plurality of cells is able
to migrate out of the tissue fragment associated with the scaffold
to proliferate and populate the scaffold.
5. The implant of claim 1, wherein the biocompatible scaffold
further comprises an adhesion agent for anchoring the suspension of
tissue fragment to the biocompatible scaffold.
6. The implant of claim 5, wherein the adhesion agent comprises an
anchoring agent selected from the group consisting of hyaluronic
acid, fibrin glue, fibrin clot, collagen gel,
gelatin-resorcin-formalin adhesive, mussel-based adhesive,
dihydroxyphenylalanine (DOPA) based adhesive, chitosan,
transglutaminase, poly(amino acid)-based adhesive, cellulose-based
adhesive, synthetic acrylate-based adhesives, platelet rich plasma
(PRP), Matrigel, Monostearoyl Glycerol co-Succinate (MGSA),
Monostearoyl Glycerol co-Succinate/polyethylene glycol (MGSA/PEG)
copolymers, laminin, elastin, proteoglycans and combinations
thereof.
7. The implant of claim 5, wherein the adhesion agent comprises a
chemical cross-linking agent selected from the group consisting of
divinyl sulfone (DVS), polyethylene glycon divinyl sulfone
(VS-PEG-VS), hydroxyethyl methacrylate divinyl sulfone
(HEMA-DIS-HEMA), formaldehyde, glutaraldehyde, aldehydes,
isocyanates, alkyl and aryl halides, imidoesters, N-substituted
maleimides, acylating compounds, carbodiimide, hydroxychloride,
N-hydroxysuccinimide, light, pH, temperature, and combinations
thereof.
8. The implant of claim 1, wherein the at least one tissue fragment
comprises tissue selected from the group consisting of cartilage
tissue, meniscal tissue, ligament tissue, tendon tissue, skin
tissue, muscle tissue, periosteal tissue, pericardial tissue,
synovial tissue, nerve tissue, kidney tissue, bone marrow, liver
tissue, bladder tissue, pancreas tissue, spleen tissue,
intervertebral disc tissue, embryonic tissue, periodontal tissue,
vascular tissue and combinations thereof.
9. The implant of claim 8, wherein the at least one tissue fragment
comprises autogeneic tissue, allogeneic tissue, xenogeneic tissue,
and combinations thereof.
10. The implant of claim 1, where in the at least one tissue
fragment comprises a bone-free tissue type selected from the group
consisting of cartilage, meniscus, tendon, ligament and
combinations thereof.
11. The implant of claim 1, wherein the biocompatible scaffold
comprises a bioabsorbable material.
12. The implant of claim 2, wherein the biocompatible scaffold
comprises a synthetic polymer selected from the group consisting of
aliphatic polyesters, poly(amino acids), poly(propylene fumarate),
copoly(ether-esters), polyalkylene oxalates, polyamides,
tyrosine-derived polycarbonates, poly(iminocarbonates),
polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters
containing amine groups, poly(anhydrides), polyphosphazenes,
polyurethanes, biosynthetic polymers and combinations thereof.
13. The implant of claim 12, wherein the biocompatible scaffold
comprises an aliphatic polyester selected from the group consisting
of homopolymers or copolymers of lactides; glycolides;
.epsilon.-caprolactone; hydroxybuterate; hydroxyvalerate;
1,4-dioxepan-2-one; 1,5,8,12-tetraoxyacyclotetradecane-7,14-dione;
1,5-dioxepan-2-one; 6,6-dimethyl-1,4-dioxan-2-one;
2,5-diketomorpholine; p-dioxanone (1,4-dioxan-2-one); trimethylene
carbonate (1,3-dioxan-2-one); alkyl derivatives of trimethylene
carbonate; .delta.-valerolactone; .beta.-butyrolactone;
.gamma.-butyrolactone, .epsilon.-decalactone, pivalolactone,
.alpha.,.alpha.-diethylpropiolactone, ethylene carbonate, ethylene
oxalate; 3-methyl-1,4-dioxane-2,5-dione; 3,3-diethyl-1,4-dioxan--
2,5-dione; 6,8-dioxabicycloctane-7-one; and combinations
thereof.
14. The implant of claim 2, wherein the biocompatible scaffold
comprises a natural polymer selected from the group consisting of a
fibrin-based material, a collagen-based material, a hyaluronic
acid-based material, a cellulose-based material, silk and
combinations thereof.
15. The implant of claim 2, wherein the biocompatible scaffold
comprises a ceramic material selected from the group consisting of
hydroxyapatite, .alpha.-tricalcium phosphate, .beta.-tricalcium
phosphate, bioglass, calcium phospate, calcium carbonate, calcium
sulfate, allograft bone graft material, xenograft bone graft
material and combinations thereof.
16. The implant of claim 1, wherein the biocompatible scaffold
comprises a polymeric foam component having pores with an open cell
pore structure.
17. The implant of claim 16, wherein the biocompatible scaffold
further comprises a reinforcing component formed of a biocompatible
mesh-containing material.
18. The implant of claim 17, wherein the foam component is
integrated with the reinforcing component such that the pores of
the foam component penetrate the mesh of the reinforcing component
and interlock with the reinforcing component.
19. The implant of claim 1, wherein the biocompatible scaffold
further comprises at least one additional biological component
applied thereto.
20. The implant of claim 19, wherein the at least one additional
biological component comprises growth factors, matrix proteins,
peptides, antibodies, enzymes, cytokines, viruses, nucleic acids,
peptides, isolated cells, platelets or combinations thereof.
21. The implant of claim 1, wherein the at least one tissue
fragment has a particle size in the range of about 0.1 to 2
mm.sup.3.
22. The implant of claim 1, wherein the at least one tissue
fragment is added to a physiological buffering solution to form a
suspension having a concentration of tissue fragments in the range
of about 1 to 100 mg/cm.sup.2.
23. The implant of claim 1, wherein the biocompatible implant
further comprises at least one additional biocompatible scaffold
selected from the group consisting of a synthetic polymer, a
natural polymer, a ceramic material, autogeneic tissue, allogeneic
tissue, xenogeneic tissue and combinations thereof, the at least
one additional biocompatible scaffold being placed in contact with
the at least one tissue fragment, such that at least a portion of
the at least one tissue fragment is disposed between at least two
biocompatible scaffolds.
24. A biocompatible implant, comprising: a biocompatible scaffold;
a suspension having at least one cartilage tissue fragment that is
associated with at least a portion of the scaffold, wherein the at
least one tissue fragment in the suspension includes an effective
amount of viable cells that can migrate out of the tissue fragment
and populate the scaffold; and a retaining element, wherein at
least a portion of the at least one tissue fragment is disposed
between the biocompatible scaffold and the retaining element.
25. The implant of claim 24, wherein the scaffold comprises a
synthetic polymer, a natural polymer, an injectable gel, a ceramic
material, autogeneic tissue, allogeneic tissue, xenogeneic tissue,
and combinations thereof.
26. The implant of claim 24, wherein the retaining element
comprises allograft tissue selected from the group consisting of
periosteum, perichondrium, fascia lata, semitendinosis tendon,
gracilis tendon, dura, mesenthera, small intestine submucosa, skin
dermis and combinations thereof.
27. The implant of claim 24, wherein the retaining element is
selected from the group consisting of autogeneic tissue, allogeneic
tissue, xenogeneic tissue, a hemostatic material, at least one
additional biocompatible scaffold and combinations thereof
28. A kit for repairing a tissue injury, comprising: a sterile
container having one or more biocompatible scaffolds; and a
harvesting tool for collecting at least one viable tissue sample
from a subject.
29. The kit of claim 28, further comprising at least one reagent
for sustaining the viability of the at least one tissue sample.
30. The kit of claim 28, wherein the scaffold is selected from the
group consisting of a synthetic polymer, a natural polymer, an
injectable gel, a ceramic material, autogeneic tissue, allogeneic
tissue, xenogeneic tissue, and combinations thereof.
31. The kit of claim 28, wherein the harvesting tool further
comprises a processing tool for dividing the tissue sample, under
sterile conditions, into at least one tissue fragment.
32. The kit of claim 28, wherein the biocompatible scaffold
comprises an adhesion agent for anchoring the tissue sample to the
biocompatible scaffold.
33. The kit of claim 32, wherein the adhesion agent comprises an
anchoring agent selected from the group consisting of hyaluronic
acid, fibrin glue, fibrin clot, collagen gel, alginate gel,
gelatin-resorcin-formalin adhesive, mussel-based adhesive,
dihydroxyphenylalanine (DOPA) based adhesive, chitosan,
transglutaminase, poly(amino acid)-based adhesive, cellulose-based
adhesive, synthetic acrylate-based adhesives, platelet rich plasma
(PRP), Matrigel, Monostearoyl Glycerol co-Succinate (MGSA),
Monostearoyl Glycerol co-Succinate/polyethylene glycol (MGSA/PEG),
copolymers, laminin, elastin, proteoglycans and combinations
thereof.
34. The kit of claim 32, wherein the adhesion agent comprises a
cross-linking agent selected from the group consisting of divinyl
sulfone (DVS), polyethylene glycon divinyl sulfone (VS-PEG-VS),
hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA),
formaldehyde, glutaraldehyde, aldehydes, isocyanates, alkyl and
aryl halides, imidoesters, N-substituted maleimides, acylating
compounds, carbodiimide, hydroxychloride, N-hydroxysuccinimide,
light, pH, temperature, and combinations thereof.
35. The kit of claim 28, wherein the at least one reagent comprises
a physiological solution selected from the group consisting of
saline, phosphate buffer solution, Hank's balanced salts, tissue
culture medium, tissue culture medium including serum and
combinations thereof.
36. A method of treating living tissue, comprising: providing a
biocompatible scaffold; providing a sample of tissue in the form of
finely divided tissue fragments; depositing the sample of tissue
upon the biocompatible scaffold to form a tissue implant; and
implanting the tissue implant in a desired position relative to the
tissue to be treated.
37. The method of claim 36, wherein the biocompatible scaffold
comprises a synthetic polymer, a natural polymer, an injectable
gel, a ceramic material, autogeneic tissue, allogeneic tissue,
xenogeneic tissue, and combinations thereof.
38. The method of claim 36, further comprising the step of affixing
the tissue implant in the desired position relative to the tissue
to be treated.
39. The method of claim 38, wherein the tissue implant is affixed
in the desired position by applying a fastener to the tissue
implant.
40. The method of claim 39, wherein the fastener comprises one or
more sutures, one or more staples, one or more suture anchors, one
or more tissue tacks, one or more darts, one or more screws, one or
more pins, one or more arrows, fibrin glue, one or more fibrin
clots, one or more biocompatible adhesives or combinations
thereof.
41. The method of claim 36, wherein prior to the step of placing
the tissue implant in the desired position relative to the tissue
to be treated, the method further includes the step of incubating
the tissue implant for a duration and under conditions effective to
allow cells within the sample of tissue to populate the scaffold
prior to reimplantation.
42. The method of claim 41, wherein the scaffold and associated
finely divided tissue fragments are incubated for a duration in the
range of about 7 days to 6 weeks.
43. The method of claim 41, wherein the scaffold and associated
finely divided tissue fragments are incubated at a temperature in
the range of about 20 to 40.degree. C. and in an atmosphere having
a high humidity.
44. The method of claim 36, wherein the finely divided tissue
fragments comprise tissue selected from the group consisting of
cartilage tissue, meniscal tissue, ligament tissue, tendon tissue,
skin tissue, muscle tissue, periosteal tissue, pericardial tissue,
synovial tissue, nerve tissue, kidney tissue, bone marrow, liver
tissue, bladder tissue, pancreas tissue, spleen tissue, and
combinations thereof.
45. The method of claim 44, wherein the finely divided tissue
fragments comprise autogeneic tissue, allogeneic tissue, xenogeneic
tissue, and combinations thereof.
46. The implant of claim 36, where in the finely divided tissue
fragments comprise a bone-free tissue type selected from the group
consisting of cartilage, meniscus, tendon, ligament and
combinations thereof.
47. The method of claim 36, wherein the finely divided tissue
fragments associated with the biocompatible scaffold comprise a
type that is the same as the tissue to be treated.
48. The method of claim 36, wherein the finely divided tissue
fragments associated with the biocompatible scaffold comprise a
type that is different from the tissue to be treated.
49. The method of claim 36, wherein the finely divided tissue
fragments include an effective amount of viable cells that can
migrate out of the tissue particles.
50. The method of claim 49, wherein the effective amount of cells
migrate out of the tissue particles and populate an outer surface
of the biocompatible scaffold.
51. The method of claim 49, wherein the effective amount of cells
migrate out of the tissue particles and populate at least a portion
of an interior region of the scaffold, such that the cells are
embedded within the scaffold.
52. The method of claim 36, wherein the method further comprises,
prior to placing the tissue implant in the desired position
relative to the tissue to be treated, the additional step of
providing at least one additional biocompatible scaffold and
placing the at least one additional biocompatible scaffold over the
deposited finely divided tissue fragments, such that at least a
portion of the finely divided tissue fragments is disposed between
at least two biocompatible scaffolds.
53. The method of claim 36, wherein the biocompatible scaffold
further comprises an adhesion agent for anchoring the sample of
living tissue to the biocompatible scaffold.
54. The method of claim 53, wherein the adhesion agent comprises an
anchoring agent selected from the group consisting of hyaluronic
acid, fibrin glue, fibrin clot, collagen gel, alginate gel,
gelatin-resorcin-formalin adhesive, mussel-based adhesive,
dihydroxyphenylalanine (DOPA) based adhesive, chitosan,
transglutaminase, poly(amino acid)-based adhesive, cellulose-based
adhesive, synthetic acrylate-based adhesives, platelet rich plasma
(PRP), Matrigel, Monostearoyl Glycerol co-Succinate (MGSA),
Monostearoyl Glycerol co-Succinate/polyethylene glycol (MGSA/PEG)
copolymers, laminin, elastin, proteoglycans and combinations
thereof.
55. The method of claim 53, wherein the adhesion agent comprises a
cross-linking agent selected from the group consisting of divinyl
sulfone (DVS), polyethylene glycon divinyl sulfone (VS-PEG-VS),
hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA),
formaldehyde, glutaraldehyde, aldehydes, isocyanates, alkyl and
aryl halides, imidoesters, N-substituted maleimides, acylating
compounds, carbodiimide, hydroxychloride, N-hydroxysuccinimide,
light, pH, temperature, and combinations thereof.
56. The method of claim 37, wherein the biocompatible scaffold
comprises a bioabsorbable material.
57. The method of claim 37, wherein the biocompatible scaffold
comprises a synthetic polymer selected from the group consisting of
aliphatic polyesters, poly(amino acids), copoly(ether-esters),
polyalkylene oxalates, polyamides, tyrosine-derived polycarbonates,
poly(iminocarbonates), polyorthoesters, polyoxaesters,
polyamidoesters, polyoxaesters containing amine groups,
poly(anhydrides), polyphosphazenes, polyurethanes, biosynthetic
polymers and combinations thereof.
