U.S. patent application number 09/740086 was filed with the patent office on 2001-10-25 for porous tissue scaffoldings for the repair or regeneration of tissue.
Invention is credited to Gorky, David V., Roller, Mark B., Scopelianos, Angelo George, Vyakarnam, Murty N., Zimmerman, Mark C..
Application Number | 20010033857 09/740086 |
Document ID | / |
Family ID | 25682420 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010033857 |
Kind Code |
A1 |
Vyakarnam, Murty N. ; et
al. |
October 25, 2001 |
Porous tissue scaffoldings for the repair or regeneration of
tissue
Abstract
The present patent describes a three-dimensional interconnected
open cell porous foams that have a gradient in composition and/or
microstructure through one or more directions. These foams can be
made from a blend of absorbable and biocompatible polymers that are
formed into foams having a compositional gradient transitioning
from predominately one polymeric material to predominately a second
polymeric material. These gradient foams are particularly well
suited to tissue engineering applications and can be designed to
mimic tissue transition or interface zones.
Inventors: |
Vyakarnam, Murty N.;
(Edison, NJ) ; Zimmerman, Mark C.; (East
Brunswick, NJ) ; Scopelianos, Angelo George;
(Whitehouse Station, NJ) ; Roller, Mark B.; (North
Brunswick, NJ) ; Gorky, David V.; (Flemington,
NJ) |
Correspondence
Address: |
Philip S. Johnson, Esq.
Johnson & Johnson
One Johnson & Johnson Plaza
New Brunswick
NJ
08933-7003
US
|
Family ID: |
25682420 |
Appl. No.: |
09/740086 |
Filed: |
December 19, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09740086 |
Dec 19, 2000 |
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09345096 |
Jun 30, 1999 |
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Current U.S.
Class: |
424/443 |
Current CPC
Class: |
A61L 27/56 20130101;
A61L 27/3817 20130101; A61F 2002/30766 20130101; A61F 2002/30004
20130101; A61F 2250/0018 20130101; A61L 31/06 20130101; A61L 31/06
20130101; A61F 2002/30784 20130101; A61F 2250/003 20130101; A61F
2002/30062 20130101; A61F 2002/3092 20130101; A61L 27/18 20130101;
A61F 2/30756 20130101; A61F 2250/0029 20130101; A61F 2002/30677
20130101; A61F 2002/30032 20130101; A61F 2002/30014 20130101; A61L
27/18 20130101; A61L 31/146 20130101; A61F 2250/0067 20130101; A61F
2/06 20130101; A61F 2002/30011 20130101; A61F 2/28 20130101; A61F
2250/0014 20130101; A61F 2210/0004 20130101; C08L 67/04 20130101;
C08L 67/04 20130101; A61F 2250/0023 20130101; Y10S 514/945
20130101; Y10S 623/915 20130101 |
Class at
Publication: |
424/443 |
International
Class: |
A61K 009/70 |
Claims
We claim:
1. A biocompatible gradient foam comprising a biocompatible
gradient foam having a first location and a second location wherein
the biocompatible gradient foam has a substantially continuous
transition in at least one characteristic selected from the group
consisting of composition, stiffness, flexibility, bioabsorption
rate and pore architecture from the first location to the second
location of said biocompatible gradient foam.
2. The biocompatible gradient foam of claim 1 wherein the
biocompatible gradient foam is bioabsorbable.
3. The biocompatible gradient foam of claim 1 wherein the
biocompatible gradient foam is made from a bioabsorbable polymer
selected from the group consisting of aliphatic polyesters,
poly(amino acids), copoly(ether-esters), polyalkylenes oxalates,
polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters,
polyamidoesters, polyoxaesters containing amine groups
poly(anhydrides), polyphosphazenes, biopolymers and blends
thereof.
4. The biocompatible gradient foam of claim 3 wherein the
bioabsorable polymer is an aliphatic polyester.
5. The biocompatible gradient foam of claim 4 wherein the aliphatic
polyester is selected from the group consisitng of homopolymers and
copolymers of lactide, lactic acid, glycolide, 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,
1,5,8,12-tetraoxacyclotetradecane-7,- 14-dione),
1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one and polymer
blends thereof.
6. The biocompatible gradient foam of claim 3 wherein the aliphatic
polyester is an elastomer.
7. The biocompatible gradient foam of claim 5 wherein the elastomer
is selected from the group consisting of copolymers of
.epsilon.-caprolactone and glycolide; copolymers of
.epsilon.-caprolactone and (L) lactide, copolymers of p-dioxanone
(1,4-dioxan-2-one) and (L) lactide, copolymers of
.epsilon.-caprolactone and p-dioxanone, copolymers of p-dioxanone
and trimethylene carbonate, copolymers of trimethylene carbonate
and glycolide, copolymer of trimethylene carbonate and (L) lactide
and blends thereof.
8. The biocompatible gradient foam of claim 4 wherein additionally
present as a constituent of the biocompatible gradient foam is a
second aliphatic polyester.
9. The biocompatible gradient foam of claim 3 wherein the
biocompatible gradient foam has a substantially continuous
transition in composition from the first location to the second
location.
10. The biocompatible gradient foam of claim 9 wherein the
biocompatible gradient foam has a substantially continuous
transition in composition from a first ratio of at least two
different aliphatic polyesters to a second ratio of said at least
two different aliphatic polyesters from the first surface to the
second surface.
11. The biocompatible gradient foam of claim 3 wherein the
biocompatible gradient foam has a substantially continuous
transition in stiffness from the first location to the second
location.
12. The biocompatible gradient foam of claim 3 wherein the
biocompatible gradient foam has a substantially continuous
transition in bioabsorption rate from the first location to the
second location.
13. The biocompatible gradient foam of claim 3 wherein the
biocompatible gradient foam has a substantially continuous
transition in flexibility from the first location to the second
location.
14. The biocompatible gradient foam of claim 3 wherein the
biocompatible gradient foam has a substantially continuous
transition in architecture from the first location to the second
location.
15. The biocompatible gradient foam of claim 14 wherein the
biocompatible gradient foam has a substantially continuous
transition in architecture from a substantially spherical pore
shape to a columnar pore shape from the first location to the
second location.
16. The biocompatible gradient foam of claim 14 wherein the
substantially spherical pore's size is from about 30 .mu.m to about
150 .mu.m.
17. The biocompatible gradient foam of claim 14 wherein the
columnar pore's diameter is from about 100 .mu.m to about 400 .mu.m
with a length to diameter ratio of at least 2.
18. The biocompatible gradient foam of claim 1 wherein also present
in the biocompatible gradient foam is a therapeutic agent.
19. The biocompatible gradient foam of claim 1 wherein additionally
present is an agent is selected from the group consisting of
antiinfectives, hormones, analgesics, anti-inflammatory agents,
growth factors, chemotherapeutic agents, anti-rejection agents
prostaglandins, RDG peptides and combinations thereof.
20. The biocompatible gradient foam of claim 19 wherein the growth
factor is selected from the group consisting of bone morphogenic
proteins, bone morphogenic-like proteins, epidermal growth factor,
fibroblast growth factors, platelet derived growth factor, insulin
like growth factor, transforming growth factors, vascular
endothelial growth factor and combinations thereof.
21. The biocompatible gradient foam of claim 1 wherein the
biocompatible gradient foam is filled with a biocomptible material
selected from the group consisting of bioabsorbable synthetic
polymers, biocompatible, bioabsorbable biopolymers, biocompatible
ceramic materials and combinations thereof.
22. A biocompatible foam comprising a biocompatible foam having a
first surface and a second surface with interconnecting pores and
channels.
23. The biocompatible foam of claim 22 wherein the channels have an
average length of at least 200 .mu.m.
24. The biocompatible foam of claim 22 wherein the channels extend
substantially from said first surface to said second surface.
25. The biocompatible foam of claim 22 wherein the biocompatible
foam is bioabsorbable.
26. The biocompatible foam of claim 22 wherein the biocompatible
foam is made from a bioabsorbable polymer selected from the group
consisting of aliphatic polyesters, poly(amino acids),
copoly(ether-esters), polyalkylenes oxalates, polyamides,
poly(iminocarbonates), polyorthoesters, polyoxaesters,
polyamidoesters, polyoxaesters containing amine groups
poly(anhydrides), polyphosphazenes, biopolymers and blends
thereof.
27. The biocompatible foam of claim 26 wherein the bioabsorable
polymer is an aliphatic polyester.
28. The biocompatible foam of claim 27 wherein the aliphatic
polyester is selected from the group consisitng of homopolymers and
copolymers of lactide, lactic acid, glycolide, 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,
1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,
6,6-dimethyl-1,4-dioxan-2-one and polymer blends thereof.
29. The biocompatible foam of claim 26 wherein the aliphatic
polyester is an elastomer.
30. The biocompatible foam of claim 29 wherein the elastomer is
selected from the group consisting of copolymers of
.epsilon.-caprolactone and glycolide; copolymers of
.epsilon.-caprolactone and (L) lactide, copolymers of p-dioxanone
(1,4-dioxan-2-one) and (L) lactide, copolymers of
.epsilon.-caprolactone and p-dioxanone, copolymers of p-dioxanone
and trimethylene carbonate, copolymers of trimethylene carbonate
and glycolide, copolymer of trimethylene carbonate and (L) lactide
and blends thereof.
31. The biocompatible foam of claim 27 wherein additionally present
as a constituent of the foam is a second aliphatic polyester.
32. The biocompatible foam of claim 26 wherein the biocompatible
foam has a substantially continuous transition in composition from
the first surface to the second surface.
33. The biocompatible foam of claim 32 wherein the biocompatible
foam has a substantially continuous transition in composition from
a first ratio of at least two different aliphatic polyesters to a
second ratio of said at least two different aliphatic polyesters
from the first surface to the second surface.
34. The biocompatible foam of claim 26 wherein the biocompatible
foam has a substantially continuous transition in stiffness from
the first surface to the second surface.
35. biocompatible foam of claim 26 wherein the biocompatible foam
has a substantially continuous transition in bioabsorption rate
from the first surface to the second surface.
36. The biocompatible foam of claim 26 wherein the biocompatible
foam has a substantially continuous transition in flexibility from
the first surface to the second surface.
37. The biocompatible foam of claim 26 wherein the biocompatible
foam has a substantially continuous transition in architecture from
the first surface to the second surface.
38. The biocompatible foam of claim 37 wherein the subsantially
spherical pore's size is from about 30 .mu.m to about 150
.mu.m.
39. The biocompatible foam of claim 22 wherein also present in the
biocompatible foam is a therapeutic agent.
40. The biocompatible foam of claim 22 wherein additonally present
is an agent selected from the group consisting of antiinfectives,
hormones, analgesics, anti-inflammatory agents, growth factors,
chemotherapeutic agents, anti-rejection agents, prostaglandins, RDG
peptides and combinations thereof.
41. The biocompatible foam of claim 40 wherein the growth factor is
selected from the group consisting of bone morphogenic proteins,
bone morphogenic-like proteins, epidermal growth factor, fibroblast
growth factors, platelet derived growth factor, insulin like growth
factor, transforming growth factors, vascular endothelial growth
factor and combinations thereof.
42. The biocompatible foam of claim 22 wherein the biocompatible
foam is filled with a biocomptible material selected from the group
consisting of bioabsorbable synthetic polymers, biocompatible,
bioabsorbable biopolymers, biocompatible ceramic materials and
combinations thereof.
43. A biocompatible foam comprising a biocompatible foam having
interconnected pores formed from a composition containing in the
range of from about 30 weight percent to about 99 weight percent
.epsilon.-caprolactone repeating units.
44. The biocompatible foam of claim 43 wherein the
.epsilon.-caprolactone repeating units are copolymerized with a
comonomer selected from the group consisting of lactide, lactic
acid, glycolide, glycolic acid), 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,
1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,
6,6-dimethyl-1,4-dioxan-2-one and polymer blends thereof.
45. The biocompatible foam of claim 43 having a first location and
a second location wherein the biocompatible foam has a
substantially continuous transition in at least one characteristic
selected from the group consisting of composition, stiffness,
flexibility, bioabsorption rate and pore architecture from the
first location to the second location of said biocompatible
foam.
46. The biocompatible foam of claim 43 wherein the interconnecting
pores have a pore size in the range from about 10 .mu.m to about
200 .mu.m.
47. The biocompatible foam of claim 43 wherein the biocompatible
foam has a porosity of in the range of from about 20 to about 98
percent.