58. The method of claim 57, wherein the biocompatible scaffold
comprises an aliphatic polyester selected from the group consisting
of homopolymers or copolymers of lactides; glycolides;
.epsilon.-caprolactone; hydroxybuterate; hydroxyvalerate;
1,4-dioxepan-2-one; 1,5,8,12-tetraoxyacyclotetradecane-7,14-dione;
1,5-dioxepan-2-one; 6,6-dimethyl-1,4-dioxan-2-one;
2,5-diketomorpholine; p-dioxanone (1,4-dioxan-2-one); trimethylene
carbonate (1,3-dioxan-2-one); alkyl derivatives of trimethylene
carbonate; .delta.-valerolactone; .beta.-butyrolactone;
.gamma.-butyrolactone; .epsilon.-decalactone; pivalolactone;
.alpha.,.alpha.-diethylpropiolactone; ethylene carbonate; ethylene
oxalate; 3-methyl-1,4-dioxane-2,5-dione; 3,3-diethyl-1,4-dioxan--
2,5-dione; 6,8-dioxabicycloctane-7-one; and combinations
thereof.
59. The method of claim 37, wherein the biocompatible scaffold
comprises a natural polymer selected from the group consisting of a
fibrin-based material, a collagen-based material, a hyaluronic
acid-based material, a cellulose-based material, silk and
combinations thereof.
60. The method of claim 37, wherein the biocompatible scaffold
comprises a ceramic selected from the group consisting of
hydroxyapatite, .alpha.-tricalcium phosphate, .beta.-tricalcium
phosphate, calcium phosphate, calcium carbonate, calcium sulfate,
bioglass, allogeneic bone graft material, xenogeneic bone graft
material and combinations thereof.
61. The method of claim 36, wherein the biocompatible scaffold
comprises a polymeric foam component having pores with an open cell
pore structure.
62. The method of claim 61, wherein the biocompatible scaffold
further comprises a reinforcing component formed of a biocompatible
mesh-containing material.
63. The method of claim 62, wherein the foam component is
integrated with the reinforcing component such that the pores of
the foam component penetrate the mesh of the reinforcing component
and interlock with the reinforcing component.
64. The method of claim 36, wherein the biocompatible scaffold
further comprises at least one additional biological component
applied thereto.
65. The method of claim 64, wherein the at least one additional
biological component comprises growth factors, matrix proteins,
enzymes, cytokines, viruses, nucleic acids, peptides, isolated
cells, platelets or combinations thereof.
66. The method of claim 36, wherein the finely divided, minced
tissue particles further comprise a plurality of cells, and at
least a portion of the plurality of cells are transfected or
transduced using a vector including at least one gene.
67. The method of claim 65, wherein the vector comprises a viral
vector or a non-viral vector.
68. The method of claim 66, wherein the at least one gene encodes a
gene product of interest.
69. The method of claim 68, wherein the gene product of interest
comprises proteins, polypeptides, interference ribonucleic acid
(iRNA) or combinations thereof.
70. The method of claim 36, wherein the method of treating tissue
is a tissue treatment technique selected from the group consisting
of tissue repair, tissue bulking, cosmetic treatment, therapeutic
treatment, tissue augmentation, and tissue sealing.
71. A method of preparing a tissue implant, comprising: providing a
bioimplantable scaffold; obtaining a sample of tissue; processing
the sample of tissue under aseptic conditions to form at least one
tissue fragment and a physiological buffering solution; and
depositing the tissue fragment on the bioimplantable scaffold to
yield a tissue implant.
72. The method of claim 71, wherein the bioimplantable scaffold
comprises a synthetic polymer, a natural polymer, an injectable
gel, a ceramic material, autogeneic tissue, allogeneic tissue,
xenogeneic tissue, and combinations thereof.
73. The method of claim 71, wherein the method further comprises
the step of incubating the tissue implant for a duration and under
conditions effective to allow cells within the at least one tissue
fragment to populate the scaffold.
74. The method of claim 71, wherein the tissue implant is incubated
for a duration in the range of about 7 days to 6 weeks.
75. The method of claim 74, wherein the tissue implant is incubated
at a temperature in the range of about 20 to 40.degree. C. and in
an atmosphere having high humidity.
76. The method of claim 71, wherein the at least one tissue
fragment comprises tissue selected from the group consisting of
cartilage tissue, meniscal tissue, ligament tissue, tendon tissue,
skin tissue, muscle tissue, periosteal tissue, pericardial tissue,
synovial tissue, nerve tissue, kidney tissue, bone marrow, liver
tissue, bladder tissue, pancreas tissue, spleen tissue, and
combinations thereof.
77. The method of claim 76, wherein the at least one tissue
fragment comprises autologous tissue.
78. The implant of claim 71, where in the at least one tissue
fragment comprises a bone-free tissue type selected from the group
consisting of cartilage, meniscus, tendon, ligament and
combinations thereof.
79. The method of claim 71, wherein the at least one tissue
fragment comprises an effective amount of viable cells that can
migrate out of the tissue fragment.
80. The method of claim 79, wherein the effective amount of cells
migrate out of the tissue fragment and populate an outer surface of
the bioimplantable scaffold.
81. The method of claim 79, wherein the effective amount of cells
migrate out of the tissue fragment and populate at least a portion
of an interior region of the scaffold, such that the cells are
embedded within the scaffold.
82. The method of claim 71, further comprising the additional step
of providing at least one additional bioimplantable scaffold and
placing the at least one additional bioimplantable scaffold over
the deposited at least one tissue fragment, such that at least a
portion of the at least one tissue fragment is disposed between at
least two bioimplantable scaffolds.
83. The method of claim 71, wherein the bioimplantable scaffold
further comprises an adhesion agent for anchoring the at least one
minced tissue fragment to the bioimplantable scaffold.
84. The method of claim 83, wherein the adhesion agent comprises an
anchoring agent selected from the group consisting of hyaluronic
acid, fibrin glue, fibrin clot, collagen gel, alginate gel,
gelatin-resorcin-formalin adhesive, mussel-based adhesive,
dihydroxyphenylalanine (DOPA) based adhesive, chitosan,
transglutaminase, poly(amino acid)-based adhesive, cellulose-based
adhesive, synthetic acrylate-based adhesives, platelet rich plasma
(PRP), Matrigel, Monostearoyl Glycerol co-Succinate (MGSA),
Monostearoyl Glycerol co-Succinate/polyethylene glycol (MGSA/PEG)
copolymers, laminin, elastin, proteoglycans and combinations
thereof.
85. The method of claim 83, wherein the adhesion agent comprises a
cross-linking agent selected from the group consisting of divinyl
sulfone (DVS), polyethylene glycon divinyl sulfone (VS-PEG-VS),
hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA),
formaldehyde, glutaraldehyde, aldehydes, isocyanates, alkyl and
aryl halides, imidoesters, N-substituted maleimides, acylating
compounds, carbodiimide, hydroxychloride, N-hydroxysuccinimide,
light, pH, temperature, and combinations thereof.
86. The method of claim 71, wherein the bioimplantable scaffold
comprises a bioabsorbable material.
87. The method of claim 72, wherein the bioimplantable scaffold
comprises a synthetic polymer selected from the group consisting of
aliphatic polyesters, poly(amino acids), poly(propylene fumarate),
copoly(ether-esters), polyalkylene oxalates, polyamides,
tyrosine-derived polycarbonates, poly(iminocarbonates),
polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters
containing amine groups, poly(anhydrides), polyphosphazenes,
polyurethanes, biosynthetic polymers and combinations thereof.
88. The method of claim 87, wherein the biocompatible scaffold
comprises an aliphatic polyester selected from the group consisting
of homopolymers or copolymers of lactides; glycolides;
.epsilon.-caprolactone; hydroxybuterate; hydroxyvalerate;
1,4-dioxepan-2-one; 1,5,8,12-tetraoxyacyclotetradecane-7,14-dione;
1,5-dioxepan-2-one; 6,6-dimethyl-1,4-dioxan-2-one;
2,5-diketomorpholine; p-dioxanone (1,4-dioxan-2-one); trimethylene
carbonate (1,3-dioxan-2-one); alkyl derivatives of trimethylene
carbonate; .delta.-valerolactone; .beta.-butyrolactone;
.gamma.-butyrolactone; .epsilon.-decalactone; pivalolactone;
.alpha.,.alpha.-diethylpropiolactone; ethylene carbonate; ethylene
oxalate; 3-methyl-1,4-dioxane-2,5-dione; 3,3-diethyl-1,4-dioxan--
2,5-dione; 6,8-dioxabicycloctane-7-one; and combinations
thereof.
89. The method of claim 72, wherein the bioimplantable scaffold
comprises a natural polymer selected from the group consisting of a
fibrin-based material, a collagen-based material, a hyaluronic
acid-based material, a cellulose-based material, silk and
combinations thereof.
90. The method of claim 72, wherein the bioimplantable scaffold
comprises a ceramic selected from the group consisting of
hydroxyapatite, .alpha.-tricalcium phosphate, .beta.-tricalcium
phosphate, bioglass, allogeneic bone graft material, xenogeneic
bone graft material and combinations thereof.
91. The method of claim 71, wherein the bioimplantable scaffold
comprises a polymeric foam component having pores with an open cell
pore structure.
92. The method of claim 91, wherein the bioimplantable scaffold
further comprises a reinforcing component formed of a biocompatible
mesh-containing material.
93. The method of claim 92, wherein the foam component is
integrated with the reinforcing component such that the pores of
the foam component penetrate the mesh of the reinforcing component
and interlock with the reinforcing component.
94. The method of claim 71, wherein the bioimplantable scaffold
further comprises at least one additional biological component
applied thereto.
95. The method of claim 94, wherein the at least one additional
biological component comprises growth factors, matrix proteins,
enzymes, cytokines, viruses, nucleic acids, peptides, isolated
cells, platelets or combinations thereof.
96. A method for measuring the effect of a substance on living
tissue, comprising the steps of: (a) creating a tissue construct by
providing a biocompatible scaffold, obtaining a sample of tissue,
processing the sample of tissue to form at least one tissue
fragment, depositing the at least one tissue fragment on the
biocompatible scaffold such that the at least one tissue fragment
is associated with the biocompatible scaffold, thereby forming a
tissue construct, and incubating the tissue construct for a
duration and under conditions that are effective to allow cells
within the tissue fragment to populate the scaffold; (b) contacting
the tissue construct with a substance; and (c) determining the
effects of the substance on the tissue construct.
97. The method of claim 96, wherein the substance comprises a drug,
a pharmaceutical composition, a chemical, a microbe, an element, a
cytokine, a growth factor, a hormone, an antibody, a peptide, a
ligand, an antagonist of membrane-bound receptors, or combinations
thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/420,093 filed on Oct. 18, 2002 and entitled
"Biocompatible Scaffold With Tissue Fragments," and to U.S.
Provisional Patent Application No. 60/419,539 filed on Oct. 18,
2002 and entitled "Biocompatible Scaffold for Ligament or Tendon
Repair."
FIELD OF THE INVENTION
[0002] The present invention relates to biocompatible tissue
implant devices for use in the repair of tissue injuries, as well
as methods for making and using such biocompatible tissue implant
devices.
BACKGROUND OF THE INVENTION
[0003] Injuries to soft tissue, such as cartilage, skin, muscle,
bone, tendon and ligament, where the tissue has been injured or
traumatized frequently require surgical intervention to repair the
damage and facilitate healing. Such surgical repairs can include
suturing or otherwise repairing the damaged tissue with known
medical devices, augmenting the damaged tissue with other tissue,
using an implant, a graft or any combination of these
techniques.
[0004] One common tissue injury involves damage to cartilage, which
is a non-vascular, resilient, flexible connective tissue. Cartilage
typically acts as a "shock-absorber" at articulating joints, but
some types of cartilage provide support to tubular structures, such
as for example, the larynx, air passages, and the ears. In general,
cartilage tissue is comprised of cartilage cells, known as
chondrocytes, located in an extracellular matrix, which contains
collagen, a structural scaffold, and aggrecan, a space-filling
proteoglycan. Several types of cartilage can be found in the body,
including hyaline cartilage, fibrocartilage and elastic cartilage.
Hyaline cartilage can appear in the body as distinct pieces, or
alternatively, this type of cartilage can be found fused to the
articular ends of bones. Hyaline cartilage is generally found in
the body as articular cartilage, costal cartilage, and temporary
cartilage (i.e., cartilage that is ultimately converted to bone
through the process of ossification). Fibrocartilage is a
transitional tissue that is typically located between tendon and
bone, bone and bone, and/or hyaline cartilage and hyaline
cartilage. Elastic cartilage, which contains elastic fibers
distributed throughout the extracellular matrix, is typically found
in the epliglottis, the ears and the nose.
[0005] One common example of hyaline cartilage injury is a
traumatic focal articular cartilage defect to the knee. A strong
impact to the joint can result in the complete or partial removal
of a cartilage fragment of various size and shape. Damaged
articular cartilage can severely restrict joint function, cause
debilitating pain and may result in long term chronic diseases such
as osteoarthritis, which gradually destroys the cartilage and
underlying bone of the joint. Injuries to the articular cartilage
tissue will not heal spontaneously and require surgical
intervention if symptomatic. The current modality of treatment
consists of lavage, removal of partially or completely unattached
tissue fragments. In addition, the surgeon will often use a variety
of methods such as abrasion, drilling or microfractures, to induce
bleeding into the cartilage defect and formation of a clot. It is
believed that the cells coming from the marrow will form a
scar-like tissue called fibrocartilage that can provide temporary
relief to some symptoms. Unfortunately, the fibrocartilage tissue
does not have the same mechanical properties as hyaline cartilage
and degrades faster over time as a consequence of wear. Patients
typically have to undergo repeated surgical procedures which can
lead to the complete deterioration of the cartilage surface. More
recently, experimental approaches involving the implantation of
autologous chondrocytes have been used with increasing frequency.
The process involves the harvest of a small biopsy of articular
cartilage in a first surgical procedure, which is then transported
to a laboratory specialized in cell culture for amplification. The
tissue biopsy is treated with enzymes that will release the
chondrocyte cells from the matrix, and the isolated cells will be
grown for a period of 3 to 4 weeks using standard tissue culture
techniques. Once the cell population has reached a target number,
the cells are sent back to the surgeon for implantation during a
second surgical procedure. This manual labor-intense process is
extremely costly and time consuming. Although, the clinical data
suggest long term benefit for the patient, the prohibitive cost of
the procedure combined with the traumatic impact of two surgical
procedures to the knee, has hampered adoption of this
technique.
[0006] One common example of cartilage injury is damage to the
menisci of a knee joint. There are two menisci of the knee joint, a
medial and a lateral meniscus. Each meniscus is a biconcave,
fibrocartilage tissue that is interposed between the femur and
tibia of the leg. In addition to the menisci of the knee joint,
meniscal cartilage can also be found in the acromioclavicular
joint, i.e., the joint between the clavicle and the acromion of the
scapula, in the sternoclavicular joint, i.e., the joint between the
clavicle and the sternum, and in the temporomandibular joint, i.e.,
the joint of the lower jaw. The primary functions of meniscal
cartilage are to bear loads, to absorb shock and to stabilize a
joint. If not treated properly, an injury to the meniscus, such as
a "bucket-handle tear" in the knee joint, may lead to the
development of osteoarthritis. Current conventional treatment
modalities for damaged meniscal cartilage include the removal
and/or surgical repair of the damaged cartilage.
[0007] Another common form of tissue injury involves damage to the
ligaments and/or tendons. Ligaments and tendons are cords or bands
of fibrous tissue that contains soft collagenous tissue. Ligaments
connect bone to bone, while tendons connect muscle to bone. Tendons
are fibrous cords or bands of variable length that have
considerable strength but are virtually devoid of elasticity.