48. The biocompatible foam of claim 43 wherein the biocompatible
foam has channels.
49. The biocompatible foam of claim 48 wherein the channels have an
average length of at least 200 .mu.m.
50. The biocompatible foam of claim 43 wherein additionally present
as a constituent of the biocompatible foam is a second aliphatic
polyester.
51. The biocompatible foam of claim 45 wherein the biocompatible
foam has a substantially continuous transition in composition from
the first location to the second location.
52. The biocompatible foam of claim 51 wherein the biocompatible
foam has a substantially continuous transition in composition from
a first ratio of at least two different aliphatic polyesters to a
second ratio of said at least two different aliphatic polyesters
from the first location to the second location.
53. The biocompatible foam of claim 45 wherein the biocompatible
foam has a substantially continuous transition in stiffness from
the first location to the second location.
54. The biocompatible foam of claim 45 wherein the biocompatible
foam has a substantially continuous transition in bioabsorption
rate from the first location to the second location.
55. The biocompatible foam of claim 45 wherein the biocompatible
foam has a substantially continuous transition in flexibility from
the first location to the second location.
56. The biocompatible foam of claim 45 wherein the biocompatible
foam has a substantially continuous transition in architecture from
the first location to the second location.
57. The biocompatible foam of claim 56 wherein the biocompatible
foam has a substantially continuous transition in architecture from
a substantially spherical pore shape to a columnar pore shape from
the first location to the second location.
58. The biocompatible foam of claim 56 wherein the substantially
spherical pore's size is from about 30 .mu.m to about 150
.mu.m.
59. The biocompatible foam of claim 56 wherein the columnar pore's
diameter is from about 30 .mu.m to about 400 .mu.m with a length to
diameter ratio of at least 2.
60. The biocompatible foam of claim 43 wherein also present in the
biocompatible foam is a therapeutic agent.
61. The biocompatible foam of claim 43 wherein additionally present
is an agent selected from the group consisting of antiinfectives,
hormones, analgesics, anti-inflammatory agents, growth factors,
agents, anti-rejection agents, prostaglandins, RDG peptides and
combinations thereof.
62. The biocompatible foam of-claim 61 wherein the growth factor is
selected from the group consisting of bone morphogenic proteins,
bone morphogenic-like proteins, epidermal growth factor, fibroblast
growth factors, platelet derived growth factor, insulin like growth
factor, transforming growth factors, vascular endothelial growth
factor and combinations thereof.
63. The biocompatible foam of claim 43 wherein the biocompatible
foam is filled with a biocomptible material selected from the group
consisting of bioabsorbable synthetic polymers, biocompatible,
bioabsorbable biopolymers, biocompatible ceramic materials and
combinations thereof.
64. The biocompatible gradient foam of claim 1 wherein the
biocompatible gradient foam is formed with an insert within the
biocompatible gradient foam.
65. The biocompatible gradient foam of claim 64 wherein the insert
is selected from the group consisting of films, scrims, woven
textiles, knitted textiles, braided textiles, orthopedic implants,
tubes and combinations thereof.
66. The biocompatible gradient foam of claim 22 wherein the
biocompatible foam is formed with an insert within the
biocompataible foam.
67. The biocompatible foam of claim 66 wherein the insert is
selected from the group consisting of films, scrims, woven
textiles, knitted textiles, braided textiles, orthopedic implants,
tubes and combinations thereof.
68. The biocompatible foam of claim 43 wherein the biocompatible
foam is formed with an insert within the biocompatible foam.
69. The biocompatible foam of claim 68 wherein the insert is
selected from the group consisting of films, scrims, woven
textiles, knitted textiles, braided textiles, orthopedic implants,
tubes and combinations thereof.
70. The biocompatible gradient foam of claim 1 wherein the
biocompatible gradient foam is formed into a three-dimensional
shaped structure.
71. The biocompatible gradient foam of claim 70 wherein the
three-dimensional shaped structure is selected from the group
consisting of tubular shapes, branched tubular shapes, spherical
shapes, hemispherical shapes, three-dimensional polygonal shapes,
ellipsoidal shapes, toroidal shapes, conical shapes, frusta conical
shapes, pyramidal shapes, both as solid and hollow constructs and
combination thereof.
72. The biocompatible foam of claim 22 wherein the biocompatible
foam is formed into a three-dimensional shaped structure.
73. The biocompatible foam of claim 72 wherein the
three-dimensional shaped structure is selected from the group
consisting of tubular shapes, branched tubular shapes, spherical
shapes, hemispherical shapes, three-dimensional polygonal shapes,
ellipsoidal shapes, toroidal shapes, conical shapes, frusta conical
shapes, pyramidal shapes, both as solid and hollow constructs and
combination thereof.
74. The biocompatible foam of claim 43 wherein the biocompatible
foam is formed into a three-dimensional shaped structure.
75. The biocompatible foam of claim 74 wherein the
three-dimensional shaped structure is selected from the group
consisting of tubular shapes, branched tubular shapes, spherical
shapes, hemispherical shapes, three-dimensional polygonal shapes,
ellipsoidal shapes, toroidal shapes, conical shapes, frusta conical
shapes, pyramidal shapes, both as solid and hollow constructs and
combination thereof.
76. A method for the repair or regeneration of tissue comprising
contacting cells with a biocompatible gradient foam having a
biocompatible gradient foam having a first location and a second
location wherein the biocompatible gradient foam has a
substantially continuous transition in at least one characteristic
selected from the group consisting of composition, stiffness,
flexibility, bioabsorption rate and pore architecture from the
first location to the second location of said biocompatible
gradient foam.
77. The method of claim 76 wherein the biocompatible gradient foam
is bioabsorbable.
78. The method of claim 76 wherein the biocompatible gradient foam
is made from a bioabsorbable polymer selected from the group
consisting of aliphatic polyesters, poly(amino acids),
copoly(ether-esters), polyalkylenes oxalates, polyamides, poly
(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters,
polyoxaesters containing amine groups poly(anhydrides),
polyphosphazenes, biopolymers and blends thereof.
79. The method foam of claim 78 wherein the bioabsorable polymer is
an aliphatic polyester.
80. The method foam of claim 79 wherein the aliphatic polyester is
selected from the group consisting of homopolymers and copolymers
of lactide, lactic acid, glycolide, 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,
1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,
6,6-dimethyl-1,4-dioxan-2-one and polymer blends thereof.
81. The biocompatible gradient foam of claim 80 wherein the
aliphatic polyester is an elastomer.
82. The method of claim 76 wherein cells are seeded onto the
biocompatible gradient foam.
83. The method of claim 80 wherein cells are seeded onto the
biocompatible gradient foam.
84. The method of claim 76 wherein the biocompatible gradient foam
is implanted in an animal and contacted with cells.
85. The method of claim 80 wherein the biocompatible gradient foam
is implanted in an animal and contacted with cells.
86. The method of claim 76 wherein the biocompatible gradient foam
is seeded with cells and the biocompatible gradient foam and cells
are placed in a cell culturing device and the cells are allowed to
multiply on the biocompatible gradient foam.
87. The method of claim 80 wherein the biocompatible gradient foam
is seeded with cells and the biocompatible gradient foam and cells
are placed in a cell culturing device and the cells are allowed to
multiply on the biocompatible gradient foam.
88. The method of claim 80 wherein the cells are selected from the
group consisting of pluripotent cells, stem cells, precursor cells
and combinations thereof.
89. The method of claim 80 wherein the cells are selected from the
group consisting of myocytes, adipocytes, fibromyoblasts,
ectodermal cell, muscle cells, osteoblast, chondrocyte, endothelial
cells, fibroblast, pancreatic cells, hepatocyte, bile duct cells,
bone marrow cells, neural cells, genitourinary cells and
combinations thereof.
90. The method of claim 82 wherein the cells are selected from the
group consisting of pluripotent cells, stem cells, precursor cells
and combinations thereof.
91. The method of claim 82 wherein the cells are selected from the
group consisting of myocytes, adipocytes, fibromyoblasts,
ectodermal cell, muscle cells, osteoblast, chondrocyte, endothelial
cells, fibroblast, pancreatic cells, hepatocyte, bile duct cells,
bone marrow cells, neural cells, genitourinary cells and
combinations thereof.
92. The method of claim 76 wherein the biocompatible gradient foam
contains an agent selected from the group consisting of
antiinfectives, hormones, analgesics, anti-inflammatory agents,
growth factors, chemotherapeutic agents, anti-rejection agents,
prostaglandins, RDG peptides and combinations thereof.
93. A method for the repair or regeneration of tissue comprising
contacting cells with a biocompatible foam having a first surface
and a second surface with interconnecting pores and channels.
94. The method of claim 93 wherein the biocompatible foam is
bioabsorbable.
95. The method of claim 93 wherein the biocompatible foam is made
from a bioabsorbable polymer selected from the group consisting of
aliphatic polyesters, poly(amino acids), copoly(ether-esters),
polyalkylenes oxalates, polyamides, poly(iminocarbonates),
polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters
containing amine groups poly(anhydrides), polyphosphazenes,
biopolymers and blends thereof.
96. The method of claim 95 wherein the bioabsorable polymer is an
aliphatic polyester.
97. The method of claim 96 wherein the aliphatic polyester is
selected from the group consisting of homopolymers and copolymers
of lactide, lactic acid, glycolide, 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,
1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,
6,6-dimethyl-1,4-dioxan-2-one and polymer blends thereof.
98. The method of claim 97 wherein the aliphatic polyester is an
elastomer.
99. The method of claim 93 wherein cells are seeded onto the
biocompatible foam.
100. The method of claim 97 wherein cells are seeded onto the
biocompatible foam.
101. The method of claim 93 wherein the biocompatible foam is
implanted in an animal and contacted with cells.
102. The method of claim 97 wherein the biocompatible foam is
implanted in an animal and contacted with cells.
103. The method of claim 93 wherein the biocompatible foam is
seeded with cells and the biocompatible foam and cells are placed
in a cell culturing device and the cells are allowed to multiply on
the biocompatible foam.
104. The method of claim 97 wherein the biocompatible foam is
seeded with cells and the biocompatible foam and cells are placed
in a cell culturing device and the cells are allowed to multiply on
the biocompatible foam.
105. The method of claim 97 wherein the cells are selected from the
group consisting of pluripotent cells, stem cells, precursor cells
and combinations thereof.
106. The method of claim 97 wherein the cells are selected from the
group consisting of myocytes, adipocytes, fibromyoblasts,
ectodermal cell, muscle cells, osteoblast, chondrocyte, endothelial
cells, fibroblast, pancreatic cells, hepatocyte, bile duct cells,
bone marrow cells, neural cells, genitourinary cells and
combinations thereof.
107. The method of claim 99 wherein the cells are selected from the
group consisting of pluripotent cells, stem cells, precursor cells
and combinations thereof.
108. The method of claim 99 wherein the cells are selected from the
group consisting of myocytes, adipocytes, fibromyoblasts,
ectodermal cell, muscle cells, osteoblast, chondrocyte, endothelial
cells, fibroblast, pancreatic cells, hepatocyte, bile duct silo
cells, bone marrow cells, neural cells, genitourinary cells and
combinations thereof.
109. The method of claim 93 wherein the biocompatible foam contains
an agent selected from the group consisting of antiinfectives,
hormones, analgesics, anti-inflammatory agents, growth factors,
chemotherapeutic agents, anti-rejection agents, prostaglandins, RDG
peptides and combinations thereof.
110. A method for the repair or regeneration of tissue comprising
contacting cells with a biocompatible having interconnected pores
formed from a composition containing in the range of from about 30
weight percent to about 99 weight percent .epsilon.-caprolactone
repeating units.
111. The method foam of claim 110 wherein the
.epsilon.-caprolactone repeating units are polymerized with a
comonomer selected from the group consisting of homopolymers and
copolymers of lactide, lactic acid, glycolide, glycolic acid),
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, 1,5,8,12-tetraoxacyclotetradecane-7,-
14-dione), 1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one and
polymer blends thereof.
112. The biocompatible gradient foam of claim ill wherein the
aliphatic polyester is an elastomer.
113. The method of claim 110 wherein cells are seeded onto the
biocompatible foam.
114. The method of claim 111 wherein cells are seeded onto the
biocompatible foam.
115. The method of claim 110 wherein the biocompatible foam is
implanted in an animal and contacted with cells.
116. The method of claim 111 wherein the biocompatible foam is
implanted in an animal and contacted with cells.