Ligaments, in contrast, are generally pliant and flexible, to allow
the ligament tissue to have freedom of movement, and simultaneously
strong and inextensible, to prevent the ligament tissue from
readily yielding under applied force. Ligaments and tendons are
comprised of fascicles, which contain the basic fibril of the
ligament or tendon, as well as the cells that produce the ligament
or tendon, known as fibroblasts. The fascicles of the tendon are
generally comprised of very densely arranged collagenous fibers,
parallel rows of elongated fibroblasts, and a proteoglycan matrix.
The fascicles of ligaments also contain a proteoglycan matrix,
fibroblasts and collagen fibrils, but the fibrils found in ligament
tissue are generally less dense and less structured than the
fibrils found in tendon tissue.
[0008] One example of a common ligament injury is a torn anterior
cruciate ligament (ACL), which is one of four major ligaments of
the knee. The primary function of the ACL is to constrain anterior
translation, rotary laxity and hyperextension. The lack of an ACL
causes instability of the knee joint and leads to degenerative
changes in the knee such as osteoarthritis. The most common repair
technique is to remove and discard the ruptured ACL and reconstruct
a new ACL using autologous bone-patellar, tendon-bone or hamstring
tendons. Although this technique has shown long-term clinical
efficacy, there is morbidity associated with the harvest site of
the tissue graft. Synthetic prosthetic devices have been clinically
evaluated in the past with little long-term success. The advantages
of a synthetic implant are that the patient does not suffer from
the donor site morbidity that is associated with autograft
procedures, and that patients having a synthetic implant are able
to undergo faster rehabilitation of the knee. These synthetic
devices were composed of non-resorbable materials and were designed
to be permanent prosthetic implants. A number of problems were
found during the clinical trials of these implants, such as for
example, synovitis, bone tunnel enlargement, wear debris, and
elongation and rupture of the devices. For this reason, autograft
reconstruction is still the widely accepted solution for repairing
a ruptured ACL.
[0009] A common tendon injury is a damaged or torn rotator cuff,
which is the portion of the shoulder joint that facilitates
circular motion of the humerus bone relative to the scapula. The
most common injury associated with the rotator cuff is a strain or
tear to the supraspinatus tendon. This tear can occur at the
insertion site of the supraspinatus tendon, where the tendon
attaches to the humerus, thereby partially or fully releasing the
tendon (depending upon the severity of the injury) from the bone.
Additionally, the strain or tear can occur within the tendon
itself. Treatment for a strained tendon usually involves rest and
reduced use of the tendon. However, depending upon the severity of
the injury, a torn tendon may require surgical intervention, such
as for example, in the case of a full tear of the supraspinatus
tendon from the humerus. In the case of severe tendon damage,
surgical intervention can involve the repair and/or reattachment of
torn tissue, which typically requires a healing and recovery
period.
[0010] There is a continuing need in this art for novel surgical
techniques for the surgical treatment of damaged tissue (e.g.,
cartilage, meniscal cartilage, ligaments, tendons and skin) that
can effect a more reliable tissue repair and can facilitate the
healing of injured tissue. Various surgical implants are known and
have been used in surgical procedures to help achieve these
benefits. For example, it is known to use various devices and
techniques for creating implants having isolated cells loaded onto
a delivery vehicle. Such cell-seeded implants are used in an in
vitro method of making and/or repairing cartilage by growing
cartilaginous structures that consist of chondrocytes seeded onto
biodegradable, biocompatible fibrous polymeric matrices. Such
methods require the initial isolation of chondrocytes from
cartilaginous tissue prior to the chondrocytes being seeded onto
the polymeric matrices. Other techniques for repairing damaged
tissue employ implants having stem or progenitor cells that are
used to produce the desired tissue. For example, it is known to use
stem or progenitor cells, such as the cells within fatty tissue,
muscle, or bone marrow, to regenerate bone and/or cartilage in a
patient. The stem cells are removed from the patient and placed in
an environment favorable to cartilage formation, thereby inducing
the fatty tissue cells to proliferate and to create a different
type of cell, such as for example, cartilage cells.
[0011] There continues to exist a need in this art for novel
devices and methods for making and/or repairing damaged tissue and
for hastening the healing of the damaged tissue.
SUMMARY OF THE INVENTION
[0012] This invention relates to biocompatible tissue implants for
use in treating tissue, and the methods for making and using these
devices. For example, the tissue implants can be used for the
repair and/or regeneration of diseased or damaged tissue. Further,
the tissue implants can be used for tissue bulking, cosmetic
treatments, therapeutic treatments, tissue augmentation, and tissue
repair. The implants include a biocompatible scaffold that is
associated with a suspension containing at least one minced tissue
fragment. The biocompatible tissue implants can also include an
additional biological agent and/or an optional retaining element
placed over the suspension of minced tissue.
[0013] The invention also relates to a method of preparing such
biocompatible tissue implants. The implants are made by providing
at least one biocompatible scaffold and a sample of minced tissue,
processing the tissue sample to create a suspension of viable
tissue having at least one minced tissue fragment, and depositing
the tissue sample upon the biocompatible scaffold. In one
embodiment, the method of producing these implants can include the
further step of incubating the tissue-laden scaffold in a suitable
environment for a duration and under conditions that are sufficient
to effectively allow cells within the tissue sample to populate the
scaffold.
[0014] The invention is also directed to a kit to assist in the
preparation of the tissue implants of the present invention. The
kits of the present invention include a sterile container which
houses at least one biocompatible scaffold, a harvesting tool for
collecting a tissue sample from a subject, and one or more reagents
for sustaining the viability of the tissue sample. The kit can also
include a processing tool for mincing the tissue into tissue
particles, or alternatively, the harvesting tool can be adapted to
collect the tissue sample and to process the sample into finely
divided tissue particles. The kit can, optionally, also include a
delivery device for transferring the scaffold from the sterile
container to a subject for implantation.
[0015] The invention also relates to methods of treating tissue
using the biocompatible tissue implants of the present invention.
Tissue treatment according to these methods can be performed by
providing a biocompatible scaffold and a sample of minced tissue,
depositing the tissue sample upon the biocompatible scaffold, and
placing the tissue-laden scaffold in a desired position relative to
the tissue to be treated. In one embodiment, tissue repair can be
achieved by providing a biocompatible scaffold and a sample of
minced tissue, depositing the tissue sample in a desired position
relative to the tissue injury, and placing the biocompatible
scaffold over the tissue. In another embodiment, the method of
producing these implants can include the further step of incubating
the tissue-laden scaffold in a suitable environment for a duration
and under conditions that are effective to allow cells within the
tissue sample to populate the scaffold. In yet another embodiment,
the methods of treating tissue can also include the additional step
of affixing the scaffold in a desired position relative to the
tissue to be treated, such as, for example, by fastening the
tissue-laden scaffold in place.
[0016] The present invention is also directed to methods for
measuring the effect(s) of a substance on living tissue. According
to this aspect of the invention, the bioimplantable tissue implants
of the present invention can be used to create tissue constructs
that can be contacted with a test substance so that the effects of
the substance on living tissue can be observed and measured. Thus,
the bioimplantable tissue constructs of the present invention can
be used as a biological screening assay to measure the effects of a
test substance on living tissue by examining the effect on various
biological responses, such as for example, the effect on cell
migration, cell proliferation and differentiation and maintenance
of cell phenotype.
[0017] In embodiments in which the implant is used for tissue
repair, the tissue repair implant can be used to treat a variety of
injuries, such as for example, injuries occurring within the
musculoskeletal system, such as rotator cuff injuries, ACL
ruptures, or meniscal tears, as well as injuries occurring in other
connective tissues, such as skin and cartilage. Furthermore, such
implants can be used in other orthopaedic surgical procedures, such
as hand and foot surgery, to repair tissues such as ligaments,
nerves, and tendons.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The invention will be more fully understood by reference to
the following detailed description when considered in conjunction
with the accompanying drawings, in which:
[0019] FIG. 1A is photomicrograph that demonstrates that cells in a
cartilage tissue sample migrate extensively into a polymer
scaffold;
[0020] FIG. 1B is a photomicrograph that demonstrates that the
migrating cells of FIG. 1A retain their phenotype and the migrating
cells produce cellular matrix that stains positive for sulfated
glycosaminoglycan using the Safranin O stain;
[0021] FIG. 2A is a photomicrograph that demonstrates that cells
within the minced tissue loaded on the biocompatible scaffolds,
following implantation into SCID mice, have proliferated and filled
the entire scaffold;
[0022] FIG. 2B is a photomicrograph that demonstrates that cells
within the minced tissue, following implantation into SCID mice,
are chondrocyte-like and are surrounded by an abundant matrix that
stains positive for Safranin O;
[0023] FIG. 3A is a photomicrograph that illustrates a scaffold
loaded with minced tissue;
[0024] FIG. 3B is a photomicrograph that illustrates a scaffold
loaded with minced tissue and platelet rich plasma (PRP) and
demonstrates that growth factors in the PRP are beneficial in
promoting the migration of chondrocyte cells from the minced tissue
and in promoting maintenance of differentiated phenotype of the
chondrocyte cells within the scaffolds;
[0025] FIG. 4 is a photomicrograph that demonstrates that
autologous cell dispersion (derived from skin) is present
histologically as keratinocyte islands;
[0026] FIG. 5A is a photomicrograph that demonstrates the extensive
migration of cells into the polymer scaffolds after incubating for
6 weeks in culture the biocompatible scaffolds having minced
anterior cruciate tissue fragments that have been treated with
collagenase;
[0027] FIG. 5B is a photomicrograph that demonstrates the extensive
migration of cells into the polymer scaffolds after incubating for
6 weeks in culture the biocompatible scaffolds having minced
anterior cruciate tissue fragments treated without collagenase;
[0028] FIG. 6A is a graph that demonstrates that cells in a
meniscal explant sample migrate extensively into a polymer
scaffold;
[0029] FIG. 6B is a photomicrograph that illustrates the histology
of cross sections of the associated meniscal explant and
biocompatible scaffolds, which demonstrates that cells in the
meniscal explant sample migrate into the polymer scaffold.
[0030] FIGS. 7A-7C are photomicrographs of histological sections of
explant samples obtained following the procedure of Example 7,
demonstrating the distribution and nature of tissue formed within a
scaffold and grown from minced cartilage tissue fragments.
[0031] FIGS. 8A-8C are photomicrographs of histological sections of
explant samples obtained following the procedure of Example 7,
demonstrating the distribution and nature of tissue formed within a
scaffold and grown from bone cartilage paste.
[0032] FIG. 9 is a graph comparing the numbers of cells obtained
for different sizes of minced cartilage tissue fragments.
[0033] FIGS. 10A-10C are photomicrographs of histological sections
of explant samples obtained following the procedure of Example 8,
demonstrating the uniformity of the cartilage-like tissue obtained
with minced cartilage tissue fragments of different sizes.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The biocompatible tissue implants of the present invention
are used in the treatment of various types of tissue for various
purposes. For example, the implants can be used for the repair
and/or regeneration of diseased or damaged tissue, or they can be
used for tissue bulking, tissue augmentation, cosmetic treatments,
therapeutic treatments, and for tissue sealing. The tissue implants
include a biocompatible scaffold and a suspension of minced tissue
having at least one minced tissue fragment, wherein the minced
tissue suspension is associated with the scaffold. The minced
tissue in the suspension of the present invention includes at least
one viable cell that can migrate from the tissue fragment and onto
the scaffold.
[0035] Although the implants are sometimes referred to herein as
"tissue repair implants" and the methods of using the implants are
sometimes characterized as tissue repair techniques, it is
understood that the implants can be used for a variety of tissue
treatments, including but not limited to tissue repair, tissue
bulking, cosmetic treatments, therapeutic treatments, tissue
augmentation, and tissue sealing.
[0036] The biocompatible tissue implant of the present invention
includes a biocompatible scaffold having at least a portion in
contact with the minced tissue suspension. The minced tissue
suspension can be disposed on the outer surface of the scaffold, on
an inner region of the scaffold, and any combination thereof, or
alternatively, the entire scaffold can be in contact with the
minced tissue suspension. The scaffold can be formed using
virtually any material or delivery vehicle that is biocompatible,
bioimplantable, easily sterilized and that has sufficient
structural integrity and physical and/or mechanical properties to
effectively provide for ease of handling in an operating room
environment and to permit it to accept and retain sutures or other
fasteners without substantially tearing. Alternatively, the
scaffold could be in the form of an injectable gel that would set
in place at the defect site. Sufficient strength and physical
properties are developed in the scaffold through the selection of
materials used to form the scaffold, and the manufacturing process.
Preferably, the scaffold is also pliable so as to allow the
scaffold to adjust to the dimensions of the target site of
implantation. In some embodiments, the scaffold can be a
bioresorbable or bioabsorbable material.
[0037] In one embodiment of the present invention, the scaffold can
be formed from a biocompatible polymer. A variety of biocompatible
polymers can be used to make the biocompatible tissue implants or
scaffold devices according to the present invention. The
biocompatible polymers can be synthetic polymers, natural polymers
or combinations thereof. As used herein the term "synthetic
polymer" refers to polymers that are not found in nature, even if
the polymers are made from naturally occurring biomaterials. The
term "natural polymer" refers to polymers that are naturally
occurring. In embodiments where the scaffold includes at least one
synthetic polymer, suitable biocompatible synthetic polymers can
include polymers selected from the group consisting of aliphatic
polyesters, poly(amino acids), poly(propylene fumarate),
copoly(ether-esters), polyalkylenes oxalates, polyamides, tyrosine
derived polycarbonates, poly(iminocarbonates), polyorthoesters,
polyoxaesters, polyamidoesters, polyoxaesters containing amine
groups, poly(anhydrides), polyphosphazenes, and blends thereof.
Suitable synthetic polymers for use in the present invention can
also include biosynthetic polymers based on sequences found in
collagen, elastin, thrombin, fibronectin, starches, poly(amino
acid), gelatin, alginate, pectin, fibrin, oxidized cellulose,
chitin, chitosan, tropoelastin, hyaluronic acid, ribonucleic acids,
deoxyribonucleic acids, polypeptides, proteins, polysaccharides,
polynucleotides and combinations thereof.
[0038] For the purpose of this invention aliphatic polyesters
include, but are not limited to, homopolymers and copolymers of
lactide (which includes lactic acid, D-,L- and meso lactide);
glycolide (including glycolic acid); .epsilon.-caprolactone;
p-dioxanone (1,4-dioxan-2-one); trimethylene carbonate
(1,3-dioxan-2-one); alkyl derivatives of trimethylene carbonate;
.delta.-valerolactone; .beta.-butyrolactone; .gamma.-butyrolactone;
.epsilon.-decalactone; hydroxybutyrate; hydroxyvalerate;
1,4-dioxepan-2-one (including its dimer
1,5,8,12-tetraoxacyclotetradecane-7,14-dione); 1,5-dioxepan-2-one;
6,6-dimethyl-1,4-dioxan-2-one; 2,5-diketomorpholine; pivalolactone;
.alpha.,.alpha. diethylpropiolactone; ethylene carbonate; ethylene
oxalate; 3-methyl-1,4-dioxane-2,5-dione;
3,3-diethyl-1,4-dioxan-2,5-dione- ; 6,6-dimethyl-dioxepan-2-one;
6,8-dioxabicycloctane-7-one and polymer blends thereof. Aliphatic
polyesters used in the present invention can be homopolymers or
copolymers (random, block, segmented, tapered blocks, graft,
triblock, etc.) having a linear, branched or star structure.
Poly(iminocarbonates), for the purpose of this invention, are
understood to include those polymers as described by Kemnitzer and
Kohn, in the Handbook of Biodegradable Polymers, edited by Domb,
et. al., Hardwood Academic Press, pp. 251-272 (1997).
Copoly(ether-esters), for the purpose of this invention, are
understood to include those copolyester-ethers as described in the
Journal of Biomaterials Research, Vol. 22, pages 993-1009, 1988 by
Cohn and Younes, and in Polymer Preprints (ACS Division of Polymer
Chemistry), Vol. 30(1), page 498, 1989 by Cohn (e.g., PEO/PLA).
Polyalkylene oxalates, for the purpose of this invention, include
those described in U.S. Pat. Nos. 4,208,511; 4,141,087; 4,130,639;
4,140,678; 4,105,034; and 4,205,399. Polyphosphazenes, co-, ter-
and higher order mixed monomer based polymers made from L-lactide,
D,L-lactide, lactic acid, glycolide, glycolic acid, para-dioxanone,
trimethylene carbonate and .epsilon.-caprolactone such as are
described by Allcock in The Encyclopedia of Polymer Science, Vol.
13, pages 31-41, Wiley Intersciences, John Wiley & Sons, 1988
and by Vandorpe, et al in the Handbook of Biodegradable Polymers,
edited by Domb, et al., Hardwood Academic Press, pp. 161-182
(1997). Polyanhydrides include those derived from diacids of the
form HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.m--O--C.-
sub.6H.sub.4--COOH, where "m" is an integer in the range of from 2
to 8, and copolymers thereof with aliphatic alpha-omega diacids of
up to 12 carbons. Polyoxaesters, polyoxaamides and polyoxaesters
containing amines and/or amido groups are described in one or more
of the following U.S. Pat. Nos. 5,464,929; 5,595,751; 5,597,579;
5,607,687; 5,618,552; 5,620,698; 5,645,850; 5,648,088; 5,698,213;
5,700,583; and 5,859,150. Polyorthoesters such as those described
by Heller in Handbook of Biodegradable Polymers, edited by Domb, et
al., Hardwood Academic Press, pp. 99-118 (1997).
[0039] As used herein, the term "glycolide" is understood to
include polyglycolic acid. Further, the term "lactide" is
understood to include L-lactide, D-lactide, blends thereof, and
lactic acid polymers and copolymers.
[0040] Elastomeric copolymers are also particularly useful in the
present invention. Suitable elastomeric polymers include those with
an inherent viscosity in the range of about 1.2 dL/g to 4 dL/g,
more preferably about 1.2 dL/g to 2 dL/g and most preferably about
1.4 dL/g to 2 dL/g as determined at 25.degree. C. in a 0.1 gram per
deciliter (g/dL) solution of polymer in hexafluoroisopropanol
(HFIP). Further, suitable elastomers exhibit a high percent
elongation and a low modulus, while possessing good tensile
strength and good recovery characteristics. In the preferred
embodiments of this invention, the elastomer exhibits a percent
elongation greater than about 200 percent and preferably greater
than about 500 percent. In addition to these elongation and modulus
properties, suitable elastomers should also have a tensile strength
greater than about 500 psi, preferably greater than about 1,000
psi, and a tear strength of greater than about 50 lbs/inch,
preferably greater than about 80 lbs/inch.
[0041] Exemplary biocompatible elastomers that can be used in the
present invention include, but are not limited to, elastomeric
copolymers of .epsilon.-caprolactone and glycolide (including
polyglycolic acid) with a mole ratio of .epsilon.-caprolactone to
glycolide of from about 35:65 to about 65:35, more preferably from
45:55 to 35:65; elastomeric copolymers of .epsilon.-caprolactone
and lactide (including L-lactide, D-lactide, blends thereof, and
lactic acid polymers and copolymers) where the mole ratio of
.epsilon.-caprolactone to lactide is from about 35:65 to about
65:35 and more preferably from 45:55 to 30:70 or from about 95:5 to
about 85:15; elastomeric copolymers of p-dioxanone
(1,4-dioxan-2-one) and lactide (including L-lactide, D-lactide,
blends thereof, and lactic acid polymers and copolymers) where the
mole ratio of p-dioxanone to lactide is from about 40:60 to about
60:40; elastomeric copolymers of .epsilon.-caprolactone and
p-dioxanone where the mole ratio of .epsilon.-caprolactone to
p-dioxanone is from about from 30:70 to about 70:30; elastomeric
copolymers of p-dioxanone and trimethylene carbonate where the mole
ratio of p-dioxanone to trimethylene carbonate is from about 30:70
to about 70:30; elastomeric copolymers of trimethylene carbonate
and glycolide (including polyglycolic acid) where the mole ratio of
trimethylene carbonate to glycolide is from about 30:70 to about
70:30; elastomeric copolymers of trimethylene carbonate and lactide
(including L-lactide, D-lactide, blends thereof, and lactic acid
polymers and copolymers) where the mole ratio of trimethylene
carbonate to lactide is from about 30:70 to about 70:30; and blends
thereof. Examples of suitable biocompatible elastomers are
described in U.S. Pat. Nos. 4,045,418; 4,057,537 and 5,468,253.
[0042] In one embodiment, the elastomer is a copolymer of 35:65
.epsilon.-caprolactone and glycolide, formed in a dioxane solvent
and including a polydioxanone mesh. In another embodiment, the
elastomer is a copolymer of 40:60 .epsilon.-caprolactone and
lactide with a polydioxanone mesh. In yet another embodiment, the
elastomer is a 50:50 blend of a 35:65 copolymer of
.epsilon.-caprolactone and glycolide and 40:60 copolymer of
.epsilon.-caprolactone and lactide. The polydioxanone mesh may be
in the form of a one layer thick two-dimensional mesh or a
multi-layer thick three-dimensional mesh.
[0043] The scaffold of the present invention can, optionally, be
formed from a bioresorbable or bioabsorbable material that has the
ability to resorb in a timely fashion in the body environment. The
differences in the absorption time under in vivo conditions can
also be the basis for combining two different copolymers when
forming the scaffolds of the present invention. For example, a
copolymer of 35:65 .epsilon.-caprolactone and glycolide (a
relatively fast absorbing polymer) can be blended with 40:60
.epsilon.-caprolactone and L-lactide copolymer (a relatively slow
absorbing polymer) to form a biocompatible scaffold. Depending upon
the processing technique used, the two constituents can be either
randomly inter-connected bicontinuous phases, or the constituents
could have a gradient-like architecture in the form of a laminate
type composite with a well integrated interface between the two
constituent layers. The microstructure of these scaffolds can be
optimized to regenerate or repair the desired anatomical features
of the tissue that is being regrown.
[0044] In one embodiment, it is desirable to use polymer blends to
form scaffolds which transition from one composition to another
composition in a gradient-like architecture. Scaffolds having this
gradient-like architecture are particularly advantageous in tissue
engineering applications to repair or regenerate the structure of
naturally occurring tissue such as cartilage (articular, meniscal,
septal, tracheal, auricular, costal, etc.), tendon, ligament,
nerve, esophagus, skin, bone, and vascular tissue. For example, by
blending an elastomer of .epsilon.-caprolactone-co-glycolide with
.epsilon.-caprolactone-co-lactid- e (e.g., with a mole ratio of
about 5:95) a scaffold may be formed that transitions from a softer
spongy material to a stiffer more rigid material, for example, in a
manner similar to the transition from cartilage to bone. Clearly,
one of ordinary skill in the art will appreciate that other polymer
blends may be used for similar gradient effects, or to provide
different gradients (e.g., different absorption profiles, stress
response profiles, or different degrees of elasticity). For
example, such design features can establish a concentration
gradient for the suspension of minced tissue associated with the
scaffolds of the present invention, such that a higher
concentration of the tissue fragments is present in one region of
the implant (e.g., an interior portion) than in another region
(e.g., outer portions).
[0045] The biocompatible scaffold of the tissue repair implant of
the present invention can also include a reinforcing material
comprised of any absorbable or non-absorbable textile having, for
example, woven, knitted, warped knitted (i.e., lace-like),
non-woven, and braided structures. In one embodiment, the
reinforcing material has a mesh-like structure. In any of the above
structures, mechanical properties of the material can be altered by
changing the density or texture of the material, the type of knit
or weave of the material, the thickness of the material, or by
embedding particles in the material. The mechanical properties of
the material may also be altered by creating sites within the mesh
where the fibers are physically bonded with each other or
physically bonded with another agent, such as, for example, an
adhesive or a polymer. The fibers used to make the reinforcing
component can be monofilaments, yarns, threads, braids, or bundles
of fibers. These fibers can be made of any biocompatible material
including bioabsorbable materials such as polylactic acid (PLA),
polyglycolic acid (PGA), polycaprolactone (PCL), polydioxanone
(PDO), trimethylene carbonate (TMC), copolymers or blends thereof.
These fibers can also be made from any biocompatible materials
based on natural polymers including silk and collagen-based
materials. These fibers can also be made of any biocompatible fiber
that is nonresorbable, such as, for example, polyethylene,
polyethylene terephthalate, poly(tetrafluoroethylene),
polycarbonate, polypropylene and poly(vinyl alcohol). In one
embodiment, the fibers are formed from 95:5 copolymer of lactide
and glycolide.
[0046] In another embodiment, the fibers that form the reinforcing
material can be made of a bioabsorbable glass. Bioglass, a silicate
containing calcium phosphate glass, or calcium phosphate glass with
varying amounts of solid particles added to control resorption time
are examples of materials that could be spun into glass fibers and
used for the reinforcing material. Suitable solid particles that
may be added include iron, magnesium, sodium, potassium, and
combinations thereof.
[0047] The biocompatible scaffolds as well as the reinforcing
material may also be formed from a thin, perforation-containing
elastomeric sheet with pores or perforations to allow tissue
ingrowth. Such a sheet could be made of blends or copolymers of
polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone
(PCL), and polydioxanone (PDO).
[0048] In one embodiment, filaments that form the biocompatible
scaffolds or the reinforcing material may be co-extruded to produce
a filament with a sheath/core construction. Such filaments are
comprised of a sheath of biodegradable polymer that surrounds one
or more cores comprised of another biodegradable polymer. Filaments
with a fast-absorbing sheath surrounding a slower-absorbing core
may be desirable in instances where extended support is necessary
for tissue ingrowth.
[0049] One of ordinary skill in the art will appreciate that one or
more layers of the reinforcing material may be used to reinforce
the tissue implant of the invention. In addition, biodegradable
textile scaffolds, such as, for example, meshes, of the same
structure and chemistry or different structures and chemistries can
be overlaid on top of one another to fabricate biocompatible tissue
implants with superior mechanical strength.
[0050] In embodiments where the scaffold includes at least one
natural polymer, suitable examples of natural polymers include, but
are not limited to, fibrin-based materials, collagen-based
materials, hyaluronic acid-based materials, glycoprotein-based
materials, cellulose-based materials, silks and combinations
thereof. By way of nonlimiting example, the biocompatible scaffold
can be constructed from a collagen-based small intestine
submucosa.
[0051] In another embodiment of the present invention, the
biocompatible scaffold can be formed from a biocompatible ceramic
material. Suitable biocompatible ceramic materials include, for
example, hydroxyapatite, .alpha.-tricalcium phosphate,
.beta.-tricalcium phosphate, bioactive glass, calcium phosphate,
calcium sulfate, calcium carbonate, xenogeneic and allogeneic bone
material and combinations thereof. Suitable bioactive glass
materials for use in the present invention include silicates
containing calcium phosphate glass, or calcium phosphate glass with
varying amounts of solid particles added to control resorption
time. Suitable compounds that may be incorporated into the calcium
phosphate bioactive glass include, but are not limited to,
magnesium oxide, sodium oxide, potassium oxide, and combinations
thereof.
[0052] In yet another embodiment of the tissue implants of the
present invention, the scaffold can be formed using tissue grafts,
such as may be obtained from autogeneic tissue, allogeneic tissue
and xenogeneic tissue. By way of non-limiting example, tissues such
as skin, cartilage, ligament, tendon, periosteum, perichondrium,
synovium, fascia, mesenter and sinew can be used as tissue grafts
to form the biocompatible scaffold. In some embodiments where an
allogeneic tissue is used, tissue from a fetus or newborns can be
used to avoid the immunogenicity associated with some adult
tissues.
[0053] In another embodiment, the scaffold could be in the form of
an injectable gel that would set in place at the defect site. The
gel can be a biological or synthetic hydrogel, including alginate,
cross-linked alginate, hyaluronic acid, collagen gel, fibrin glue,
fibrin clot, poly(N-isopropylacrylamide), agarose, chitin,
chitosan, cellulose, polysaccharides, poly(oxyalkylene), a
copolymer of poly(ethylene oxide)-poly(propylene oxide), poly(vinyl
alcohol), polyacrylate, platelet rich plasma (PRP) clot, platelet
poor plasma (PPP) clot, Matrigel, or blends thereof.
[0054] In still yet another embodiment of the tissue implants, the
scaffold can be formed from a polymeric foam component having pores
with an open cell pore structure. The pore size can vary, but
preferably, the pores are sized to allow tissue ingrowth. More
preferably, the pore size is in the range of about 50 to 1000
microns, and even more preferably, in the range of about 50 to 500
microns. The polymeric foam component can, optionally, contain a
reinforcing component, such as for example, the textiles disclosed
above. In some embodiments where the polymeric foam component
contains a reinforcing component, the foam component can be
integrated with the reinforcing component such that the pores of
the foam component penetrate the mesh of the reinforcing component
and interlock with the reinforcing component.
[0055] The foam component of the tissue implant may be formed as a
foam by a variety of techniques well known to those having ordinary
skill in the art. For example, the polymeric starting materials may
be foamed by lyophilization, supercritical solvent foaming (i.e.,
as described in EP 464,163), gas injection extrusion, gas injection
molding or casting with an extractable material (e.g., salts, sugar
or similar suitable materials).
[0056] In one embodiment, the foam component of the engineered
tissue repair implant devices of the present invention may be made
by a polymer-solvent phase separation technique, such as
lyophilization. Generally, however, a polymer solution can be
separated into two phases by any one of the four techniques: (a)
thermally induced gelation/crystallization; (b) non-solvent induced
separation of solvent and polymer phases; (c) chemically induced
phase separation, and (d) thermally induced spinodal decomposition.
The polymer solution is separated in a controlled manner into
either two distinct phases or two bicontinuous phases. Subsequent
removal of the solvent phase usually leaves a porous structure with
a density less than the bulk polymer and pores in the micrometer
ranges. See Microcellular Foams Via Phase Separation, J. Vac. Sci.
Technol., A. T. Young, Vol. 4(3), May/June 1986.
[0057] The steps involved in the preparation of these foams include
choosing the right solvents for the polymers to be lyophilized and
preparing a homogeneous solution. Next, the polymer solution is
subjected to a freezing and vacuum drying cycle. The freezing step
phase separates the polymer solution and vacuum drying step removes
the solvent by sublimation and/or drying, leaving a porous polymer
structure or an interconnected open cell porous foam.