117. The method of claim 110 wherein the biocompatible foam is
seeded with cells and the biocompatible foam and cells are placed
in a cell culturing device and the cells are allowed to multiply on
the biocompatible foam.
118. The method of claim 111 wherein the biocompatible gradient
foam is seeded with cells and the biocompatible foam and cells are
placed in a cell culturing device and the cells are allowed to
multiply on the biocompatible foam.
119. The method of claim 110 wherein the cells are selected from
the group consisting of pluripotent cells, stem cells, precursor
cells and combinations thereof.
120. The method of claim 110 wherein the cells are selected from
the group consisting of myocytes, adipocytes, fibromyoblasts,
ectodermal cell, muscle cells, osteoblast, chondrocyte, endothelial
cells, fibroblast, pancreatic cells, hepatocyte, bile duct cells,
bone marrow cells, neural cells, genitourinary cells and
combinations thereof.
121. The method of claim 110 wherein the cells are selected from
the group consisting of pluripotent cells, stem cells, precursor
cells and combinations thereof.
122. The method of claim 111 wherein the cells are selected from
the group consisting myocytes, adipocytes, fibromyoblasts,
ectodermal cell, muscle cells, osteoblast, chondrocyte, endothelial
cells, fibroblast, pancreatic cells, hepatocyte, bile duct cells,
bone marrow cells, neural cells, genitourinary cells and
combinations thereof.
123. The method of claim 110 wherein the biocompatible gradient
foam contains an agent selected from the group consisting of
antiinfectives, hormones, analgesics, anti-inflammatory agents,
growth factors, chemotherapeutic agents, anti-rejection agents,
prostaglandins, RDG peptides and combinations thereof.
124. The method of claim 86 wherein after the cells are allowed to
multiply on the biocompatible foam, the biocompatible foam and the
cells are implanted into an animal.
125. The method of claim 103 wherein after the cells are allowed to
multiply on the biocompatible foam, the biocompatible foam and the
cells are implanted into an animal.
126. The method of claim 118 wherein after the cells are allowed to
multiply on the biocompatible foam, the biocompatible foam and the
cells are implanted into an animal.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of
tissue repair and regeneration. More particularly the present
invention relates to porous biocompatible bioabsorbable foams that
have a gradient in composition and/or microstructure that serve as
a template for tissue regeneration, repair or augmentation.
BACKGROUND OF THE INVENTION
[0002] Open cell porous biocompatible foams have been recognized to
have significant potential for use in the repair and regeneration
of tissue. Early efforts in tissue repair focused on the use of
amorphous biocompatible foam as porous plugs to fill voids in bone.
Brekke, et al. (U.S. Pat. No. 4,186,448) described the use of
porous mesh plugs composed of polyhydroxy acid polymers such as
polylactide for healing bone voids. Several attempts have been made
in the recent past to make TE scaffolds using different methods,
for example U.S. Pat. No. 5,522,895 (Mikos) and 5,514,378 (Mikos,
et al.) using leachables; U.S. Pat. No. 5,755,792 (Brekke) and U.S.
Pat. No. 5,133,755 (Brekke) using vacuum foaming techniques; U.S.
Pat. No. 5,716,413 (Walter, et al.) and U.S. Pat. No. 5,607,474
(Athanasiou, et al.) using precipitated polymer gel masses; U.S.
Pat. No. 5,686,091 (Leong, et al.) and U.S. Pat. No. 5,677,355
(Shalaby, et al.) using polymer melts with fugitive compounds that
sublimate at temperatures greater than room temperature; and U.S.
Pat. No. 5,770,193 (Vacanti, et al.) U.S. Pat. No. 5,769,899
(Schwartz, et al.) and U.S. Pat. No. 5,711,960 (Shikinami) using
textile-based fibrous scaffolds. Hinsch et al. (EPA 274,898)
described a porous open cell foam of polyhydroxy acids with pore
sizes from about 10 to about 200 .mu.m for the in-growth of blood
vessels and cells. The foam described by Hincsh could also be
reinforced with fibers, yarns, braids, knitted fabrics, scrims and
the like. Hincsh's work also described the use of a variety of
polyhydroxy acid polymers and copolymers such as poly-L-lactide,
poly-DL-lactide, polyglycolide, and polydioxanone. The Hincsh foams
had the advantage of having regular pore sizes and shapes that
could be controlled by the processing conditions, solvents
selected, and the additives.
[0003] However, the above techniques have limitations in producing
a scaffold with a gradient structure. Most of the scaffolds are
isotropic in form and function and lack the anisotropic features of
natural tissues.
[0004] Further, it is the limitation of prior art to make 3D
scaffolds that have the ability to control the spatial distribution
of various pore shapes. The process that is described to fabricate
the microstructure controlled foams is a low temperature process
that offers many advantages over other conventional techniques. For
example the process allows the incorporation of thermally sensitive
compounds like proteins, drugs and other additives with the
thermally and hydrolytically unstable absorbable polymers.
[0005] Athanasiou et al. (U.S. Pat. No. 5,607,474) have more
recently proposed using a two layer foam device for repairing
osteochondral defects at a location where two dissimilar types of
tissue are present. The Athanasiou device is composed of a first
and second layer, prepared in part separately, and joined together
at a subsequent step. Each of the scaffold layers is designed to
have stiffness and compressibility corresponding to the respective
cartilage and bone tissue. Since cartilage and bone often form
adjacent layers in the body this approach is an attempt to more
clearly mimic the structure of the human body. However, the
interface between the cartilage and bone in the human body is not a
discrete junction of two dissimilar materials with an abrupt change
in anatomical features and/or the mechanical properties. The
cartilage cells have distinctly different cell morphology and
orientation depending on the location of the cartilage cell in
relation to the underlying bone structure. The difference in
cartilage cell morphology and orientation provides a continuous
transition from the outer surface of the cartilage to the
underlying bone cartilage interface. Thus the two layer system of
Athanasiou, although an incremental improvement, does not mimic the
tissue interfaces present in the human body.
[0006] Another approach to make three-dimensional laminated foams
is proposed by Mikos et al. (U.S. Pat. No. 5,514,378). In this
technique which is quite cumbersome, a porous membrane is first
prepared by drying a polymer solution containing leachable salt
crystals. A three-dimensional structure is then obtained by
laminating several membranes together, which are cut to a contour
drawing of the desired shape.
[0007] One of the major weaknesses of the prior art regarding
three-dimensional porous scaffolds used for the regeneration of
biological tissue like cartilage is that their microstructure is
random. These scaffolds, unlike natural tissue, do not vary in
morphology or structure. Further, current scaffolds do not provide
adequate nutrient and fluid transport for many applications.
Finally, the laminated structures are not completely integrated and
subjected to delamination under in vivo conditions.
[0008] Therefore, it is an object of the present invention to
provide a biocompatible, bioabsorbable foam that provides a
continuous transitional gradient of morphological, structural
and/or materials. Further, it is preferred that foams used in
tissue engineering have a structure that provides organization at
the microstructure level that provides a template that facilitates
cellular invasion, proliferation and differentiation that will
ultimately result in regeneration of functional tissue.
SUMMARY OF INVENTION
[0009] The present invention provides a biocompatible gradient foam
that has a substantially continuous transition in at least one
characteristic selected from the group consisting of composition,
stiffness, flexibility, bioabsorption rate pore architecture and/or
microstructure. This gradient foam can be made from a blend of
absorbable polymers that form compositional gradient transitions
from one polymeric material to a second polymeric material. In
situations where a single chemical composition is sufficient for
the application, the invention provides a biocompatible foam that
may have microstructural variations in the structure across one or
more dimensions that may mimic the anatomical features of the
tissue (e.g. cartilage, skin, bone etc.).
[0010] The present invention further provides biocompatible foam
having interconnecting pores and channels to facilitate the
transport of nutrients and/or invasion of cells into the scaffold.
These biocompatible foams are especially well adapted for
facilitating the ingrowth of tissue as is described in Example
7.
[0011] In yet another embodiment of the present invention
biocompatible foams having interconnecting pores formed from a
composition containing in the range of from about 30 weight percent
to about 99 weight .epsilon.-caprolactone repeating units are
disclosed. These biocompatible foams are especially well adapted
for facilitating the growth of osteoblasts as is described in
Example 6.
[0012] The present invention also provides a method for the repair
or regeneration of tissue contacting a first tissue with a gradient
foam at a location on the foam that has appropriate properties to
facilitate the growth of said tissue. The concept of a continuous
transition in physical properties, chemical composition and/or
microstructural features in the porous scaffold (foam) can
facilitate the growth or regeneration of tissue. These foam
structures are particularly useful for the generation of tissue
junctions between two or more different types of tissues. For a
multi-cellular system in the simplest case, one cell type could be
present on one side of the scaffold and a second cell type on the
other side of the scaffold. Examples of such regeneration can be
(a) skin: with fibroblasts on one side to regenerate dermis, and
keratinocytes on the other to regenerate epidermis; (b) vascular
grafts: with an endothelial layer on the inside of the graft and a
smooth muscle cell layer on the outside.
BRIEF DESCRIPTION OF FIGURES
[0013] FIG. 1 is a scanning electron micrograph of the cross
section of a random microstructure foam made from 5% solution of
35/65 .epsilon.-caprolactone-co-glycolide copolymer.
[0014] FIG. 2 is a scanning electron micrograph of the cross
section of a foam with vertical open channels made from 10%
solution of 35/65 .epsilon.-caprolactone-co-glycolide
copolymer.
[0015] FIG. 3 is a scanning electron micrograph of the cross
section of a foam with architectural gradient made from 10%
solution of 35/65 .epsilon.-caprolactone-co-glycolide
copolymer.
[0016] FIG. 4 is a scanning electron micrograph of the cross
section of a gradient foam made from a 50/50 blend of 40/60
.epsilon.-caprolactone-co-- (L) lactide copolymer and 35/65
.epsilon.-caprolactone-co-glycolide copolymer.
[0017] FIG. 5 is a scanning electron micrograph of a cross section
of the top portion of a gradient foam made from a 50/50 blend of
40/60 .epsilon.-caprolactone-co-(L) lactide copolymer and 35/65
.epsilon.-caprolactone-co-glycolide copolymer.
[0018] FIG. 6 is a scanning electron micrograph of a cross section
of the bottom portion of a gradient foam made from a 50/50 blend of
40/60 .epsilon.-caprolactone-co-(L) lactide 1.5 copolymer and 35/65
.epsilon.-caprolactone-co-glycolide copolymer.
[0019] FIG. 7 is a graphical presentation of cell culture data, 7A,
7B and 7C.
[0020] FIG. 8 is an anatomical sketch of cartilage tissue.
[0021] FIGS. 9A, 9B, and 9C are scanning electron micrographs of a
0.5 mm foam made from a 50/50 blend of a 35/65
.epsilon.-caprolactone-co-glycoli- de copolymer and a 40/60
.epsilon.-caprolactone-co-(L) lactide copolymer with architecture
suitable for use as a skin scaffold.
[0022] FIG. 9A shows the porosity of the surface of the scaffold
that preferably would face the wound bed.
[0023] FIG. 9B shows the porosity of the surface of the scaffolding
that would preferably face away from the wound bed.
[0024] FIG. 9C shows a cross section of the scaffold with channels
running through the thickness of the foam.
[0025] FIG. 10 is a dark field 40.times. photomicrograph of a
trichrome stained sample illustrating the cellular invasion of the
foam shown in FIG. 9, eight days after implantation in a swine
model.
[0026] FIG. 11 is a 100.times. composite photomicrograph of a
trichrome stained sample illustrating the cellular invasion of the
foam shown in FIG. 9 which also contained PDGF, eight days after
implantation in a swine model.
DETAILED DESCRIPTION OF THE INVENTION
[0027] This invention describes porous bioabsorbable polymer foams
that have novel microstructures. The features of such foams can be
controlled to suit a desired application by choosing the
appropriate conditions to form the foam during lyophilization.
These features in absorbable polymers have distinct advantages over
the prior art where the scaffolds are typically isotropic or random
structures. However, it is preferred that foams used in tissue
engineering (i.e. repair or regeneration) have a structure that
provides organization at the microstructural level that provides a
template that facilitates cellular organization and regeneration of
tissue that has the anatomical, biomechanical, and biochemical
features of normal tissues. These foams can be used to repair or
regenerate tissue (including organs) in animals such as domestic
animals, primates and humans.