[0058] Suitable solvents that may be used in the preparation of the
foam component include, but are not limited to, formic acid, ethyl
formate, acetic acid, hexafluoroisopropanol (HFIP), cyclic ethers
(e.g., tetrahydrofuran (THF), dimethylene fluoride (DMF), and
polydioxanone (PDO)), acetone, acetates of C2 to C5 alcohols (e.g.,
ethyl acetate and t-butylacetate), glyme (e.g., monoglyme, ethyl
glyme, diglyme, ethyl diglyme, triglyme, butyl diglyme and
tetraglyme), methylethyl ketone, dipropyleneglycol methyl ether,
lactones (e.g., .gamma.-valerolactone, .delta.-valerolactone,
.beta.-butyrolactone, .gamma.-butyrolactone), 1,4-dioxane,
1,3-dioxolane, 1,3-dioxolane-2-one (ethylene carbonate),
dimethlycarbonate, benzene, toluene, benzyl alcohol, p-xylene,
naphthalene, tetrahydrofuran, N-methyl pyrrolidone,
dimethylformamide, chloroform, 1,2-dichloromethane, morpholine,
dimethylsulfoxide, hexafluoroacetone sesquihydrate (HFAS), anisole
and mixtures thereof. Among these solvents, a preferred solvent is
1,4-dioxane. A homogeneous solution of the polymer in the solvent
is prepared using standard techniques.
[0059] The applicable polymer concentration or amount of solvent
that may be utilized will vary with each system. Generally, the
amount of polymer in the solution can vary from about 0.5% to about
90% and, preferably, will vary from about 0.5% to about 30% by
weight, depending on factors such as the solubility of the polymer
in a given solvent and the final properties desired in the
foam.
[0060] In one embodiment, solids may be added to the
polymer-solvent system to modify the composition of the resulting
foam surfaces. As the added particles settle out of solution to the
bottom surface, regions will be created that will have the
composition of the added solids, not the foamed polymeric material.
Alternatively, the added solids may be more concentrated in desired
regions (i.e., near the top, sides, or bottom) of the resulting
tissue implant, thus causing compositional changes in all such
regions. For example, concentration of solids in selected locations
can be accomplished by adding metallic solids to a solution placed
in a mold made of a magnetic material (or vice versa).
[0061] A variety of types of solids can be added to the
polymer-solvent system. Preferably, the solids are of a type that
will not react with the polymer or the solvent. Generally, the
added solids have an average diameter of less than about 1.0 mm and
preferably will have an average diameter of about 50 to about 500
microns. Preferably, the solids are present in an amount such that
they will constitute from about 1 to about 50 volume percent of the
total volume of the particle and polymer-solvent mixture (wherein
the total volume percent equals 100 volume percent).
[0062] Exemplary solids include, but are not limited to, particles
of demineralized bone, calcium phosphate particles, bioglass
particles, calcium sulfate, or calcium carbonate particles for bone
repair, leachable solids for pore creation and particles of
bioabsorbable polymers not soluble in the solvent system that are
effective as reinforcing materials or to create pores as they are
absorbed, and non-bioabsorbable materials.
[0063] Suitable leachable solids include nontoxic leachable
materials such as salts (e.g., sodium chloride, potassium chloride,
calcium chloride, sodium tartrate, sodium citrate, and the like),
biocompatible mono and disaccharides (e.g., glucose, fructose,
dextrose, maltose, lactose and sucrose), polysaccharides (e.g.,
starch, alginate, chitosan), water soluble proteins (e.g., gelatin
and agarose). The leachable materials can be removed by immersing
the foam with the leachable material in a solvent in which the
particle is soluble for a sufficient amount of time to allow
leaching of substantially all of the particles, but which does not
dissolve or detrimentally alter the foam. The preferred extraction
solvent is water, most preferably distilled-deionized water. Such a
process is described in U.S. Pat. No. 5,514,378. Preferably the
foam will be dried after the leaching process is complete at low
temperature and/or vacuum to minimize hydrolysis of the foam unless
accelerated absorption of the foam is desired.
[0064] Suitable non-bioabsorbable materials include biocompatible
metals such as stainless steel, cobalt chrome, titanium and
titanium alloys, and bioinert ceramic particles (e.g., alumina,
zirconia, and calcium sulfate particles). Further, the
non-bioabsorbable materials may include polymers such as
polyethylene, polyvinylacetate, polymethylmethacrylate,
polypropylene, poly(ethylene terephthalate), silicone, polyethylene
oxide, polyethylene glycol, polyurethanes, polyvinyl alcohol,
natural polymers (e.g., cellulose particles, chitin, and keratin),
and fluorinated polymers and copolymers (e.g., polyvinylidene
fluoride, polytetrafluoroethylene, and hexafluoropropylene).
[0065] It is also possible to add solids (e.g., barium sulfate)
that will render the tissue implants radio opaque. The solids that
may be added also include those that will promote tissue
regeneration or regrowth, as well as those that act as buffers,
reinforcing materials or porosity modifiers.
[0066] As noted above, porous, reinforced tissue repair implant
devices of the present invention are made by injecting, pouring, or
otherwise placing, the appropriate polymer solution into a mold
set-up comprised of a mold and the reinforcing elements of the
present invention. The mold set-up is cooled in an appropriate bath
or on a refrigerated shelf and then lyophilized, thereby providing
a reinforced scaffold. A biological component can be added either
before or after the lyophilization step. In the course of forming
the foam component, it is believed to be important to control the
rate of freezing of the polymer-solvent system. The type of pore
morphology that is developed during the freezing step is a function
of factors such as the solution thermodynamics, freezing rate,
temperature to which it is cooled, concentration of the solution,
and whether homogeneous or heterogenous nucleation occurs. One of
ordinary skill in the art can readily optimize the parameters
without undue experimentation.
[0067] The required general processing steps include the selection
of the appropriate materials from which the polymeric foam and the
reinforcing components are made. If a mesh reinforcing material is
used, the proper mesh density must be selected. Further, the
reinforcing material must be properly aligned in the mold, the
polymer solution must be added at an appropriate rate and,
preferably, into a mold that is tilted at an appropriate angle to
avoid the formation of air bubbles, and the polymer solution must
be lyophilized.
[0068] In embodiments that utilize a mesh reinforcing material, the
reinforcing mesh has to be of a certain density. That is, the
openings in the mesh material must be sufficiently small to render
the construct sutureable or otherwise fastenable, but not so small
as to impede proper bonding between the foam and the reinforcing
mesh as the foam material and the open cells and cell walls thereof
penetrate the mesh openings. Without proper bonding the integrity
of the layered structure is compromised leaving the construct
fragile and difficult to handle. Because the density of the mesh
determines the mechanical strength of the construct, the density of
the mesh can vary according to the desired use for tissue repair.
In addition, the type of weave used in the mesh can determine the
directionality of the mechanical strength of the construct, as well
as the mechanical properties of the reinforcing material, such as
for example, the elasticity, stiffness, burst strength, suture
retention strength and ultimate tensile strength of the construct.
By way of non-limiting example, the mesh reinforcing material in a
foam-based biocompatible scaffold of the present invention can be
designed to be stiff in one direction, yet elastic in another, or
alternatively, the mesh reinforcing material can be made
isotropic.
[0069] During the lyophilization of the reinforced foam, several
parameters and procedures are important to produce implants with
the desired integrity and mechanical properties. Preferably, the
reinforcement material is substantially flat when placed in the
mold. To ensure the proper degree of flatness, the reinforcement
(e.g., mesh) is pressed flat using a heated press prior to its
placement within the mold. Further, in the event that reinforcing
structures are not isotropic it is desirable to indicate this
anisotropy by marking the construct to indicate directionality.
This can be accomplished by embedding one or more indicators, such
as dyed markings or dyed threads, within the woven reinforcements.
The direction or orientation of the indicator will indicate to a
surgeon the dimension of the implant in which physical properties
are superior.
[0070] As noted above, the manner in which the polymer solution is
added to the mold prior to lyophilization helps contribute to the
creation of a tissue implant with adequate mechanical integrity.
Assuming that a mesh reinforcing material will be used, and that it
will be positioned between two thin (e.g., 0.75 mm) shims it should
be positioned in a substantially flat orientation at a desired
depth in the mold. The polymer solution is poured in a way that
allows air bubbles to escape from between the layers of the foam
component. Preferably, the mold is tilted at a desired angle and
pouring is effected at a controlled rate to best prevent bubble
formation. One of ordinary skill in the art will appreciate that a
number of variables will control the tilt angle and pour rate.
Generally, the mold should be tilted at an angle of greater than
about 1 degree to avoid bubble formation. In addition, the rate of
pouring should be slow enough to enable any air bubbles to escape
from the mold, rather than to be trapped in the mold.
[0071] If a mesh material is used as the reinforcing component, the
density of the mesh openings is an important factor in the
formation of a resulting tissue implant with the desired mechanical
properties. A low density, or open knitted mesh material, is
preferred. One preferred material is a 90:10 copolymer of glycolide
and lactide, sold under the tradename VICRYL (Ethicon, Inc.,
Somerville, N.J.). One exemplary low density, open knitted mesh is
Knitted VICRYL VKM-M, available from Ethicon, Inc., Somerville,
N.J. Other preferred materials are polydioxanone or 95:5 copolymer
of lactide and glycolide.
[0072] The density or "openness" of a mesh material can be
evaluated using a digital photocamera interfaced with a computer.
In one evaluation, the density of the mesh was determined using a
Nikon SMZ-U Zoom with a Sony digital photocamera DKC-5000
interfaced with an IBM 300PL computer. Digital images of sections
of each mesh magnified to 20.times. were manipulated using
Image-Pro Plus 4.0 software in order to determine the mesh density.
Once a digital image was captured by the software, the image was
thresholded such that the area accounting for the empty spaces in
the mesh could be subtracted from the total area of the image. The
mesh density was taken to be the percentage of the remaining
digital image. Implants with the most desirable mechanical
properties were found to be those with a mesh density in the range
of about 12 to 80% and more preferably about 45 to 80%.
[0073] In one embodiment, the preferred scaffold for cartilage
repair is a mesh reinforced foam. More preferably, the foam is
reinforced with a mesh that includes polydioxanone (PDO) and the
foam composition is a copolymer of 35:65 .epsilon.-caprolactone and
glycolide. For articular cartilage, the preferred structure to
allow cell and tissue ingrowth is one that has an open pore
structure and is sized to sufficiently allow cell migration. A
suitable pore size is one in which an average diameter is in the
range of about 50 to 1000 microns, and more preferably, between
about 50 to 500 microns. The mesh layer has a thickness in the
range of 1 micron to 1000 microns. Preferably, the foam has a
thickness in the range of about 300 microns to 2 mm, and more
preferably, between about 500 microns and 1.5 mm. Preferably, the
mesh layer has a mesh density in the range of about 12 to 80% and
more preferably about 45 to 80%.
[0074] In another embodiment, the preferred scaffold for cartilage
repair is a nonwoven structure. More preferably, the composition of
the nonwoven structure are PANACRYL, a 95:5 copolymer of lactide
and glycolide, VICRYL, a 90:10 copolymer of glycolide and lactide,
or a blend of polydioxanone and VICRYL sold under the tradename
ETHISORB (Johnson & Johnson International, Belgium). For
articular cartilage, the preferred structure to allow cell and
tissue ingrowth is one that has an open pore structure and is sized
to sufficiently allow cell migration. A suitable pore size for the
nonwoven scaffold is one in which an average diameter is in the
range of about 50 to 1000 microns and more preferably between about
100 to 500 microns. The nonwoven scaffold has a thickness between
about 300 microns and 2 mm, and more preferably, between about 500
microns and 1.5 mm.
[0075] In one embodiment, the preferred scaffold for meniscus
repair is a mesh reinforced foam. More preferably, the foam is
reinforced foam with a mesh that includes polydioxanone (PDO) and
the foam composition is a copolymer of 35:65 .epsilon.-caprolactone
and glycolide. The preferred structure to allow cell and tissue
ingrowth is one that has an open pore structure and is sized to
sufficiently allow cell migration. A suitable pore size is one in
which an average diameter is in the range of about 50 to 1000
microns, and more preferably, between about 50 to 500 microns. The
mesh layer has a thickness in the range of about 1 micron to 1000
microns. Preferably, the foam has a thickness in the range of about
300 microns to 2 mm, and more preferably, between about 500 microns
and 1.5 mm. In this embodiment, the preferred method of use is to
surround the minced cartilage tissue with this scaffold material.
Preferably, the mesh layer has a mesh density in the range of about
12 to 80% and more preferably about 45 to 80%.
[0076] In one embodiment, the preferred scaffold for tendon or
ligament repair is a mesh reinforced foam. More preferably, the
foam is reinforced with a mesh that includes polydioxanone (PDO)
and the foam composition is a copolymer of 35:65
.epsilon.-caprolactone and glycolide. The preferred structure to
allow cell and tissue ingrowth is one that has an open pore
structure and is sized to sufficiently allow cell migration. A
suitable pore size is one in which an average diameter is in the
range of about 50 to 1000 microns, and more preferably, between
about 50 to 500 microns. The mesh layer has a thickness in the
range of about 1 micron to 1000 microns. Preferably, the foam has a
thickness in the range of about 300 microns to 2 mm, and more
preferably, between about 500 microns and 1.5 mm. Preferably, the
mesh layer has a mesh density in the range of about 12 to 80% and
more preferably about 45 to 80%.
[0077] In another embodiment, the preferred scaffold for tendon or
ligament repair is constructed from a polymer that has a slow
resorption profile (e.g., at least three months, and preferably, at
least six months) and high mechanical strength. More preferably,
the tensile strength and elastic modulus of the scaffold must be
similar to that of native ligament. The preferred tensile strength
of the scaffold is between about 500N and 4000N, and more
preferably, between about 1000N and 2500N. The preferred elastic
modulus of the scaffold is between about 100 N/m and 300 N/m, and
more preferably, between about 150 N/m and 200 N/m. The preferred
structure of this scaffold is a cylindrical-shaped or
elliptically-shaped scaffold or a scaffold with a high aspect ratio
(i.e., ratio of length to width). Preferably, the aspect ratio is
greater than 1, and more preferably it is greater than 2 and less
than 100. Further, the scaffold preferably has a diameter or width
in the range of about 3 mm and 12 mm, and more preferably, between
about 4 mm and 10 mm. By way of non-limiting example, the scaffold
for ligament repair can include a 95:5 copolymer of lactide and
glycolide. In one embodiment, the scaffold for ligament repair can
be formed as a composite structure including a 95:5 copolymer of
lactide and glycolide and other polymers, such as for example,
polylactide, polyglycolide, polydioxanone, polycaprolactone and
combinations thereof. The scaffold may be formed of a woven, knit
or braided material. Optionally, the polymers from which the
scaffold is made can be formed as a nonwoven, textile structure,
such as for example, a weave or a mesh structure, or alternatively
these polymers can be formed as a foam. In another embodiment, the
composite structure can include natural polymers, such as for
example, collagen, fibrin, or silk. In this embodiment, the natural
polymer can act as a coating to the composite structure, or
alternatively, the natural polymer can be formed as a foam. The
composite structure can also optionally include strips of collagen
or silk to reside within the whole scaffold or just the periphery
of the scaffold.
[0078] In one embodiment, the scaffold useful for ligament or
tendon repair is formed of a plurality of filaments, a majority of
the fibers of which are aligned in the longitudinal direction.
[0079] One of ordinary skill in the art will appreciate that the
selection of a suitable material for forming the biocompatible
scaffold of the present invention depends on several factors. These
factors include in vivo mechanical performance; cell response to
the material in terms of cell attachment, proliferation, migration
and differentiation; biocompatibility; and optionally,
bioabsorption (or bio-degradation) kinetics. Other relevant factors
include the chemical composition, spatial distribution of the
constituents, the molecular weight of the polymer, and the degree
of crystallinity.
[0080] In addition to the biocompatible scaffold, the tissue repair
implants of the present invention further include at least one
sample of viable tissue that is associated with at least a portion
of the scaffold. The term "viable," as used herein, refers to a
tissue sample having one or more viable cells. Virtually any type
of tissue can be used to construct the tissue repair implants of
the present invention. Preferably, the tissue used is selected from
cartilage tissue, meniscal tissue, ligament tissue, tendon tissue,
skin tissue, bone tissue, muscle tissue, periosteal tissue,
pericardial tissue, synovial tissue, nerve tissue, fat tissue,
kidney tissue, bone marrow, liver tissue, bladder tissue, pancreas
tissue, spleen tissue, intervertebral disc tissue, embryonic
tissue, periodontal tissue, vascular tissue, blood and combinations
thereof. In one embodiment useful for cartilage repair, the tissue
is free of bone tissue and is selected from the group consisting of
cartilage tissue, meniscal tissue, ligament tissue and tendon
tissue. The tissue used to construct the tissue implant can be
autogeneic tissue, allogeneic tissue, or xenogeneic tissue.
[0081] In one embodiment useful for meniscal repair, the tissue
used in the tissue repair implant can be selected from the group
consisting of meniscal tissue, cartilage tissue, skin, synovial
tissue, periosteal tissue, pericardial tissue, fat tissue, bone
marrow, blood, tendon tissue, ligament tissue, or combinations
thereof. In one embodiment useful for ligament repair, the tissue
used in the tissue repair implant can be selected from the group
consisting of tendon tissue, ligament tissue of the same type that
is to be repaired, ligament tissue of a different type than the
tissue that is to be repaired, synovial tissue, periosteal tissue,
fascia, skin, and combinations thereof.
[0082] The tissue can be obtained using any of a variety of
conventional techniques, such as for example, by biopsy or other
surgical removal. Preferably, the tissue sample is obtained under
aseptic conditions. Once a sample of living tissue has been
obtained, the sample can then be processed under sterile conditions
to create a suspension having at least one minced, or finely
divided, tissue particle. The particle size of each tissue fragment
can vary, for example, the tissue size can be in the range of about
0.1 and 3 mm.sup.3, in the range of about 0.5 and 1 mm.sup.3, in
the range of about 1 to 2 mm.sup.3, or in the range of about 2 to 3
mm.sup.3, but preferably the tissue particle is less than 1
mm.sup.3.
[0083] Preferably, the minced tissue has at least one viable cell
that can migrate from the tissue fragment onto the scaffold. More
preferably, the tissue contains an effective amount of cells that
can migrate from the tissue fragment and begin populating the
scaffold. In an optional embodiment, the minced tissue fragments
may be contacted with a matrix-digesting enzyme to facilitate cell
migration out of the extracellular matrix surrounding the cells.
The enzymes are used to increase the rate of cell migration out of
the extracellular matrix and into the scaffold material. Suitable
matrix-digesting enzymes that can be used in the present invention
include, but are not limited to, collagenase, chondroitinase,
trypsin, elastase, hyaluronidase, petidase, thermolysin and
protease.
[0084] In one embodiment, the minced tissue particles can be formed
as a suspension in which the tissue particles are associated with a
physiological buffering solution. Suitable physiological buffering
solutions include, but are not limited to, saline, phosphate buffer
solution, Hank's balanced salts, Tris buffered saline, Hepes
buffered saline and combinations thereof. In addition, the tissue
can be minced in any standard cell culture medium known to those
having ordinary skill in the art, either in the presence or absence
of serum. Prior to depositing the suspension of minced tissue on
the scaffold or at the site of tissue injury, the minced tissue
suspension can be filtered and concentrated, such that only a small
quantity of physiological buffering solution remains in the
suspension to prevent the tissue particles from drying out, and the
minced tissue particles can be directly applied to the scaffold or
site of injury. Preferably, the minced tissue particles are loaded
at a concentration in the range of approximately 1 to 100
mg/cm.sup.2, and more preferably in the range of about 1 to 20
mg/cm.sup.2.
[0085] The suspension of minced living tissue can be used to create
a tissue repair implant according to the present invention by
depositing the suspension of living tissue upon a biocompatible
scaffold, such that the tissue and the scaffold become associated.
Preferably, the tissue is associated with at least a portion of the
scaffold. The tissue repair implant can be implanted in a subject
immediately, or alternatively, the construct can be incubated under
sterile conditions for a duration and under conditions that are
effective to maintain the viability of the tissue sample. In
embodiments where the construct is incubated, the incubation
conditions can vary, but preferably, the construct is incubated for
a duration in the range of 1 hour to 6 weeks, and more preferably
between about 1 week and 6 weeks, at a temperature in the range of
about 20 to 40.degree. C., and in an atmosphere containing between
about 5 and 10% carbon dioxide (CO.sub.2) and high humidity, e.g.,
approximately 100% humidity.
[0086] A kit can be used to assist in the preparation of the tissue
repair implants of the present invention. According to the present
invention, the kit includes a sterile container that houses one or
more biocompatible scaffolds, a harvesting tool for collecting the
living tissue sample from a subject, and one or more reagents for
sustaining the viability of the tissue sample. Suitable reagents
for sustaining the viability of the tissue sample include a
physiological solution, such as for example, saline, phosphate
buffering solution, Hank's balanced salts, standard cell culture
medium, Dulbecco's modified Eagle's medium, ascorbic acid, HEPES,
nonessential amino acid, L-proline, fetal bovine serum, autologous
serum, and combinations thereof. The kit can also include a
processing tool for dividing the tissue into minced tissue
particles, or alternatively, the harvesting tool can be adapted to
collect the tissue sample and to process the sample into finely
divided tissue particles. The kit can, optionally, also include a
delivery device for transferring the scaffold from the sterile
container to a subject for implantation.
[0087] A biological component may, optionally, be incorporated
within the tissue repair implants of the present invention.
Preferably, the biological component is incorporated within, or
coated on, the scaffolds disclosed above. In embodiments where the
biological component is coated onto the scaffold, the biological
component is preferably associated with at least a portion of the
scaffold. By way of nonlimiting example, the biocompatible scaffold
can include an adhesion agent for anchoring the suspension of
minced tissue fragments to the scaffold. Preferably, the adhesion
agent is an anchoring agent, a cross-linking agent (i.e., chemical
or physical), and combinations thereof.
[0088] Suitable anchoring agents include, but are not limited to,
hyaluronic acid, fibrin glue, fibrin clot, collagen gel, alginate
gel, gelatin-resorcin-formalin adhesive, mussel-based adhesive,
dihydroxyphenylalanine (DOPA) based adhesive, chitosan,
transglutaminase, poly(amino acid)-based adhesive, cellulose-based
adhesive, polysaccharide-based adhesive, synthetic acrylate-based
adhesives, platelet rich plasma (PRP), platelet poor plasma (PPP),
clot of PRP, clot of PPP, Matrigel, Monostearoyl Glycerol
co-Succinate (MGSA), Monostearoyl Glycerol
co-Succinate/polyethylene glycol (MGSA/PEG) copolymers, laminin,
elastin, proteoglycans, and combinations thereof.
[0089] Suitable cross-linking agents include, for example, divinyl
sulfone (DVS), polyethylene glycol divinyl sulfone (VS-PEG-VS),
hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA),
formaldehyde, glutaraldehyde, aldehydes, isocyanates, alkyl and
aryl halides, imidoesters, N-substituted maleimides, acylating
compounds, carbodiimide, hydroxychloride, N-hydroxysuccinimide,
light (e.g., blue light and UV light), pH, temperature, and
combinations thereof.
[0090] The biological components used in the present invention can
also be selected from among a variety of effectors that, when
present at the site of injury, promote healing and/or regeneration
of the affected tissue. In addition to being compounds or agents
that actually promote or expedite healing, the effectors may also
include compounds or agents that prevent infection (e.g.,
antimicrobial agents and antibiotics), compounds or agents that
reduce inflammation (e.g., anti-inflammatory agents), compounds
that prevent or minimize adhesion formnation, such as oxidized
regenerated cellulose (e.g., INTERCEED and Surgicel.RTM., available
from Ethicon, Inc.), hyaluronic acid, and compounds or agents that
suppress the immune system (e.g., immunosuppressants).
[0091] By way of example, other types of effectors present within
the implant of the present invention can include heterologous or
autologous growth factors, proteins (including matrix proteins),
peptides, antibodies, enzymes, platelets, glycoproteins, hormones,
cytokines, glycosaminoglycans, nucleic acids, analgesics, viruses,
virus particles, and cell types. It is understood that one or more
effectors of the same or different functionality may be
incorporated within the implant.
[0092] Examples of suitable effectors include the multitude of
heterologous or autologous growth factors known to promote healing
and/or regeneration of injured or damaged tissue. These growth
factors can be incorporated directly into the biocompatible
scaffold, or alternatively, the biocompatible scaffold can include
a source of growth factors, such as for example, platelets.
Exemplary growth factors include, but are not limited to,
TGF-.beta., bone morphogenic protein, cartilage-derived morphogenic
protein, fibroblast growth factor, platelet-derived growth factor,
vascular endothelial cell-derived growth factor (VEGF), epidermal
growth factor, insulin-like growth factor, hepatocyte growth
factor, and fragments thereof. Suitable effectors likewise include
the agonists and antagonists of the agents noted above. The growth
factor can also include combinations of the growth factors listed
above. In addition, the growth factor can be autologous growth
factor that is supplied by platelets in the blood. In this case,
the growth factor from platelets will be an undefined cocktail of
various growth factors.
[0093] The proteins that may be present within the implant include
proteins that are secreted from a cell or other biological source,
such as for example, a platelet, which is housed within the
implant, as well as those that are present within the implant in an
isolated form. The isolated form of a protein typically is one that
is about 55% or greater in purity, i.e., isolated from other
cellular proteins, molecules, debris, etc. More preferably, the
isolated protein is one that is at least 65% pure, and most
preferably one that is at least about 75 to 95% pure.
Notwithstanding the above, one of ordinary skill in the art will
appreciate that proteins having a purity below about 55% are still
considered to be within the scope of this invention. As used
herein, the term "protein" embraces glycoproteins, lipoproteins,
proteoglycans, peptides, and fragments thereof. Examples of
proteins useful as effectors include, but are not limited to,
pleiotrophin, endothelin, tenascin, fibronectin, fibrinogen,
vitronectin, V-CAM, I-CAM, N-CAM, selectin, cadherin, integrin,
laminin, actin, myosin, collagen, microfilament, intermediate
filament, antibody, elastin, fibrillin, and fragments thereof.
[0094] Glycosaminoglycans, highly charged polysaccharides which
play a role in cellular adhesion, may also serve as effectors
according to the present invention. Exemplary glycosaminoglycans
useful as effectors include, but are not limited to, heparan
sulfate, heparin, chondroitin sulfate, dermatan sulfate, keratan
sulfate, hyaluronan (also known as hyaluronic acid), and
combinations thereof.
[0095] The biocompatible scaffolds of the present invention can
also have cells incorporated therein. Suitable cell types that can
serve as effectors according to this invention include, but are not
limited to, osteocytes, osteoblasts, osteoclasts, fibroblasts, stem
cells, pluripotent cells, chondrocyte progenitors, chondrocytes,
endothelial cells, macrophages, leukocytes, adipocytes, monocytes,
plasma cells, mast cells, umbilical cord cells, stromal cells,
mesenchymal stem cells, epithelial cells, myoblasts, tenocytes,
ligament fibroblasts, neurons, and bone marrow cells. Cells
typically have at their surface receptor molecules which are
responsive to a cognate ligand (e.g., a stimulator). A stimulator
is a ligand which when in contact with its cognate receptor induce
the cell possessing the receptor to produce a specific biological
action. For example, in response to a stimulator (or ligand) a cell
may produce significant levels of secondary messengers, like
Ca.sup.+2, which then will have subsequent effects upon cellular
processes such as the phosphorylation of proteins, such as (keeping
with our example) protein kinase C. In some instances, once a cell
is stimulated with the proper stimulator, the cell secretes a
cellular messenger usually in the form of a protein (including
glycoproteins, proteoglycans, and lipoproteins). This cellular
messenger can be an antibody (e.g., secreted from plasma cells), a
hormone, (e.g., a paracrine, autocrine, or exocrine hormone), a
cytokine, or natural or synthetic fragments thereof.
[0096] The tissue implants of the invention can also be used in
gene therapy techniques in which nucleic acids, viruses, or virus
particles deliver a gene of interest, which encodes at least one
gene product of interest, to specific cells or cell types.
Accordingly, the biological effector can be a nucleic acid (e.g.,
DNA, RNA, or an oligonucleotide), a virus, a virus particle, or a
non-viral vector. The viruses and virus particles may be, or may be
derived from, DNA or RNA viruses. The gene product of interest is
preferably selected from the group consisting of proteins,
polypeptides, interference ribonucleic acids (iRNA) and
combinations thereof.
[0097] Once the applicable nucleic acids and/or viral agents (i.e.,
viruses or viral particles) are incorporated into the biocompatible
scaffold of the tissue repair implant, the implant can then be
implanted into a particular site to elicit a type of biological
response. The nucleic acid or viral agent can then be taken up by
the cells and any proteins that they encode can be produced locally
by the cells. In one embodiment, the nucleic acid or viral agent
can be taken up by the cells within the tissue fragment of the
minced tissue suspension, or, in an alternative embodiment, the
nucleic acid or viral agent can be taken up by the cells in the
tissue surrounding the site of the injured tissue. One of ordinary
skill in the art will recognize that the protein produced can be a
protein of the type noted above, or a similar protein that
facilitates an enhanced capacity of the tissue to heal an injury or
a disease, combat an infection, or reduce an inflammatory response.
Nucleic acids can also be used to block the expression of unwanted
gene product that may impact negatively on a tissue repair process
or other normal biological processes. DNA, RNA and viral agents are
often used to accomplish such an expression blocking function,
which is also known as gene expression knock out.
[0098] One of ordinary skill in the art will appreciate that the
identity of the biological component may be determined by a
surgeon, based on principles of medical science and the applicable
treatment objectives.
[0099] The biological component or effector of the issue repair
implant can be incorporated within the scaffold before or after
manufacture of the scaffold, or before or after the surgical
placement of the implant.
[0100] Prior to surgical placement, the biocompatible scaffold can
be placed in a suitable container comprising the biological
component. After an appropriate time and under suitable conditions,
the scaffold will become impregnated with the biological component.
Alternatively, the biological component can be incorporated within
the scaffold by, for example, using an appropriately gauged syringe
to inject the biological agent(s) into the scaffold. Other methods
well known to those of ordinary skill in the art can be applied in
order to load a scaffold with an appropriate biological component,
such as mixing, pressing, spreading, centrifuging and placing the
biological component into the scaffold. Alternatively, the
biological component can be mixed with a gel-like carrier prior to
injection into the scaffold. The gel-like carrier can be a
biological or synthetic hydrogel, including an alginate, a
cross-linked alginate, hyaluronic acid, collagen gel,
poly(N-isopropylacrylamide), poly(oxyalkylene), a copolymer of
poly(ethylene oxide)-poly(propylene oxide), poly(vinyl alcohol) and
blends thereof.
[0101] Following surgical placement, an implant wherein the
biocompatible scaffold is devoid of any biological component can be
infused with biological agent(s), or an implant wherein the
scaffold includes at least one biological component can be
augmented with a supplemental quantity of the biological component.
One method of incorporating a biological component within a
surgically installed implant is by injection using an appropriately
gauged syringe.