[0028] The features of such foams can be controlled to suit desired
application by selecting the appropriate conditions for
lyophilization to obtain one or more of the following properties:
(1) interconnecting pores of sizes ranging from about 10 to about
200 .mu.m (or greater) that provide pathways for cellular ingrowth
and nutrient diffusion; (2) a variety of porosities ranging from
about 20% to about 98% and preferably ranging from about 80% to
about 95%; (3) gradient in the pore size across one direction for
preferential cell culturing; (4) channels that run through the foam
for improved cell invasion, vascularization and nutrient diffusion;
(5) micro-patterning of pores on the surface for cellular
organization; (6) tailorability of pore shape and/or orientation
(e.g. substantially spherical, ellipsoidal, columnar); (7)
anisotropic mechanical properties; (8) composite foams with a
polymer composition gradient to elicit or take advantage of
different cell response to different materials; (9) blends of
different polymer compositions to create structures that have
portions that will break down at different rates; (10) foams
co-lyophilized or coated with pharmaceutically active compounds
including but not limited to biological factors such as RGD's,
growth factors (PDGF, TGF-.beta., VEGF, BMP, FGF etc.) and the
like; (11) ability to make 3 dimensional shapes and devices with
preferred microstructures; and (12) lyophilization with other parts
or medical devices to provide a composite structure. These
controlled features in absorbable polymers have distinct advantages
over the prior art where the scaffolds are typically isotropic or
random structures with no preferred morphology at the pore level.
However, it is preferred that foams used in tissue scaffolds have a
structure that provides organization at the microstructure level
and provides a template that facilitates cellular organization that
may mimic natural tissue. The cells will adhere, proliferate and
differentiate along and through the contours of the structure. This
wilt ultimately result in a cultured tissue that may mimic the
anatomical features of real tissues to a large extent.
[0029] For example, as shown in FIG. 3 the orientation of the major
axis of the pores may be changed from being in the same plane as
the foam to being oriented perpendicular to the plane of the foam.
As can be seen from FIG. 3 the pore size can be varied from a small
pore size generally between about 30 .mu.m and about 50 .mu.m to a
larger size of from about 100 .mu.m to about 200 .mu.m in porous
gradient foams. Ideally the foam structure could be created to
facilitate the repair or regeneration of human tissue junctions
such as the cartilage to bone junction present in joints. This foam
would progress from a small (i.e. about 30 .mu.m to about 150 .mu.m
in diameter) round pores to larger column-like pores (i.e. about 30
.mu.m to about 400 .mu.m in diameter, preferably about 100 .mu.m to
about 400 .mu.m in diameter, in most cases with a length to
diameter ratio of at least 2). Foams with channels are illustrated
in FIG. 2 and FIG. 3. The channels formed by this process generally
begin on one surface of the foam and may traverse the thickness of
the foam. The channel's length is generally at least two times the
average pore diameter and preferably are at least four times the
average pore diameter and most preferably at least eight times the
average pore diameter. Channels for most applications will be at
least 200 microns in length and may extend through the thickness of
the foam. The diameter of the channel will be at least one time the
size of the average pore diameter and preferably at least 2 to 3
times the average pore diameter. The channel size and diameter of
course will be selected based on the desired functionality of the
channel such as cellular invasion, nutrient diffusion or as an
avenue for vascularization.
[0030] There are a number of biological tissues that demonstrate
gradient architectures. Examples of tissues where a gradient
scaffold could be used, include, but are not limited to: bone,
spine disc, articular cartilage, meniscus, fibrocartilage, tendons,
ligaments, dura, skin, vascular grafts, nerves, liver, and
pancreas. The examples below only highlight a few tissues where
gradient scaffolds could be used. The design of tissue engineered
scaffolds to facilitate development of these organ structures would
benefit greatly from the ability to process or create a gradient
architecture in the scaffold.
[0031] Cartilage
[0032] Articular cartilage covers the ends of all bones that form
articulating joints in humans and animals. The cartilage acts in
the joint as a mechanism for force distribution and as a bearing
surface between different bones. Without articular cartilage,
stress concentration and friction would occur to the degree that
the joint would not permit ease of motion. Loss of the articular
cartilage usually leads to painful arthritis and decreased joint
motion. A schematic showing the morphological features of a healthy
cartilage is shown in FIG. 8.
[0033] Articular cartilage is an excellent example of a naturally
occurring gradient structure. Articular cartilage is composed of
four different zones that include the superficial or tangential
zone within the first 10-20% of the structure (this includes the
articular surface), the middle zone which is 40-60% of the middle
structure, and the deep zone that is adjacent to the tide mark, and
a transition zone between the bone and cartilage that is composed
of calcified cartilage. Subchondral bone is located adjacent to the
tide mark and this transitions into cancellous bone. In the
superficial or tangential zone, the collagen fibrils are parallel
to the surface. The fibers are oriented to resist shear forces
generated during normal joint articulation. The middle zone has a
randomly arranged organization of much larger diameter collagen
fibers. Finally, in the deep zone there are larger collagen fiber
bundles, which are perpendicular to the surface, and they insert
into the calcified cartilage. The cells are speroidiol and tend to
arrange themselves in a columnar manner. The calcified cartilage
zone has smaller cells with relatively little cytoplasm.
[0034] A preferred embodiment of this invention would be to
generate a gradient foam structure that could act as a template for
multiple distinct zones. These foam structures could be fabricated
in a variety of shapes to regenerate or repair osteochondrial
defects and cartilage. One potential foam structure would be
cylindrical in shape with an approximate dimensions of 10 mm in
diameter and 10 mm in depth. The top surface is would be
approximately 1 mm thick and would be a low porosity layer to
control the fluid permeability. By adopting a suitable processing
method the surface porosity of the foam could be controlled. The
porosity of this skin like surface can be varied from completely
impervious to completely porous. Fluid permeability would be
controlled by surface porosity. Below such a skin the structure
would consist of three zones. An upper porous zone which lies
adjacent to cartilage tissue, a lower porous zone which lies
adjacent to bone tissue, and a transition zone between the upper
and is lower porous zones. For articular cartilage, it is currently
preferred that the stiffness (modulus) of the upper and lower
porous layers at the time of implantation be at least as stiff, as
the corresponding adjacent tissue. In such a case the porous layers
will be able to support the environmental loading and thereby
protect the invading cells until they have differentiated and
consolidated into tissue that is capable of sustaining load. For
example the porous structure used for the superficial tangential
zone could have elongated pores and the orientation of the
structure could be parallel to the surface of the host cartilage.
However, the deep zone may have a porosity of about 80 to about 95%
with pores that are of the order of 100 .mu.m (about 80 .mu.m to
about 120 .mu.m). It is expected that chondrocytes will invade this
zone. Below this, would be a zone with larger pores (about 100
.mu.m to about 200 .mu.m) and a porosity in the range of about 50
to about 80%. Such 100 .mu.m to about 200 .mu.m porous foam would
have a structure such that the struts or walls of the pores are
larger and vertical to the load, similar to the naturally occurring
structure and to bear the loads. Finally, at the bottom of this
structure there is a need for larger pores (about 150 .mu.m to
about 300 .mu.m) with higher stiffness to be structurally
compatible with cancellous bone. The foam in this section could be
reinforced with ceramic particles or fibers made up of calcium
phosphates and the like.
[0035] Recent data generated in our laboratories support the
hypothesis that cell invasion can be controlled by pore size. In
these studies, a scaffold made of 95/5 mole percent poly (L)
lactide-co-.epsilon.-caprolac- tone) with an approximate pore size
of about 80 .mu.m had chondrocyte invasion of about 30
cells/mm.sup.2 of the scaffold (under static conditions). Scaffolds
made of 40/60 mole percent poly(.epsilon.-caprolac- tone-co-(L)
lactide) with a larger approximate pore size of about 100 .mu.m had
a statistically significantly greater cellular invasion of
cells/mm.sup.2 (under static conditions). In both cases the cells
were bovine chondrocytes. A very simple gradient structure with a
variation of pore sized from about 80 .mu.m to about 150 .mu.m
would provide a structure where chondrocytes would more easily
invade the area with larger pores. The area with smaller pores
would be void of chondrocytes or would be filled with a second cell
types (e.g., fibroblasts).
[0036] In a compositionally gradient foam a blend of two or more
elastomeric copolymers or in combination with high modulus
semi-crystalline polymers along with additives such as growth
factors or particulates can be chosen such that first a desired
pore gradient is developed with a preferred spatial organization of
the additives. Then using a variety of the approaches referred to
in the preferred methods of making gradient foams, a compositional
gradient can be superimposed primarily due to the differences in
the polymer-solvent phase separation behavior of each system. Such
a gradient foam structure would elicit a favorable response to
chondrocytes or osteoblasts depending on the spatial location.
[0037] Further, the purpose of a functional gradient is to more
evenly distribute the stresses across a region through which
mechanical and/or physical properties are varying and thereby
alleviate the stress concentrating effects of a sudden interface.
This more closely resembles the actual biological tissues and
structures, where structural transitions between differing tissues
such as cartilage and bone are gradual. Therefore, it is an object
of the present invention to provide an implant with a functional
gradient between material phases. The present invention provides a
multi-phasic functionally graded bioabsorbable implant with
attachment means for use in surgical repair of osteochondral
defects or sites of osteoarthritis. Several patents have proposed
systems for repairing cartilage that could be used with the present
inventive porous scaffolds. For example, U.S. Pat. No. 5,769,899
describes a device for repairing cartilage defects and U.S. Pat.
No. 5,713,374 describes securing cartilage repair devices with bone
anchors (both hereby incorporated herein by reference).
[0038] Bone
[0039] Gradient structures naturally occur for the bone/cartilage
interface. In a study in our laboratories, we have demonstrated
that material differences significantly influence cell function. In
initial and long-term response of primary osteoblasts to polymer
films (95/5 L-lactide-co-glycolide copolymer, 90/10
glycolide-co-(L) lactide copolymer, 95/5
L-lactide-co-.epsilon.-caprolactone copolymer, 75/25
glycolide-co-(L) lactide copolymer and 40/60
.epsilon.-caprolactone-co-(L- ) lactide copolymer and knitted
meshes (95/5 (L) lactide-co-glycolide and 90/10 glycolide-co-(L)
lactide copolymers) were evaluated in vitro. The results
demonstrated that osteoblasts attached and proliferated well on all
the biodegradable polymer films and meshes following 6-day
incubation. None of the tested polymer films, except a 40/60
.epsilon.-caprolactone-co-(L) lactide copolymer film, demonstrated
significant enhancement in differentiation of primary rat
osteoblasts as compared to tissue culture polystyrene (control).
Films made of 40/60 .epsilon.-caprolactone-co-(L) lactide promoted
enhanced differentiation of cultured osteoblasts as demonstrated by
increased alkaline phosphatase activity and osteoclacin mRNA
expression as compared to the other films and TCPS. Hence, it is
clear that different absorbable materials will significantly alter
cell function and differentiation. By identifying the optimal
materials for cell growth and differentiation a composite materials
with a gradient composition could be utilized to optimize tissue
regeneration with different cell types in the same scaffold.
[0040] Therefore, for bone repair or regeneration devices or
scaffoldings, a device made from a homopolymer, copolymer (random,
block, segmented block, tappered blocks, graft, triblock, etc.)
having a linear, branched or star structure containing
.epsilon.-caprolactone is especially preferred. Currently preferred
are aliphatic polyester copolymers containing in the range of from
about 30 weight percent to about 99 weight percent
.epsilon.-caprolactone. Suitable repeating units that may be
copolymerized with .epsilon.-caprolactone are well known in the
art. Suitable comonomers that may be copolymerized with
.epsilon.-caprolactone include, but are not limited to lactic acid,
lactide (including L-, D-, meso and D,L mixtures), glycolic acid,
glycolide, p-dioxanone (1,4-dioxan-2-one), trimethylene carbonate
(1,3-dioxan-2-one), .delta.-valerolactone, .beta.-butyrolactone,
.epsilon.-decalactone, 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, .gamma.-butyrolactone,
1,4-dioxepan-2-one, 1,5-dioxepan-2-one,
6,6-dimethyl-dioxepan-2-one, 6,8-dioxabicycloctane-7-one and
combinations thereof.
[0041] Preferred medical devices or tissue scaffoldings for bone
tissue repair and/or regeneration containing bioabsorbable polymers
made from .epsilon.-caprolactone include but are not limited to the
porous foam scaffoldings (such as described in this application),
fibrous three dimensional, spun, nonwoven, woven, knitted, or
braided tissue scaffoldings, composite containing reinforcing
fibers, matrices and combinations thereof.