[0102] The amount of the biological component included with a
biocompatible scaffold will vary depending on a variety of factors,
including the size of the scaffold, the material from which the
scaffold is made, the porosity of the scaffold, the identity of the
biologically component, and the intended purpose of the tissue
repair implant. One of ordinary skill in the art can readily
determine the appropriate quantity of biological component to
include within a biocompatible scaffold for a given application in
order to facilitate and/or expedite the healing of tissue. The
amount of biological component will, of course, vary depending upon
the identity of the biological component and the given
application.
[0103] In another embodiment, the tissue repair implant can include
an additional retaining element that is placed over the
tissue-laden scaffold. Preferably, in this embodiment, at least a
portion of the tissue suspension is associated with at least a
portion of the outer surface of the scaffold, such that the tissue
suspension is "sandwiched" between the biocompatible scaffold and
the retaining element. The retaining element can be formed from
virtually any biocompatible material, and in one embodiment, the
retaining element can be formed using tissue grafts, including
grafts obtained from allogeneic tissue, autogeneic tissue, and
xenogeneic tissue, an additional biocompatible scaffold selected
from the biocompatible scaffolds disclosed above, and combinations
thereof. In another embodiment, the retaining element can be a
porous mesh, a porous mesh-like material, such as for example, a
knit, a weave, a nonwoven, or a thin, perforated elastomeric sheet
having pores or perforations to allow tissue ingrowth. The thin,
perforated elastomeric sheets are preferably constructed from
collagen or silk or blends or copolymers of polylactic acid (PLA),
polyglycolic acid (PGA), polycaprolactone (PCL) and polydioxanone
(PDO). The type of retaining element used can vary according to the
desired tissue repair. By way of non-limiting example, in one
embodiment for meniscus repair, the retaining element can be a
mesh-reinforced foam. In embodiments for ACL and cartilage repair,
the retaining element can be a mesh structure. In embodiments where
the retaining element is an allograft or an autograft, preferably
the allograft or autograft is selected from periosteum,
perichondrium, iliotibial band or fascia lata, gracilis tendon,
semitendinosis tendon, patellar tendon, synovium and combinations
thereof. In embodiments where the retaining element is a xenograft,
the xenograft is preferably selected from the corresponding
anatomical structure for small intestine, periosteum,
perichondrium, iliotibial band or fascia lata, gracilis tendon,
semitendonous tendon, patellar tendon, synovium, and combinations
thereof. These retaining elements can be placed over the
biocompatible scaffold, or alternatively, the retaining element can
be affixed, such as for example, by suturing or stapling, the
implant to act as a retaining element. One of ordinary skill in the
art will appreciate that additional processing of the retaining
element, such as for example, the placement of holes within the
retaining element, may be determined by a surgeon, based on
principles of medical science and the applicable treatment
objectives.
[0104] In yet another embodiment, an electrostatically spun fabric
barrier may be added to the implant to act as a barrier to
hyperplasia and tissue adhesion, thus reducing the possibility of
postsurgical adhesions. The fabric barrier is preferably in the
form of dense fibrous fabric that is added to the implant.
Preferably, the fibrous fabric is comprised of small diameter
fibers that are fused to the top and/or bottom surface of the
biocompatible scaffold. This enables certain surface properties of
the structure, such as porosity, permeability, degradation rate and
mechanical properties, to be controlled.
[0105] One of ordinary skill in the art will appreciate that the
fibrous fabric can be produced via an electrostatic spinning
process in which a fibrous layer can be built up on lyophilized
foam and nonwoven surfaces. This electrostatic spinning process may
be conducted using a variety of fiber materials. Exemplary fiber
materials include aliphatic polyesters. A variety of solvents may
be used as well, including those identified above that are useful
to prepare the polymer solution that forms the foam component.
[0106] The composition, thickness, and porosity of the fibrous
layer may be controlled to provide the desired mechanical and
biological characteristics. For example, the bioabsorption rate of
the fibrous layer may be selected to provide a longer or shorter
bioabsorption profile as compared to the underlying biocompatible
scaffold. Additionally, the fibrous layer may provide greater
structural integrity to the composite so that mechanical force may
be applied to the fibrous side of the structure. In one embodiment
the fibrous layer could allow the use of sutures, staples or
various fixation devices to hold the composite in place. Generally,
the fibrous layer has a thickness in the range of about 1 micron to
1000 microns. However, for some applications such as rotator cuff
and meniscus injury repair, the fibrous layer has a thickness
greater than about 1.5 mm.
[0107] The tissue repair implants of the present invention can be
used in a variety of surgical and non-surgical applications. In
some surgical applications, such as for use in the repair of a
variety of tissues including a torn ligament, tendon, rotator cuff,
nerve, skin, cartilage, or meniscus, the tissue implants of the
invention must be able to be handled in the operating room, and
they must be able to be sutured or otherwise fastened without
tearing. Additionally, the implants should have a burst strength
adequate to reinforce the tissue, and the structure of the implant
can be suitable to encourage tissue ingrowth. By way of
non-limiting example, the scaffolds of the present invention can be
highly porous to allow cell growth therein. Preferably, the median
pore size is in the range of about 100 to 500 microns. In these
embodiments, the scaffold should be sufficiently pliable to
accommodate tissue growth within the interior region of the
scaffold, so that the geometry of the scaffold can be remodeled as
tissue ingrowth increases. Accordingly, in the present invention,
tissue can be grown on the surface of the biocompatible scaffold,
or alternatively, tissue can be grown into and on the surface of
the biocompatible scaffold, such that the tissue becomes embedded
in and integrated with the scaffold.
[0108] In one embodiment of the present invention, the tissue
repair implant is used in the treatment of a tissue injury, such as
injury to a ligament, tendon, nerve, skin, cartilage or meniscus.
Repairing tissue injuries involves the steps of obtaining a sample
of living tissue by any of the variety of techniques known to those
having ordinary skill in the art, processing that sample of living
tissue under sterile conditions, such as for example by cutting the
tissue, to create at least one minced, finely divided tissue
particle, depositing the tissue sample upon the biocompatible
scaffold, such that the tissue sample becomes associated with the
scaffold to form a tissue repair implant, and placing the tissue
repair implant in a desired position relative to the tissue injury.
Repairing tissue injuries may also involve placing the scaffold at
the site of tissue injury and then depositing the fine tissue
particles onto the scaffold. The cells in the tissue particles
associated with the scaffold can migrate to the scaffold and begin
proliferating and integrating with surrounding tissue at the site
of implantation, thereby repairing the tissue injury. This method
for repairing tissue injuries can include an additional, optional
step. Prior to the step of placing the tissue repair implant in a
desired position relative to the tissue injury, the scaffold and
associated tissue particles can be incubated for a duration and
under conditions effective to allow cells within the tissue
particles to migrate from the tissue and begin populating the
scaffold.
[0109] The tissue samples used in the present invention are
obtained from a donor (autogenic, allogeneic, or xenogeneic) using
appropriate harvesting tools. The tissue samples can be finely
minced and divided into small particles either as the tissue is
collected, or alternatively, the tissue sample can be minced after
it is harvested and collected outside the body. In embodiments,
where the tissue sample is minced after it is harvested, the tissue
samples can be weighed and then washed three times in phosphate
buffered saline. Approximately 300 to 500 mg of tissue can then be
minced in the presence of a small quantity, such as, for example,
about 1 ml, of a physiological buffering solution, such as, for
example, phosphate buffered saline, or a matrix digesting enzyme,
such as, for example, 0.2% collagenase in Hams F12. Mincing the
tissue divides the tissue into particles or small pieces of
approximately 1 mm.sup.3. Mincing the tissue can be accomplished by
a variety of methods. In one embodiment, the mincing is
accomplished with two sterile scalpels using a parallel direction,
and in another embodiment, the tissue can be minced by a processing
tool that automatically divides the tissue into particles of a
desired size. In one embodiment, the minced tissue can be separated
from the physiological fluid and concentrated using any of a
variety of methods known to those having ordinary skill in the art,
such as for example, sieving, sedimenting or centrifuging. In
embodiments where the minced tissue is filtered and concentrated,
the suspension of minced tissue preferably retains a small quantity
of fluid in the suspension to prevent the tissue from drying out.
In another embodiment, the suspension of minced tissue is not
concentrated, and the minced tissue can be directly delivered to
the site of tissue repair via a high concentration tissue
suspension or other carrier such as for example, a hydrogel, fibrin
glue, or collagen. In this embodiment, the minced tissue suspension
can be covered by any of the biocompatible scaffolds described
above to retain the tissue fragments in place.
[0110] The minced tissue can then be distributed onto a scaffold
using a cell spreader so as to cover the entire scaffold. In a
preferable embodiment for meniscus and cartilage repair, the minced
tissue is spread onto 4.times.5 cm scaffolds that have been
presoaked in Dulbecco's modified Eagles medium (DMEM) so as to
cover the entire scaffold. Optionally, the tissue particles can be
adhered to the scaffolds using any of the adhesive agents described
above, such as, for example, fibrin glue or platelet rich plasma.
In embodiments using fibrin glue or platelet rich plasma, a few
microliters of thrombin can be placed on the scaffolds, prior to
distribution of fibrinogen or platelet rich plasma on the
scaffolds, and allowed to set. Once the tissue particles and any
additional agents have been deposited on the scaffold, the tissue
repair implant can then implanted immediately, or alternatively,
the implant can be cultured in vitro for a duration and under
conditions sufficient to allow the cells in the tissue particles to
migrate from the tissue particles onto the scaffold. In an
embodiment where the tissue repair implant is incubated prior to
implantation, the implant is preferably cultured in vitro for
approximately 1-3 weeks in a chondrocyte growth medium, such as for
example, DMEM-high glucose, supplemented with 20% fetal calf serum
(FCS), 10 mM HEPES, 0.1 mM nonessential amino acids, 20 mg/ml of
L-proline, 50 mg/ml ascorbic acid, 100 mg/ml penicillin, 100 mg/ml
of streptomycin and 0.25 mg/ml of amphotericin B.
[0111] The methods of repairing tissue injuries using the tissue
implants according to the present invention can be conducted during
a surgical operation to repair the tissue injury. Alternatively,
the steps of processing the tissue sample to create minced, finely
divided tissue particles, depositing the tissue particles upon the
scaffold to form a tissue repair implant, and/or incubating the
tissue repair implant prior to implantation can be conducted at
another, sterile location prior to surgical placement of the
implant relative to the site of injury.
[0112] The implants used to repair injured tissue can be of a size
and shape such that they match the geometry and dimensions of a
desired portion or lesion of the tissue to be treated. The implant
can be sized and shaped to produce the necessary geometry by
numerous techniques including cutting, folding, rolling, or
otherwise manipulating the implant. As noted above, the biological
component may be added to the scaffold during or after manufacture
of the scaffold or before or after the implant is installed in a
patient. An additional quantity of the biological component may be
added after the implant is installed. Once access is made into the
affected anatomical site (whether by minimally invasive, open or
mini-open surgical technique), the implant can be affixed to a
desired position relative to the tissue injury, such as within a
tear or lesion. Once the implant is placed in the desired position
or lesion, it can be affixed by using a suitable technique. In one
aspect, the implant can be affixed by a chemical and/or mechanical
fastening technique. Suitable chemical fasteners include glues
and/or adhesive such as fibrin glue, fibrin clot, and other known
biologically compatible adhesives. Suitable mechanical fasteners
include sutures, staples, tissue tacks, suture anchors, darts,
screws, pins and arrows. It is understood that combinations of one
or more chemical and/or mechanical fasteners can be used.
Alternatively, one need not use any chemical and/or mechanical
fasteners. Instead, placement of the implant can be accomplished
through an interference fit of the implant with an appropriate site
in the tissue to be treated.
[0113] In another embodiment, the tissue repair implant is useful
in surgical techniques that repair ligaments, tendons, skin and/or
nerves.
[0114] In one use, the tissue repair implant can be for repair and
to augment tissue loss during tendon or ligament repair surgery or
it can be used as a stand alone device. In the case of repair,
tendon or ligament ends are approximated through appropriate
surgical techniques and the tissue repair implant is used around
the joined end to give more mechanical support and to enhance the
healing response. As a result of the healing process, the tendon or
ligament tissue grows within the implant device, eventually
maturing into a tissue with similar mechanical properties to that
of native tissue. The implant provides the mechanical support that
is initially necessary to ensure proper healing, and it also serves
as a guide for tissue regeneration. In another use as a stand alone
device, the ruptured tissue is removed, and the tissue repair
implant with minced tissue serves to replace the function of the
damaged tissue. The ruptured tissue can be the tissue source used
for healing damaged tissue.
[0115] In embodiments where the tissue repair implant is used to
repair ligament tissue, the tissue repair implant can be used for
tissue augmentation, or alternatively, as a stand-alone device. In
embodiments where the tissue repair implant is used for
augmentation, the tissue repair implant can be used in conjunction
with any of a variety of standard, established repair techniques
known to those having ordinary skill in the art. In embodiments
where the tissue repair implant is used for augmentation during ACL
repair, surgeons currently use an autograft consisting of ligament
tissue, bone-patellar tendons, tendon-bone tendons, hamstring
tendons, or iliotibial band to repair tissue, and the tissue repair
implant of the present invention can be placed either around the
autograft, surrounded by the autograft, or alongside the autograft.
In embodiments where the tissue repair element is used as a
stand-alone device, the ruptured ligament can be removed and
completely replaced by the tissue repair implant. In this case, the
tissue repair implant can be affixed to bone at each end of the
implant. In the case of ACL repair, one end of the implant can be
stabilized at the original origin site of the femur, while the
other end can be placed at the original insertion site on the
tibia.
[0116] The tissue repair implant can be utilized in a variety of
configurations. For example, the implant can be folded or stacked
in multiple laminates or it can be rolled into the shape or a
tube-like structure. Tendon or ligament ends can be joined, for
example, by suturing, stapling, clipping, adhering, or anchoring,
the implant to ends of the implant. In some embodiments where the
tissue repair implant is used to repair tendons, such as for
example, rotator cuff repair, the surgeon can use the tissue repair
implant to assist in the reapproximation of the torn rotator cuff
to a bony trough through the cortical surface of the greater
tuberosity. Often times, in older patients, the rotator cuff tissue
is thin and degenerate and/or the quality of the humerus is
osteoporotic. Therefore, in order to increase the strength of the
attachment to the bony trough, the tissue repair implant can be
placed on top of the tendon, such that the sutures would pass
through both the scaffold and tendon, or alternatively, the tissue
repair implant can be used on top of the bone bridge to prevent the
sutures from pulling out of the bone. In either embodiment, the
tissue repair implant provides suture retention strength. In
situations where the quality of the rotator cuff is so degenerate
that the tissue cannot be reapproximated to the humerus, the tissue
repair implant can serve as a bridge, wherein one end of the
implant can be joined to the remaining tendon while the other end
can be attached to the bone.
[0117] In another variation, the implant can be used to repair or
replace the sheath of a tendon. To do so, the implant is sutured or
otherwise joined to the connective tissue, such as the periosteum,
synovium, or muscle, and wrapped around the tendon. This
construction allows free gliding of the tendon within the sheath
formed by the implant. The implant provides the necessary
structural support following surgery. Over time, however, the
implant in this embodiment can be resorbed and replaced by new
tissue.