[0042] Skin
[0043] Another example of a tissue that has a gradient structure is
skin. The basic structure of skin has two distinct, but well
integrated layers where the thickness of each layer varies at
different locations of the body. The outer layer or epidermis, is
avascular and mainly consists of keratinocytes with smaller numbers
of immune cells (Langerhan cells) and pigmented cells
(melanocytes). The keratinocytes produce keratin fibers and
corneocyte envelopes, which gives the epidermis its durability and
protective capabilities. The development of these structures is
completely dependent upon the differentiation state of the
epidermis. The epidermis forms a stratified epithelium, with
different protein expression patterns, as the cells move further
away from the basement membrane. This stratified layer of
differentially expressing cells must be formed for maintenance of
epidermal function. Below the epidermis is the dermis, which is a
dense irregular connective tissue that is highly vascular. This
layer is heavily populated with collageneic and elastic fibers,
which give it its exceptional elasticity add strength. Fibroblasts
are the main cell types in this layer. Between these two layers is
the basement membrane, which serves as the site of attachment for
epidermal cells and serves also to regulate their function and
differentiation. The layer of keratinocytes, which attaches
directly to the basement membrane, are cuboidal in shape and highly
aligned. This attachment and architecture are critical requirements
driving the ultimate production of the higher squamous structures
in the epidermis. The basal layer provides a source of precursor
cells for repair and replacement of the epidermis. The squamous
layers provide strength and resistance to insult and infection.
[0044] Any material used for replacement of skin must be able to
entice invasion of fibroblasts or other cells it necessary to
produce the dermal components of the healed tissue. Additionally,
the material must not inhibit, and preferably should enhance, the
rate of re-epithelialization in such a fashion that a discreet,
epidermal basal layer is formed. Materials that permit invasion
into the scaffold by migrating keratinocytes can produce partially
differentiated cells. Consequently, control of access of particular
cell types and a porous design that facilitates the regeneration of
the natural tissue can have functional benefits. Now refer to FIGS.
9A, 9B and 9C which illustrates the microstructure of this foam
scaffold. FIGS. 10 (100.times. magnification) and 11 (40.times.
magnification composite picture) provide photomicrographic evidence
of the invasion of fibroblasts, macrophages, macrophage giant cells
and endothelial-like cells into the a 0.5 mm foam. The foam tissue
scaffolding 101 shown in both pictures was a 50:50 blend of
.epsilon.-caprolactone-co-glycolide copolymer and
.epsilon.-caprolactone-co-lactide copolymer (made as described in
Example 7). The pictures were taken at 8 days after implantation in
1.5 cm.times.1.5 cm.times.0.2 cm excisional wound model in a
Yorkshire pig model. Complete incorporation of the matrix into the
granulation tissue bed is evident in both pictures. The dense
fibrous tissue above the foam tissue scaffolding appears to provide
a suitable substrate for the over growth of epidermis. PDGF was
incorporated into the foam tissue scaffolding shown in FIG. 11. In
compromised wound healing models the addition of a growth factor
such as PDGF may in fact be necessary.
[0045] From our initial studies it appears that it is desirable to
use as a skin scaffold a foam tissue scaffold having a thickness of
from about 150 .mu.m to about 3 mm, preferably the thickness of the
foam may be in the range of from about 300 .mu.m to about 1500
.mu.m and most preferably about 500 to about 1000 .mu.m. Clearly
different skin injuries (i.e. diabetic ulcers, venous stasis
ulcers, decubitis ulcers, burns etc.) may require different foam
thickness. Additionally, the patient's condition may necessitate
the incorporation of growth factors, antibiotics and antifungal
compounds to facilitate wound healing.
[0046] Vascular Grafts
[0047] The creation of tubular structures with gradients may also
be of interest. In vascular grafts, having a tube with pores in the
outer diameter which transitions to smaller pores on the inner
surface or visa versa may be useful in the culturing of endothelial
cells and smooth muscle cells for the tissue culturing of
vessels.
[0048] Multilayered tubular structures allow the regeneration of
tissue that mimics the mechanical and/or biological characteristics
of blood vessels will have utility as a vascular grafts. Concentric
layers, made from different compositions under different processing
conditions could have tailored mechanical properties, bioabsorption
properties, and tissue ingrowth rates. The inner most, or luminal
layer would be optimized for endothelialization through control of
the porosity of the surface and the possible addition of a surface
treatment. The outermost, or adventitial layer of the vascular
graft would be tailored to induce tissue ingrowth, again by
optimizing the porosity (percent porosity, pore size, pore shape
and pore size distribution) and by incorporating bioactive factors,
pharmaceutical agents, or cells. There may or may not be a barrier
layer with low porosity between these two porous layers to increase
strength and decrease leakage.
[0049] Composition of Foams
[0050] A variety of absorbable polymers can be used to make foams.
Examples of suitable biocompatible, bioabsorbable polymers that
could be used include polymers selected from the group consisting
of aliphatic polyesters, poly(amino acids), copoly(ether-esters),
polyalkylenes oxalates, polyamides, poly(iminocarbonates),
polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters
containing amine groups, poly(anhydrides), polyphosphazenes,
biomolecules and blends thereof. 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, 6-valerolactone, .beta.-butyrolactone,
.gamma.-butyrolactone, .epsilon.-decalactone, hydroxybutyrate
(repeating units), hydroxyvalerate (repeating units),
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,S-dione,
3,3-diethyl-1,4-dioxan-2,5-dione, 6,8-dioxabicycloctane-7-one and
polymer blends thereof. Poly(iminocarbonate) for the purpose of
this invention include as described by Kemnitzer and Kohn, in the
Handbook of Biodegradable Polymers, edited by Domb, Kost and
Wisemen, Hardwood Academic Press, 1997, pages 251-272.
Copoly(ether-esters) for the purpose of this invention include
those copolyester-ethers described in "Journal of Biomaterials
Research", Vol. 22, pages 993-1009, 1988 by Cohn and Younes and
Cohn, Polymer Preprints (ACS Division of Polymer Chemistry) Vol.
30(1), page 498, 1989 (e.g. PEO/PLA). Polyalkylene oxalates for the
purpose of this invention include U.S. Pat. Nos. 4,208,511;
4,141,087; 4,130,639; 4,140,678; 4,105,034; and 4,205,399
(incorporated by reference herein). 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, Schacht, Dejardin and Lemmouchi in the Handbook of
Biodegradable Polymers, edited by Domb, Kost and Wisemen, Hardwood
Academic Press, 1997, pages 161-182 (which are hereby incorporated
by reference herein). Polyanhydrides from diacids of the form
HOOC--C.sub.6H.sub.4--O--(CH.sub.2)--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 (which are incorporated herein by reference).
Polyorthoesters such as those described by Heller in Handbook of
Biodegradable Polymers, edited by Domb, Kost and Wisemen, Hardwood
Academic Press, 1997, pages 99-118 (hereby incorporated herein by
reference).
[0051] Currently aliphatic polyesters are the absorbable polymers
that are preferred for making gradient foams. Aliphatic polyesters
can be homopolymers, copolymers (random, block, segmented, tappered
blocks, graft, triblock, etc.) having a linear, branched or star
structure. Preferred are linear copolymers. Suitable monomers for
making aliphatic homopolymers and copolymers may be selected from
the group consisting of, but are not limited, to lactic acid,
lactide (including L-, D-, meso and D,L mixtures), glycolic acid,
glycolide, .epsilon.-caprolactone, p-dioxanone (1,4-dioxan-2-one),
trimethylene carbonate (1,3-dioxan-2-one), delta-valerolactone,
beta-butyrolactone, epsilon-decalactone, 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, gamma-butyrolactone,
1,4-dioxepan-2-one, 1,5-dioxepan-2-one,
6,6-dimethyl-dioxepan-2-one, 6,8-dioxabicycloctane-7-one and
combinations thereof.
[0052] Elastomeric copolymers also are particularly useful in the
present invention. Suitable bioabsorbable biocompatible elastomers
include but are not limited to those selected from the group
consisting of elastomeric copolymers of .epsilon.-caprolactone and
glycolide (preferably having 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 or lactic acid copolymers (preferably having a mole
ratio of .epsilon.-caprolactone to lactide of 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 and
lactic acid (preferably having a mole ratio of p-dioxanone to
lactide of from about 40:60 to about 60:40) elastomeric copolymers
of .epsilon.-caprolactone and p-dioxanone (preferably having a mole
ratio of .epsilon.-caprolactone to p-dioxanone of from about from
30:70 to about 70:30) elastomeric copolymers of p-dioxanone and
trimethylene carbonate (preferably having a mole ratio of
p-dioxanone to trimethylene carbonate of from about 30:70 to about
70:30), elastomeric copolymers of trimethylene carbonate and
glycolide (preferably having a mole ratio of trimethylene carbonate
to glycolide of from about 30:70 to about 70:30), elastomeric
copolymer of trimethylene carbonate and lactide including
L-lactide, D-lactide, blends thereof or lactic acid copolymers
(preferably having a mole ratio of trimethylene carbonate to
lactide of from about 30:70 to about 70:30) and blends thereof.
[0053] Examples of suitable bioabsorbable elastomers are described
in U.S. Pat. Nos. 4,045,418; 4,057,537 and 5,468,253 all hereby
incorporated by reference. These elastomeric polymers will have an
inherent viscosity of from about 1.2 dL/g to about 4 dL/g,
preferably an inherent viscosity of from about 1.2 dL/g to about 2
dL/g and most preferably an inherent viscosity of from about 1.4
dL/g to about 2 dL/g as determined at 25.degree. C. in a 0.1 gram
per deciliter (g/dL) solution of polymer in hexafluoroisopropanol
(HFIP).
[0054] Preferably, the elastomers will 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 from which the foams
are formed will exhibit a percent elongation greater than about 200
percent and preferably greater than about 500 percent. There
properties, which measure the degree of elasticity of the
bioabsorbable elastomer, are achieved while maintaining 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.
[0055] The polymer or copolymer suitable for forming a gradient
foam for tissue regeneration depends on several factors. The
chemical composition, spatial distribution of the constituents, the
molecular weight of the polymer and the degree of crystallinity all
dictate to some extent the in-vitro and in-vivo behavior of the
polymer. However, the selection of the polymers to make gradient
foams for tissue regeneration largely depends on (but not limited
to) the following factors: (a) bio-absorption (or bio-degradation)
kinetics; (b) in-vivo mechanical performance; and (c) cell response
to the material in terms of cell attachment, proliferation,
migration and differentiation and (d) biocompatibility.
[0056] The ability of the material substrate to resorb in a timely
fashion in the body environment is critical. But the differences in
the absorption time under in-vivo conditions can also be the basis
for combining two different copolymers. For example a copolymer of
35:65 .epsilon.-caprolactone and glycolide (a relatively fast
absorbing polymer) is blended with 40:60 .epsilon.-caprolactone and
(L) lactide copolymer (a relatively slow absorbing polymer) to form
a foam. Such a foam could have several different physical
structures depending upon the processing technique used. The two
constituents can be either randomly inter-connected bicontinuous
phases, or the constituents can have a gradient through the
thickness or a laminate type composite with a well integrated
interface between the two constituent layers. The microstructure of
these foams can be optimized to regenerate or repair the desired
anatomical features of the tissue that is being engineered.
[0057] One preferred embodiment of the present invention is to use
polymer blends to form structures which transition from one
composition to another composition in a gradient like architecture.
Foams having this gradient 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 etc.),
esophaguses, skin, bone and vascular tissue. For example by
blending an elastomer of .epsilon.-caprolactone-co-glycolide with
.epsilon.-caprolactone-co-lactide (i.e. with a mole ratio of about
5:95) a foam may be formed that transitions from a softer spongy
foam to a stiffer more rigid foam similar to the transition from
cartilage to bone. Clearly other polymer blends may be used for
similar gradient effects or to provide different gradients such as
different absorption profiles, stress response profiles, or
different degrees of elasticity. Additionally, these foams can be
used for organ repair replacement or regeneration strategies that
may benefit from these unique scaffolds, including but are not
limited to, spine disc, dura, nerve tissue, liver, pancreas,
kidney, bladder, tendons, ligaments and breast tissues.
[0058] These elastomeric polymers may be foamed by lyophilization,
supercritical solvent foaming (i.e., as described in EP 464,163
B1), gas injection extrusion, gas injection molding or casting with
an extractable material (i.e., salts, sugar or any other means
known to those skilled in the art). Currently it is preferred to
prepare bioabsorbable, biocompatible elastomeric foams by
lyophilization. Suitable methods for lyophilizing elastomeric
polymers to form foams is described in the Examples and in the
copending patent application entitled, "Process for Manufacturing
Biomedical Foams", assigned to Ethicon, Inc., docket number
ETH-1352, filed Jun. 30, 1999 hereby incorporated herein by
reference herein.
[0059] The foams that are made in this invention are made by a
polymer-solvent phase separation technique with modifications to
the prior art that unexpectedly creates gradients in the foam
structure. Generally, a polymer solution can be separated into two
phases by any one of the four techniques: (a) thermally induced
gelation/crystalization; (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 of density
less than the bulk polymer and pores in the micrometer ranges (ref.
"Microcellular foams via phase separation" by A. T. Young, J. Vac.
Sci. Technolol. A 4(3), May/June 1986). The steps involved in the
preparation of these foams consists of choosing the right solvents
for the polymers that needs 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.
[0060] Suitable solvents that should be generally suited as a
starting place for selecting a solvent for the preferred absorbable
aliphatic polyesters include but are not limited to solvents
selected from a group consisting of formic acid, ethyl formate,
acetic acid, hexafluoroisopropanol (HFIP), cyclic ethers (i.e. THF,
DMF, and PDO), acetone, acetates of C2 to C5 alcohol (such as-ethyl
acetate and t-butylacetate), glyme (i.e. monoglyme, ethyl glyme,
diglyme, ethyl diglyme, triglyme, butyl diglyme and tetraglyme)
methylethyl ketone, dipropyleneglycol methyl ether, lactones (such
as .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, the preferred solvent
is 1,4-dioxane. A homogeneous solution of the polymer in the
solvent is prepared using standard techniques.
[0061] Accordingly, as will be appreciated, the applicable polymer
concentration or amount of solvent, which may be utilized, will
vary with each system. Suitable phase diagram curves for several
systems have already been developed. However, if an appropriate
curve is not available, this can be readily developed by known
techniques. For example, a suitable technique is set forth in
Smolders, van Aartsen and Steenbergen, Kolloid-Z. u. Z. Polymere,
243, 14 (1971). As a general guideline 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 to a large
extent on the solubility of the polymer inra vgiven solvent and the
final properties of the foam desired.
[0062] Additionally, solids may be added to the polymer-solvent
system. The solids added to the polymer-solvent system preferably
will not react with the polymer or the solvent. Suitable solids
include materials that promote tissue regeneration or regrowth,
buffers, reinforcing materials or porosity modifiers. Suitable
solids include, but are not limited to, particles of demineralized
bone, calcium phosphate particles, or calcium carbonate particles
for bone repair, leachable solids for pore creation and particles
of bioabsorbable polymers not soluble in the solvent system as
reinforcing or to create pores as they are absorbed. Suitable
leachable solids include but are not limited nontoxic leachable
materials selected from the group consisting of salts (i.e. sodium
chloride, potassium chloride, calcium chloride, sodium tartrate,
sodium citrate, and the like) biocompatible mono and disaccharides
(i.e. glucose, fructose, dextrose, maltose, lactose and sucrose),
polysaccharides (i.e. starch, alginate), water soluble proteins
(i.e. gelatin and agarose). Generally all of these materials will
have an average diameter of less than about 1 mm and preferably
will have an average diameter of from about 50 to about 500 .mu.m.
The particles will generally constitute from about 1 to about 56
volume percent of the total volume of the particle and
polymer-solvent mixture (wherein the total volume percent equals
100 volume percent). 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. This process is described in U.S. Pat. No. 5,514,378 hereby
incorporated herein by reference (see column 6). 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.
[0063] After the polymer solvent mixture is formed the mixture is
then solidified. For a specific polymer-solvent system, the
solidification point, the melt temperature and the apparent glass
transition of the polymer-solvent system can be determined using
standard differential scanning calorimetric (DSC) techniques. In
theory, but in no way limiting the scope of the present invention,
it is believed that as a polymer solvent system is cooled down an
initial solidification occurs at about or below the freezing point
of the solvent. This corresponds to the freezing of a substantial
portion of the solvent in the system. The initial freezing appears
as a first exothermic peak. A second freezing point occurs when the
remaining solvent associated with the polymer solidifies. The
second freezing point is marked by a second exothermic peak. The
apparent Tg is the temperature at which the fully frozen system
displays the first endothermic shift on reheating.
[0064] An important parameter to control is the rate of freezing of
the polymer-solvent system. The type of pore morphology that gets
locked in during the freezing step is a function of the solution
thermodynamics, freezing rate, temperature to which it is cooled,
concentration of the solution, homogeneous or heterogenous
nucleation etc. Detailed description of these phase separation
phenomenon can be found in the references provided herein
("Microcellular foams via phase separation" by A. T. Young, J. Vac.
Sci. Technol. A 4(3), May/June 1986; and "Thermodynamics of
Formation of Porous Poymeric Membrane from Solutions" by S.
Matsuda, Polymer J. Vol. 23, No. 5, pp 435-444, 1991).
[0065] The polymer solution previously described can be solidified
in a variety of manners such as placing or injecting the solution
in a mold and cooling the mold in an appropriate bath or on a
refrigerated shelf. Alternatively, the polymer solution can be
atomized by an atomizer and sprayed onto a cold surface causing
solidification of the spray layer by layer. The cold surface can be
a medical device or part thereof or a film. The shape of the
solidified spray will be similar to the shape of the surface it is
sprayed onto. Alternatively, the mixture after solidification can
be cut or formed to shape while frozen. Using these and other
processes the foams can be made or molded in a variety of shapes
and sizes (i.e. tubular shapes, branched tubular shapes, spherical
shapes, hemispherical shapes, three-dimensional polygonal shapes,
ellipsoidal shapes (i.e. kidney shaped), toroidal shapes, conical
shapes, frusta conical shapes, pyramidal shapes, both as solid and
hollow constructs and combination thereof).
[0066] Alternatively, another method to make shaped foamed parts is
to use a cold finger (a metal part whose surface represents the
inside of the part we want to fabricate). The cold finger is dipped
into a mixture of polymer in an appropriate solvent and removed.
This is much like dipping an ice cream pop into warm chocolate that
freezes to a hard, cold skin, or dipping a form into a latex of
rubber to form gloves or condoms. The thickness and morphology of
the foam produced are a function of the temperature, dwell time and
withdrawal rate of the cold finger in the mixture. Longer dwell,
colder finger and slower withdrawal will produce a thicker coating.
After withdrawal, the cold finger is placed on a fixture of large
thermal mass that is in contact with the refrigerated tray of the
lyophilizer. From this point the primary and secondary drying
processes are as described above. This method is particularly well
suited to making tubes, branched tubular structures or sleeves that
may be shaped to fit devices or portions of an animal's anatomy
(for repair, regeneration or augmentation of tissue).
[0067] Additionally, the polymer solution can be solidified with
various inserts incorporated with the solution such as films,
scrims, woven, nonwoven, knitted or braided textile structures.
Additionally, the solution can be prepared in association with
another structure such an orthopedic implant (e.g. screws, pins,
nails, and plates) or vascular or branched tubular construct (as a
scaffold for a vascularized or ducted organ). These inserts will be
made of at least one biocompatible material and may be
non-absorbable, absorbable or a combination thereof.
[0068] The polymer solution in a mold undergoes directional cooling
through the wall of the mold that is in contact with the freeze
dryer shelf, which is subjected to a thermal cycle. The mold and
its surface can be made from virtually any material that does not
interfere with the polymer-solvent system, though it is preferred
to have a highly conducting material. The heat transfer front moves
upwards from the lyophilizer shelf through the mold wall into the
polymer solution. The instant the temperature of the mixture goes
below the gellation and/or freezing point the mixture also phase
separates.
[0069] The morphology of this phase separated system is locked in
place during the freezing step of the lyophilization process and
the creation of the open pores is initiated by the onset of vacuum
drying resulting in the sublimation of the solvent. However, the
mixture in container or mold that is cooled from a heat sink will
solidify prior to completely freezing. Although the mixture may
appear solid, initially there appears to be some residual solvent
associated with the polymer that has not cystallized. It is
theorized, but in no way limiting the present invention, that a
freezing front it moves through the mixture from the heat sink to
complete the solidification after the mixture has apparently
solidified. The material in front of the freezing front at a given
time will not be as cold as the material behind the front and will
not be in a completely frozen state.
[0070] We have discovered that if a vacuum is applied to the
apparently solid polymer-solvent mixture immediately after it
appears to solidify, a foam with a gradient structure having
variable pore size and structure as well as channels can be
created. Therefore, timing of the onset of the sublimation process
(by pressure reduction i.e. vacuum drying) is a critical step in
the process to create transitions in the structure. The timing of
the onset of sublimation will be affected by the thickness of the
foam being made, concentration of the solution, rate of heat
transfer, and directionalities of the heat transfer. Those skilled
in the art will appreciate that this process can be monitored and
characterized for specific polymer-solvent systems by using
thermocouples and monitoring the heat transfer rates of the foams
at various depths and locations in the device being foamed. By
controlling the sublimation process, structures with a gradient in
pore morphology and anisotropy may be created. This process can
lead to the creation of microstructures that mimic tissues such as
cartilage, bone and skin. For example, is channels will generally
be formed if a vacuum is pulled immediately after the solution
apparently solidifies. However, if the same solution is allowed to
solidify further the foam will have larger pores closer to the
surface where the vacuum is being drawn (opposite the heat sink)
and smaller pores closer to the heat sink.
[0071] This process is the antitheses of the prior art process that
focused on creating foams with a uniform microstructure (randomly
interconnected pores), whereby the whole solution is completely
frozen. And vacuum drying is applied only after a considerable
amount of time is given for the completion of desired phase
separation (U.S. Pat. No. 5,755,792 (Brekke); U.S. Pat. No.
5,133,755 (Brekke); U.S. Pat. No. 5,716,413 (Walter, et al.); U.S.
Pat. No. 5,607,474 (Athanasiou, et al.); U.S. Pat. No. 5,686,091
(Leong, et al.); U.S. Pat. No. 5,677,355 (Shalaby, et al.); and
European disclosures E0274898 (Hinsch) and EPA 594148
(Totakura)).
[0072] Foams with various structures are shown in FIGS. 2, 3, and
4. For example, as shown in FIG. 3 the orientation of the major
axis of the pores may be changed from being in the same plane as
the foam to being oriented perpendicular to the plane of the foam.
By way of theory, but in no way limiting the scope of this
invention, it is believed that this in conventional foam processing
as the solvent crystallizes a freezing front moves through the
solution solidifying the solution in crystalline layers parallel to
the freezing front. However, if a vacuum is pulled before the
solution completely freezes, the morphology of the foam results in
pores being formed generally aligned parallel to the vacuum source.
As is illustrated in FIG. 3.
[0073] As can be seen from FIG. 3 the pore size can be varied from
a small pore size generally between about 10 .mu.m and about 60
.mu.m to a larger size of from about 60 .mu.m to about 200 .mu.m in
a porous gradient foam. Again this results from pulling a vacuum on
the apparently solidified solution before it is completely
solidified. The polymer concentration in the solution and the
cooling rates are also important parameters in controlling the cell
size. Ideally the foam structure could be created to serve as a
template to restore human tissue junctions such as the cartilage to
bone junction present in joints. This foam would progress form a
small round pores to larger column-like (i.e. with a diameter to
length ratio of at least 2 to 1) pores. Additionally, the stiffness
of the foam can controlled by the foams structure or blending two
different polymers with different Young's moduli.
[0074] Foams can also have channels as is illustrated in FIG. 2.
The channels formed by this process may traverse the thickness of
the foam and generally range in diameter from about 30 to about 200
.mu.m in diameter. The channels generally are at least two times
the channel's average diameter and preferably are at least four
times the channel's average diameter and most preferably at least
eight times the channel's average diameter. The channel size and
diameter of course will be selected based on the desired
functionality of the channel such as cell invasion, nutrient
diffusion or as a avenue for vascularization.