[0118] The implants of the invention can also be used for organ
repair replacement or regeneration strategies that may benefit from
these unique tissue implants. For example, these implants can be
used for spinal disc, cranial tissue, dura, nerve tissue, liver,
pancreas, kidney, bladder, uterus, esophagus, liver spleen, cardiac
muscle, skeletal muscle, skin, fascia, pelvic floor, stomach,
tendons, cartilage, ligaments, and breast tissues.
[0119] In yet another embodiment, the implants of the present
invention can be used to create a biological assay for measuring
the effect of a substance on living tissue. In this embodiment,
tissue constructs are created, as described above, by providing a
sterile, biocompatible scaffold, obtaining a sample of living
tissue, processing the sample of living tissue under sterile
conditions to form a suspension of minced tissue having minced
tissue fragments and a physiological buffering solution, and
depositing the suspension of minced tissue on the biocompatible
scaffold such that the suspension of minced tissue and the scaffold
become associated. The tissue construct is incubated under
conditions that are effective to allow cells within the minced
tissue to populate the scaffold. The tissue construct can then be
contacted with the substance that is to be tested, and the
effect(s) of that substance can be determined. These tissue
constructs can be used to determine and/or test the biological
responses to a test substance, such as for example, cell viability,
growth, migration, differentiation and maintenance of cell
phenotype, metabolic activity, induction or repression. These
biological responses can be assayed using any of a variety of
techniques known to those having ordinary skill in the art, such as
for example, proliferation assay, cell migration assay, protein
assay, gene expression assay, viability assay, calorimetric assay
or metabolic assay. By way of non-limiting example, the expression
of a selected gene(s) or gene products typically expressed by the
tissue of the tissue construct, such as for example, the expression
of type II, type IX or type XI collagen expressed by chondrocytes,
using a variety known assays, such as for example, northern blot
analysis, RNAse protection assays, polymerase chain reaction (PCR),
western blot analysis and enzyme-linked immunoabsorbant assay
(ELISA). Suitable substances that can be tested using the tissue
constructs of the present invention include, but are not limited
to, drugs, pharmaceutical compositions, chemicals, microbes,
elements, cytokines, growth factors, hormones, antibodies,
peptides, ligands, antagonists of membrane-bound receptors, and
combinations thereof.
[0120] The implants of the present invention can also be used as a
delivery device for a therapeutic, wherein the therapeutic is the
minced tissue, which includes a combination of cells, extracellular
matrix and inherent growth factors. The scaffold portion of the
implant can allow for hormones and proteins to be released into the
surrounding environment.
[0121] The methods of repairing tissue injuries using the tissue
implants according to the present invention can be conducted during
a surgical operation to repair the tissue injury. A patient is
prepared for tissue repair surgery in a conventional manner using
conventional surgical techniques. Tissue repair is performed at the
site of injured tissue using the tissue repair implants of the
present invention. The tissue sample used to form the tissue repair
implant is obtained from the patient (or another donor) using
appropriate tools and techniques. The tissue sample is finely
minced and divided into at least one tissue particle having a
particle size in the range of about 0.1 to 3 mm.sup.3. The tissue
can be minced using a conventional mincing technique such as two
sterile scalpels used in a parallel direction. Between about 300 to
500 mg of tissue is minced in the presence of about 1 ml of a
physiological buffering solution, depending on the extent of the
tissue injury at the site of repair. The minced tissue is filtered
and concentrated to separate the minced tissue particle from the
physiological buffering solution. The minced tissue can be
concentrated using any of a variety of conventional techniques,
such as for example, sieving, sedimenting or centrifuging. The
minced tissue particles are then distributed using a cell spreader
onto a 4.times.5 cm biocompatible scaffold that has been soaked in
Dulbecco's modified Eagles medium (DMEM). An adhesion agent can be
added to the biocompatible scaffold and the minced tissue
particles. The tissue repair implant is implanted at the site of
tissue injury, either immediately or after a period of in vitro
incubation. Final wound closure is performed in a conventional
manner using conventional surgical techniques.
[0122] The following examples are illustrative of the principles
and practice of this invention. Numerous additional embodiments
within the scope and spirit of the invention will become apparent
to those skilled in the art.
EXAMPLE 1
[0123] Healthy cartilage tissue from articulating joints was
obtained from bovine shoulders. The cartilage tissue, which was
substantially free of bone tissue, was minced using scalpel blades
to obtain small tissue fragments in the presence of 0.2%
collagenase. The size of the tissue fragments varied but on average
should be approximately 1.times.1 mm in dimension. The minced
tissue was then distributed uniformly on a 4.times.5 cm synthetic
bioresorbable polycaprolactone/polyglycolic acid (PCL/PGA)
scaffold. Ethylene oxide sterilized polymer scaffolds, were
pre-soaked for 4 hours in Dulbecco's Modified Eagle's Medium prior
to distribution of tissue fragments. The scaffold loaded with
minced fragments was then placed in a 10 cm cell culture dish
containing chondrocyte growth medium. The chondrocyte growth medium
consisted of Dulbecco's modified eagles medium (DMEM-high glucose)
supplemented with 20% fetal calf serum (FCS), 10 mM HEPES, 0.1 mM
nonessential amino acids, 20 mg/ml of L-proline, 50 mg/ml ascorbic
acid, 100 mg/ml penicillin, 100 mg/ml of streptomycin and 0.25
mg/ml of amphotericin B. The growth medium was replenished every
other day. Scaffolds were cultured at 37.degree. C. in a cell
culture incubator. Six weeks following culture samples were removed
and analyzed for cell distribution and migration within the
scaffolds and for production of cartilage like matrix. FIG. 1
demonstrates that cells migrate extensively into the polymer
scaffolds from the minced cartilage tissue fragments (FIG. 1A). The
migrating cells retain their phenotype and produce matrix that
stained positive for the sulfated glycosaminoglycans using the
Safranin O stain (FIG. 1B).
EXAMPLE 2
[0124] The bioresorbable scaffolds containing minced cartilage
tissue and cells from Example 1 were also implanted into SCID mice.
The objective was to evaluate the chondrocytic ingrowth of minced
cartilaginous tissues into polymer scaffolds in vivo. Polymer
scaffolds 5 mm in diameter, were subcutaneously implanted
bilaterally in the lateral thoracic region of SCID mice. The
implanted scaffold was permitted to support cell growth for four
weeks. The subcutaneous implantation sites with their overlying
skin were then excised and preserved in 10% buffered formalin
fixative. Following fixation, each implantation site was processed
for histology. Histological sections were stained with Hematoxylin
and eosin, and Safranin-O. FIGS. 2 A and B show that abundant cells
were distributed within the scaffold. The cells displayed
chondrocyte-like morphology, as evidenced by the intense positive
staining for Safranin O of the synthesized matrix.
EXAMPLE 3
[0125] Minced cartilage tissue prepared according to the method
described in Example 1 was distributed uniformly on a 4.times.5 cm
synthetic bioresorbable polycaprolactone/polyglycolic acid
(PCL/PGA) scaffold. Minced cartilage tissue fragments were adhered
to the scaffolds with 1 mL of platelet rich plasma (PRP, Human).
Sixty microliters (60 units) of thrombin were used to induce clot
formation in the PRP. Control scaffolds loaded with minced
cartilage fragments alone and scaffolds loaded with minced
cartilage fragments adhered by PRP, were cultured in vitro for 1
week, and then implanted into SCID mice as described in the Example
2. FIG. 3A is a photomicrograph of a control scaffold loaded with
minced tissue. FIG. 3B is a photomicrograph depicting a scaffold
loaded with minced tissue and PRP. FIG. 3B demonstrates that PRP is
beneficial in promoting the migration of the chondrocyte cells, and
PRP is also beneficial in promoting the maintenance of the
differentiated phenotype of the chondrocyte cells within the
scaffolds. The migrating cells retain their phenotype and produce
matrix that stained positive for the sulfated glycosaminoglycans
using the Safranin O stain (FIG. 3B).
EXAMPLE 4
[0126] Healthy full-thickness skin samples, collected from
1.times.1 cm wounds created on the dorsal side of the pigs, were
immediately placed in 50 ml conical tubes containing DMEM with
10.times. antibiotics/antimycotics. Tissue samples were rinsed once
in PBS containing 10.times. antibiotic/antimycotics followed by an
additional rinsing step with PBS containing 1.times.
antibiotics/antimycotics. The tissue was minced aseptically using a
scalpel blade in a laminar flow hood. Dispersed skin samples were
subjected to enzymatic digestion with 1 ml of 0.25%
collagenase/0.25% dispase at 37.degree. C. for 15 min (Autologous
cell dispersion #1). Another set of samples were first digested
with 500 .mu.l of 0.25% trypsin for 10 min, then washed with PBS to
remove trypsin, and then incubated with 1 ml of 0.25%
collagenase/0.25% dispase at 37.degree. C. for 15 min (Autologous
cell dispersion #2). Following digestion, the samples were
centrifuged at 2500 rpm for 5 min. The supernatant was aspirated
and discarded. Dispersed, partially digested skin samples were
washed once in PBS and then re-suspended in 500 .mu.l of PBS.
Approximately 20 .mu.l of cell suspension was distributed evenly in
the wound bed and bioresorbable scaffold was carefully applied on
the top of dispersed cells making sure not to dislodge the cell
suspension. Dispersed cells could be distributed evenly on the
scaffold and placed onto the wound bed. FIG. 4 demonstrates that
autologous cell dispersion was present histologically as
keratinocyte "islands," some of which had migrated throughout the
scaffold towards the wound surface.
EXAMPLE 5
[0127] Healthy anterior cruciate ligament tissue was obtained from
bovine knees. The ligament tissue was minced using scalpel blades
and/or scissors to obtain small tissue fragments. While the size of
the tissue fragments varied, the average particle size was
approximately 1 mm.sup.3 in dimension. In this example, the
ligament was minced with and without 0.2% collagenase. The minced
tissue was then distributed uniformly on a 4.times.5 cm synthetic
bioresorbable polycaprolactone/polyglycolic acid PGA/PCL scaffold
or polylactic acid/polyglycolic acid (PLA/PGA) scaffold. The
scaffolds were sterilized in 70% ethanol for our hour and washed
three times with sterile PBS. The scaffolds were then pre-soaked
for 1-2 hours in Dulbecco's Modified Eagle's Medium with
1.times.antibiotic-antim- ycotic prior to distribution of tissue
fragments. The scaffold loaded with minced fragments was then
placed in a 10 cm cell culture dish containing growth medium, which
consisted of Dulbecco's modified eagles medium (DMEM-high glucose)
supplemented with 20% fetal calf serum (FCS), 100 mg/ml penicillin,
100 mg/ml of streptomycin and 0.25 mg/ml of amphotericin B.
Scaffolds with the minced tissue were cultured at 37.degree. C. in
a cell culture incubator and the growth medium was exchanged every
other day. Three and six weeks following culture, samples were
removed and analyzed for cell distribution and migration within the
scaffolds. FIG. 5 demonstrates cells migrating extensively into the
polymer scaffolds after 6 weeks in culture from the minced anterior
cruciate tissue fragments treated with collagenase (FIG. 5A) and
without collagenase (FIG. 5B).
EXAMPLE 6
[0128] Menisci were harvested from adult Goat knees and 4 mm
diameter explants (2 mm thick) were taken from the white and
red/white regions. A 2 mm punch biopsy was removed from the center
of the explants. A bioresorbable scaffold polylactic
acid/polycaprolactone (PLA/PCL) 2 mm in diameter and 2 mm thick was
inserted into the center of the meniscal explant. The explants with
scaffolds were cultured for 2 and 3 weeks under standard cell
culture conditions with changes in media (DMEM containing 1% FBS,
1.times.antibiotic-antimycotic) occurring every other day. At 14
and 21 days following culture, half the samples were placed into
10% buffered formalin for histological processing. Sections were
stained with Hematoxylin to visualize the cells. From the remaining
samples the scaffolds were removed and cell number estimated by
quantitation of DNA using the CyQuant assay. FIG. 6A demonstrates
that there is cell migration into the polymer scaffolds from the
meniscal explants. FIG. 6B shows the histology of cross sections of
scaffolds demonstrating cell migration into scaffolds.
EXAMPLE 7
[0129] Healthy cartilage tissue and osteochondral plugs were
obtained from articulating joints of bovine shoulders. Minced
cartilage tissue was prepared according to the method described in
Example 1. In addition, osteochondral plugs (1.times.1 cm) were
harvested from bovine shoulders using a diamond bone saw and
morselized with bone cutters to obtain bone cartilage paste. Next,
250 mg of the sample (minced cartilage or bone cartilage paste) was
distributed on 2.times.5 cm synthetic bioresorbable (PCL/PGA)
scaffolds. The scaffold loaded with minced cartilage fragments or
osteochondral paste was then placed in a 10 cm cell culture dish
containing chondrocyte growth medium and cultured in a cell culture
incubator as described in Example 1. Three weeks following culture
the samples were removed and implanted into SCID mice as described
in Example 2. The objective was to evaluate the nature of tissue
formed within the scaffold following implantation for 4 weeks.
Histological sections were analyzed for cell distribution and for
the nature of the matrix formed, within the scaffolds, by staining
with Hematoxylin and eosin (H/E), Safranin O (SO) and Modified
Mallory's Aniline Blue (MMAB). FIGS. 7A-7C demonstrate that cells
migrate extensively into the polymer scaffolds from the minced
cartilage tissue fragments and form cartilage like matrix that
stains positive for Safranin O. This is particularly evident in
FIG. 7B in which the darker area in the center and top of the
photograph is indicative of positive staining. FIGS. 8A-8C
demonstrate that cells migrate from bone cartilage paste into
polymer scaffolds. However, the tissue that is formed comprises
cartilage as well as new bone. The appearance of the new bone is
indicated by the lighter arrows in FIG. 8C while the old bone
fragments are indicated by the darker arrows in FIGS. 8 A and
8C.
EXAMPLE 8
[0130] Healthy cartilage tissue was obtained from articulating
joints of bovine shoulders. Minced cartilage tissue was prepared
according to the method described in Example 1. Biopsy punches were
used to obtain cartilage tissue fragments 2 mm and 3 mm in
diameter. The thickness of these fragments was approximately 1 mm.
250 mg of minced cartilage or cartilage fragments 2 or 3 mm in
diameter were distributed on 2.times.5 cm synthetic bioresorbable
(PCL/PGA) scaffold. The scaffold loaded with cartilage fragments
was then placed in a 10 cm cell culture dish containing chondrocyte
growth medium and cultured in a cell culture incubator as described
in Example 1. Three weeks following culture samples were removed
and cell number estimated by quantitation of DNA content. 5 mm
biopsy punches were also implanted into SCID mice as described in
Example 2. The objective was to evaluate the optimal size of tissue
fragments for this process. FIG. 9 demonstrates that the highest
cell number was observed in scaffolds loaded with minced cartilage
tissue and the lowest in scaffolds loaded with cartilage fragments
3 mm in diameter. FIGS. 10A-10C provide histological evaluations of
scaffolds implanted into SCID mice and stained with Safranin O.
These results demonstrate that uniform cartilage-like tissue
(stained, darker areas) in scaffolds loaded with minced cartilage
tissue and cartilage fragments 2 mm in diameter (FIGS. 10A and B).
Scaffolds that were loaded with cartilage fragments 3 mm in
diameter were not uniformly filled (FIG. 10C).
[0131] One of ordinary skill in the art will appreciate further
features and advantages of the invention based on the
above-described embodiments. Accordingly, the invention is not to
be limited by what has been particularly shown and described,
except as indicated by the appended claims. All publications and
references cited herein are expressly incorporated herein by
reference in their entirety.
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