[0075] One skilled in the art can easily visualize that such a
directionality can be created in any three dimensions by designing
an appropriate mold and subjecting the walls of such a mold to
different temperatures if needed. The following types of gradient
structures can be made by variation in the pore size and/or shape
through the thickness with a uniform composition: pores of one type
and size for a certain thickness followed by another type and size
of pores, etc; compositional gradient with predominantly one
composition on one side and another one on the other with a
transition from one entity to the other; a thick skin comprising
low porosity of low pore size layer followed by a large pore size
region; foams with vertical pores with a spatial organization these
vertical pores can act as channels for nutrient diffusion the
making of these in 3D molds to create 3D foams with the desired
microstructure combinations of compositional and architectural
gradient. Generally the foams formed in containers or molds will
have a thickness in the range of from about 0.25 mm to about 100 mm
and preferably will have a thickness of from about 0.5 mm to about
50 mm. Thicker foams can be made but the lyophilization cycle times
may be quite long, the final foam structures may be more difficult
to control and the residual solvent content may be higher.
[0076] As previously described the inventive process cycle for
producing biocompatible foam is significantly reduced by performing
the sublimation step above the apparent glass transition
temperature and below the solidification temperature of the mixture
(preferably just below the solidification temperature). The
combined cycle time of (freezing+primary drying+secondary drying)
is much faster than is described in the prior art. For example, the
combined cycle for aliphatic polyesters using volatile solvents is
generally less than 72 hours, preferably less than 48 hours, more
preferably less than 24 hours and most preferably less than 10
hours. In fact the combined cycle can be performed with some
aliphatic polyesters in less than 3 hrs for foams of thickness 1 mm
or less; less than 6 hrs for foams of thickness around 2 mm and
less than 9 hrs for foams of thickness around 3 mm. Compare this
with prior art which is typically 72 hours or greater. The residual
solvent concentrations in these foams made by this process will be
very low. As described for aliphatic polyesters foams made using
1,4-dioxane as a solvent the residual concentration of 1,4-dioxane
was less than 10 ppm (parts per million) more preferably less than
1 ppm and most preferably less than 100 ppb (parts per
billion).
[0077] One skilled in the art can easily visualize that such a
directionality can be created in any three-dimensions by designing
an appropriate mold and subjecting the walls of such a mold to
different temperatures if needed. The following types of gradient
structures can be made by this invention
[0078] 1. variation in the pore size and/or shape through the
thickness with a uniform composition,
[0079] 2.pores of one type and size for a certain thickness
followed by another type and size of pores, etc
[0080] 3. compositional gradient with predominantly one compostion
on one side and another composition on the other with a transition
from one entity to the other
[0081] 4. a thick skin comprising low porosity of low pore size
layer followed by a large pore size region
[0082] 5. foams with vertical pores with a spatial organization . .
. these vertical pores can act as channels for nutrient
diffusion
[0083] 6. the making of these in three-dimensional molds to create
three-dimensional foams with the desired microstructure.
[0084] 7. combinations of compositional and architectural
gradient
[0085] Additionally, various proteins (including short chain
peptides), growth agents, chemotatic agents and therapeutic agents
(antibiotics, analgesics, anti-inflammatories, anti-rejection (e.g.
immunosuppressants) and anticancer drugs), or ceramic particles can
be added to the foams during processing, adsorbed onto the surface
or back filled into the foams after the foams are made. For
example, the pores of the foam may be partially or completely
filled with biocompatible resorbable synthetic polymers or
biopolymers (such as collagen or elastin) or biocompatible ceramic
materials (such as hydroxyapatite) and combinations thereof (that
may or may not contain materials that promote tissue growth through
the device). Suitable materials include but are not limited to
autograft, allograft, or xenograft bone, bone marrow, morphogenic
proteins (BMP's), epidermal growth factor (EGF), fibroblast growth
factor (FgF), platelet derived growth factor (PDGF), insulin
derived growth factor (IGF-I and IGF-II), transforming growth
factors (TGF-.beta.), vascular endothelial growth factor (VEGF) or
other osteoinductive or osteoconductive materials known in the art.
Biopolymers could also be used as conductive or chemotactic
materials, or as delivery vehicles for growth factors. Examples
could be recombinant or animal derived collagen or elastin or
hyaluronic acid. Bioactive coatings or surface treatments could
also be attached to the surface of the materials. For example,
bioactive peptide sequences (RGD's) could be attached to facilitate
protein adsorption and subsequent cell tissue attachment.
Therapeutic agents may also be delivered with these foams.
[0086] In another embodiment of the present invention, the polymers
and blends that are used to form the foam can contain therapeutic
agents. To form these foams, the previously described polymer would
be mixed with a therapeutic agent prior to forming the foam or
loaded into the foam after it is formed. The variety of different
therapeutic agents that can be used in conjunction with the foams
of the present invention is vast. In general, therapeutic agents
which may be administered via the pharmaceutical compositions of
the invention include, without limitation: antiinfectives such as
antibiotics and antiviral agents; chemotherapeutic agents (i.e.
anticancer agents); anti-rejection agents; analgesics and analgesic
combinations; anti-inflammatory agents; hormones such as steroids;
growth factors (bone morphogenic proteins (i.e. BMP's 1-7), bone
morphogenic-like proteins (i.e. GFD-5, GFD-7 and GFD-8), epidermal
growth factor (EGF), fibroblast growth factor (i.e. FGF 1-9),
platelet derived growth factor (PDGF), insulin like growth factor
(IGF-I and IGF-II), transforming growth factors (i.e. TGF-.beta.
I-III), vascular endothelial growth factor (VEGF)); and other
naturally derived or genetically engineered proteins,
polysaccharides, glycoproteins, or lipoproteins. These growth
factors are described in The Cellular and Molecular Basis of Bone
Formation and Repair by Vicki Rosen and R. Scott Thies, published
by R. G. Landes Company hereby incorporated herein by
reference.
[0087] Foams containing bio-active materials may be formulated by
mixing one or more therapeutic agents with the polymer used to make
the foam or with the solvent or with the polymer-solvent mixture
and foamed. Alternatively, a therapeutic agent could be coated on
to the foam preferably with a pharmaceutically acceptable
carrier.
[0088] Any pharmaceutical carrier can be used that does not
dissolve the foam. The therapeutic agents, may be present as a
liquid, a finely divided solid, or any other appropriate physical
form. Typically, but optionally, the matrix will include one or
more additives, such as diluents, carriers, excipients, stabilizers
or the like.
[0089] The amount of therapeutic agent will depend on the
particular drug being employed and medical condition being treated.
Typically, the amount of drug represents about 0.001 percent to
about 70 percent, more typically about 0.001 percent to about 50
percent, most typically about 0.001 percent to about 20 percent by
weight of the matrix. The quantity and type of polymer incorporated
into the drug delivery matrix will vary depending on the release
profile desired and the amount of drug employed.
[0090] Upon contact with body fluids the drug will be released. If
the drug is incorporated into the foam then as the foam undergoes
gradual degradation (mainly through hydrolysis) the drug will be
released. This can result in prolonged delivery (over, say 1 to
5,000 hours, preferably 2 to 800 hours) of effective amounts (say,
0.0001 mg/kg/hour to 10 mg/kg/hour) of the drug. This dosage form
can be administered as is necessary depending on the subject being
treated, the severity of the affliction, the judgment of the
prescribing physician, and the like. Following this or similar
procedures, those skilled in the art will be able to prepare a
variety of formulations.
[0091] The foam may also serve as a scaffold for the engineering of
tissue. The porous gradient structure would be conducive to growth
of cells. As outlined in previous patents (Vacanti, U.S. Pat. No.
5,770,417), cells can be harvested from a patient (before or during
surgery to repair the tissue) and the cells can be processed under
sterile conditions to provide a specific cell type (i.e.,
pluripotent cells, stem cells or precursor cells such as the
mesenchymal stem cells described in Caplan, U.S. Pat. No.
5,486,359, etc.). Suitable cell that may be contacted or seeded
into the foam scaffolds include but are not limited to myocytes,
adipocytes, fibromyoblasts, ectodermal cell, muscle cells,
osteoblast (i.e. bone cells), chondrocyte (i.e. cartilage cells),
endothelial cells, fibroblast, pancreatic cells, hepatocyte, bile
duct cells, bone marrow cells, neural cells, genitourinary cells
(including nephritic cells) and combinations thereof. Various
cellular strategies could be used with these scaffolds (i.e.,
autogenous, allogenic, xenogeneic cells etc.). The cells could also
contain inserted DNA encoding a protein that could stimulate the
attachment, proliferation or differentiation of tissue. The foam
would be placed in cell culture and the cells seeded onto or into
the structure. The foam would be maintained in a sterile
environment and then implanted into the donor patient once the
cells have invaded the microstructure of the device. The in vitro
seeding of cells could provide for a more rapid development and
differentiation process for the tissue. It is clear that cellular
differentiation and the creation of tissue specific extracellular
matrix is critical for the tissue engineering of a functional
implant.
[0092] The option for seeding different cell types into the
different pore structures would be available to investigators.
Schaufer et al., have demonstrated that different cell types (i.e.
stromal cells and chondrocytes) can be cultured on different
structures. The structures can be combined after a short period of
time and the entire structure can be placed back in cell culture so
a biphasic tissue structure can be generated for implantation. A
gradient structure would also allow for co-cultured tissue
scaffolds to be generated. (Schaefer, D. et al.). Additionally,
radio-opaque markers may be added to the foams to allow imaging
after implantation. After a defined period of in vitro development
(for example 3 weeks), the tissue engineered implant would be
harvested and implanted into the patient. If an acellular strategy
is pursued, then the sterile acellular scaffolds would be used to
replace damaged or traumatized tissue.
[0093] The foam scaffolds of the present invention may be
sterilized using conventional sterilization process such as
radiation based sterilization (i.e. gamma-ray), chemical based
sterilization (ethylene oxide) or other appropriate procedures.
Preferably the sterilization process will be with ethylene oxide at
a temperature between 52-55.degree. C. for a time of 8 hours or
less. After sterilization the foam scaffolds may be packaged in an
appropriate sterilize moisture resistant package for trio shipment
and use in hospitals and other health care facilities.
[0094] The following examples are illustrative of the principles
and practice of this invention, although not limited thereto.
Numerous additional embodiments within the scope and spirit of the
invention will become apparent to those skilled in the art.
EXAMPLES
[0095] In the examples which follow, the polymers and monomers were
characterized for chemical composition and purity (NMR, FT-IR),
thermal analysis (DSC), molecular weight (inherent viscosity), and
baseline and in vitro mechanical properties (Instron
stress/strain).
[0096] .sup.1H NMR was performed on a 300 MHz NMR using CDCl.sub.3
or HFAD (hexafluoroacetone sesqua deutrium oxide) as a solvent.
Thermal analysis of segmented polymers and monomers was performed
on a Dupont 912 Differential Scanning Calorimeter (DSC). Inherent
viscosities (I.V., dL/g) of the polymers and copolymers were
measured using a 50 bore Cannon-Ubbelhode dilution viscometer
immersed in a thermostatically controlled water bath at 25.degree.
C. utilizing chloroform or hexafluoroisopropanol (HFIP) as the
solvent at a concentration of 0.1 g/dL.
[0097] In these examples certain abbreviations are usde such as PCL
to indicate polymerized .epsilon.-caprolactone, PGA to indicate
polymerized glycolide, PLA to indicate polymerized (L) lactide.
Additionally, the percentages in front of the copolymer indicates
the respective mole percentages of each constituent.
Example 1
[0098] Preparation of a Foam With Random Microstructure (No
Preferred Architecture)
[0099] Step A. Preparing 5% wt./wt. Homogeneous Solution of 35/65
PCL/PGA in 1,4-Dioxane
[0100] A 5% wt./wt. polymer solution is prepared by dissolving 1
part of 35/65 PCL/PGA with 19 parts of the solvent 1,4-dioxane. The
35/65 PCL/PGA copolymer was made substantially as described in
Example 8. The solution is prepared in a flask with a magnetic stir
bar. For the copolymer to dissolve completely, it is recommended
that the mixture is gently heated to 60.+-.5.degree. C. and
continuously stirred for a minimum of 4 hours but not exceeding 8
hours. A clear homogeneous solution is then obtained by filtering
the solution through an extra coarse porosity filter (Pyrex brand
extraction thimble with fritted disc) using dry nitrogen to help in
the filtration of this viscous solution.
[0101] Step B. Lyophilization
[0102] A laboratory scale lyophilizer--Freezemobile 6 of VIRTIS was
used in this experiment. The freeze dryer is powered up and the
shelf chamber is maintained at 20.degree. C. under dry nitrogen for
approximately 30 minutes. Thermocouples to monitor the shelf
temperature are attached for monitoring. Carefully fill the
homogeneous polymer solution prepared in Step A. into the molds
just before the actual start of the cycle. A glass mold was used in
this example but a mold made of any material that is inert to
1,4-dioxane; has good heat transfer characteristics; and has a
surface that enables the easy removal of the foam can be used. The
glass mold or dish used in this example weighed 620 grams, was
optical glass 5.5 mm thick, and cylindrical with a 21 cm outer
diameter and a 19.5 cm inner diameter. The lip height of the dish
was 2.5 cm. Next the following steps are followed in a sequence to
make a 2 mm thick foam:
[0103] (i). The glass dish with the solution is carefully placed
(without tilting) on the shelf of the lyophilizer, which is
maintained at 20.degree. C. The cycle is started and the shelf
temperature is held at 20.degree. C. for 30 minutes for thermal
conditioning.
[0104] (ii). The solution is then cooled to -5.degree. C. by
cooling the shelf to -5.degree. C.
[0105] (iii). After 60 minutes of freezing at -5.degree. C., a
vacuum is applied to initiate primary drying of the dioxane by
sublimation. One hour of primary drying under vacuum at -5.degree.
C. is needed to remove most of the solvent. At the end of this
drying stage typically the vacuum level reached about 50 mTorr or
less.
[0106] (iv). Next, secondary drying under a 50 mTorr vacuum or less
was done in two stages to remove the adsorbed dioxane. In the first
stage, the shelf temperature was raised to 5.degree. C. and held at
that temperature for 1 hour. At the end of the first stage the
second stage of drying was begun. In the second stage of drying,
the shelf temperature was raised to 20.degree. C. and held at that
temperature for 1 hour.
[0107] (v). At the end of the second stage, the lyophilizer is
brought to room temperature and the vacuum is broken with nitrogen.
The chamber is purged with dry nitrogen for approximately 30
minutes before opening the door.
[0108] The steps described above are suitable for making foams that
are about 2 mm thick or less. As one skilled in the art would know,
the conditions described herein are typical and operating ranges
depend on several factors e.g.: concentration of the solution;
polymer molecular weights and compositions; volume of the solution;
mold parameters; machine variables like cooling rate, heating
rates; and the like. FIG. 1 shows a SEM of a cross section of the
foam produced following the process set forth in this example. Note
the random microstructure (not a preferred architecture) of this
foam.
Example 2
[0109] Preparation of a Foam With Vertical Channels
[0110] This example describes the making of a 35/65 PCL/PGA foam
with vertical channels that would provide pathways for nutrient
transport and guided tissue regeneration.
[0111] We used a FTS Dura Dry Freeze dryer with computer control
and data monitoring system to make this foam. First step in the
preparation of this foam was to generate a homogeneous solution. A
10% wt./wt. homogeneous solution of 35/65 PCL/PGA was made in a
manner similar to that described in Example 1, Step A. The polymer
solution was carefully filled into a dish just before the actual
start of the cycle. The dish weighed 620 grams, was optical glass
5.5 mm thick, and cylindrical with a 21 cm outer diameter and a
19.5 cm inner diameter. The lip height of the dish was 2.5 cm. Next
the following steps are followed in sequence to make a 2 mm thick
foam with the desired architecture:
[0112] (i). The solution filled dish was placed on the freeze dryer
shelf that was precooled to -17.degree. C. The cycle was started
and the shelf temperature was held at -17.degree. C. for 15 minutes
quenching the polymer solution.
[0113] (ii). After 15 minutes of quenching to -17.degree. C., a
vacuum was applied to initiate primary drying of the dioxane by
sublimation and held at 100 milliTorr for one hour.
[0114] (iii). Next, secondary drying was done at 5.degree. C. for
one hour and at 20.degree. C. for one hour. At each temperature the
vacuum level was maintained at 20 mTorr.
[0115] (iv). At the end of the second stage, the lyophilizer was
brought to room temperature and the vacuum was broken with
nitrogen. The chamber was purged with dry nitrogen for
approximately 30 minutes before opening the door.
[0116] FIG. 2 is a SEM picture that shows a cross section of the
foam with vertical channels. These channels run through the
thickness of the foam.
Example 3
[0117] Architecturally Gradient Foam
[0118] This example describes the making of a foam that has a
gradient in foam morphology as shown in FIG. 3 using a 10% solution
of 35/65 .epsilon.-caprolactone-co-glycolide. The method used to
make such a foam is similar to the description given in Example 2
with one difference. In step (ii) of the lyophilization process the
time for which the solution is kept at the freezing step is 30
minutes.
[0119] FIG. 3 is a scanning electron micrograph of a cross section
of this foam. Note the variation in the pore size and pore shape
through the thickness of the foam.
Example 4
[0120] Transcompositional Foam
[0121] This example describes the making of a foam that has a
compositional gradient and not necessarily a morphological
gradient. Such a foam is made from polymer solutions that have been
made from physical mixtures of two or more polymers. This example
describes a transcompositional foam made from 35/65 PCL/PGA and
40/60 PCL/PLA
[0122] Step A. Preparing a Solution Mixture of 35/65 PCL/PGA and
40/60 PCL/PLA in 1,4-Dioxane
[0123] In the preferred method the two separate solutions are first
prepared (a) a 10% wt/wt polymer solution of 35/65 PCL/PGA and (b)
a 10% wt/wt 40/60 PCL/PLA. Once these solutions are prepared as
described in Example 1, equal parts of each solution was poured
into one mixing flask. The polymers used to make these solutions
are described is in Examples 8 and 9. A homogeneous solution of
this physical mixture was obtained by gently heating to
60.+-.5.degree. C. and continuously stirring using a magnetic stir
bar for approximately 2 hours.
[0124] Step B. Lyophilization cycle
[0125] We used an FTS Dura Dry Freeze dryer with computer control
and data monitoring system to make this foam. The first step in the
preparation of such a foam was to generate a homogeneous solution
as described in Step A. The solution was carefully filled into a
dish just before the actual start of the cycle. The cylindrical
glass dish weighed 117 grams, was optical glass 2.5 mm thick and
cylindrical with a 100 mm outer diameter and a 95 mm inner
diameter. The lip height of the dish was 50 mm. Next the following
steps were followed in sequence to make a 25 mm thick foam with the
transcompositional gradient:
[0126] (i). The solution filled dish was placed on the freeze dryer
shelf and the solution conditioned at 20.degree. C. for 30 minutes.
The cycle was started and the shelf temperature was set to
-5.degree. C. with a programmed cooling rate of 0.5.degree.
C./min.
[0127] (ii). The solution was held at the freezing condition
(-5.degree. C.) for 5 hours.
[0128] (iii). Vacuum was applied to initiate primary drying of the
dioxane by sublimation and held at 100 milliTorr for 5 hours.
[0129] (iv). Next, secondary drying was done at 5.degree. C. for 5
hours and at 20.degree. C. for 10 hours. At each temperature the
vacuum level was maintained at 20 mTorr.
[0130] (v). At the end of the second stage, the lyophilizer was
brought to room temperature and the vacuum was broken with
nitrogen. The chamber was purged with dry nitrogen for
approximately 30 minutes before opening the door.
[0131] The foam has a gradient in chemical composition which is
evident from a close scrutiny of the foam wall morphology as shown
in FIGS. 4, 5 and 6. The gradient in the chemical composition was
further supported by NMR data as detailed below:
[0132] Foam sample produced by the above method and which was
approximately 25 mm thick was characterized for mole % composition.
The foam sample is composed of a physical blend of PCL/PLA and
PCL/PGA. Slices of the foam sample were prepared and analyzed to
confirm that the material was a compositional gradient. The sample
slices were identified as 1) foam IA (top slice), 2) foam IB (top
middle slice), 3) foam IC (bottom middle slice), 4) foam ID (bottom
slice). The NMR sample preparation consisted of dissolving a 5 mg
of material into 300 .mu.L hexafluoroacetone sesqua deutrium oxide
(HFAD) and then diluting with 300 .mu.L of C.sub.6D.sub.6.
1 1H NMR Results: Mole % Composition Sample ID PLA PGA PCL Foam IA
47.2 12.4 40.5 Foam IB 12.3 51.3 36.5 Foam IC 7.7 56.5 35.8 Foam ID
7.8 56.3 35.8
[0133] The NMR results indicate that the foam samples have a
gradient with respect to composition. The top layer of the foam is
high in PLA concentration (47 mole %), whereas the bottom layer of
the foam is high in PGA concentration (56 mole %). These results
suggest that the PCL/PGA copolymer and the PCL/PLA copolymer have
differences in their phase separation behaviors during the freezing
step and formed a unique compositionally gradient foam.
Example 5
[0134] Transstructural Foam
[0135] This example describes the making of a foam that has a
compositional and structural gradient and not necessarily a
morphological gradient. Such a foam is made from polymer solutions
that have been made by physical mixtures of two or more polymers.
This example describes a transcompositional foam made from 35/65
PCL/PLA (as described in Example 9) and 95/5 PLA/PCL (a random
copolymer with an IV of 1.8 in HFIP measured as described herein).
Note, 35/65 PCL/PLA is a soft elastomeric copolymer while 95/5
PLA/PCL is a relatively stiff copolymer. The combination of the two
provides a compositional as well as structural gradient. This foam
is made using the steps outlined in Example 4 starting from a
homogeneous 50/50 physical mixture of a 10% wt./wt. solution of
35/65 PCL/PLA and 10% wt./wt. Solution of 95/5 PLA/PCL in 1,4
dioxane. Such a transcompositional foam will provide a good
template for tissue junctions such as bone-cartilage
interfaces.
Example 6
[0136] Cell Culture and Differentiation Data
[0137] Films made from 95/5 PLA/PGA, 90/10 PGA/PLA, 95/5 PLA/PCL,
75/25 PGA/PCL and 40/60 PCL/PLA were tested. Tissue culture
polystyrene (TCPS) was used as a positive control for all the
assays. Before testing, polymer discs were positioned at the bottom
of a 24-well ultralow cluster dish and were pre-wetted in growth
media for 20 min.
[0138] The 95/5 PLA/PGA copolymer used in this example was a random
copolymer with an IV of 1.76 as determined in HFIP at 25.degree.
C., which is currently used in Panacryl.TM. suture (Ethicon Inc.,
Somerville, N.J.). The 90/10 PGA/PLA copolymer was a random
copolymer with an IV of 1.74 as determined in HFIP at 25.degree.
C., which is currently used in Vicyl.TM. suture (Ethicon Inc.,
Somerville, N.J.). The 95/5 PLA/PCL polymer was made as described
in Example 10, with an IV of 2.1 as determined in HFIP at
25.degree. C. The 75/25 PG/PCL copolymer is a segmented block
copolymer with an IV of 1.85 and is described in U.S. Pat. No.
5,133,739 this copolymer is currently used in Monocryl.TM. sutures
(Ethicon Inc., Somerville, N.J.). The 40/60 PCL/PLA copolymer used
in this Example was made as described in Example 9 and had an IV of
1.44.
[0139] Cell Attachment and Proliferation:
[0140] Cells were seeded at 40,000/well in 24-well ultralow cluster
dishes (Corning) containing the polymers. The ultralow cluster
dishes are coated with a layer of hydrogel polymer, which retards
protein and cell adhesion to the wells. Cell attachment to the
biopolymers was determined following 24 hrs of incubation (N=3 for
each polymer). The attached cells were released by trypsinization
and the number of cells was determined using a heamacytometer. Cell
proliferation was assessed by determining cell counts at days 3 and
6 following seeding.
[0141] Differentiation Assays:
[0142] Alkaline Phosphatase Activity:
[0143] Alkaline phosphatase activity was determined by a
calorimetric assay using p-nitrophenol phosphate substrate (Sigma
104) and following manufacturers instruction. Briefly, cells were
seeded on the films or meshes at a density of 40,000 cells/well and
incubated for 1, 6, 14, and 21 d. Once cells reached confluence at
day 6 they were fed with mineralization medium (growth medium
supplemented with 10 mM .beta.-glycerophosphate, 50 .mu.g/ml
ascorbic acid). Alkaline phosphatase activity was determined in
cell homogenates (0.05% Triton X-100) at the above time points. The
quantity of protein in cell extracts was determined by micro BCA
reagent from Pierce. Primary rat osteoblasts cultured on films and
meshes were also stained for membrane-bound alkaline phosphatase
using a histochemical staining kit (Sigma). For all the films and
meshes three samples per group were tested.
[0144] Osteocalin ELISA:
[0145] Osteocalcin secreted into the medium by osteoblasts cultured
on various films was quantified by ELISA (Osteocalcin ELISA kit,
Biomedical Technologies Inc, Boston). Aliquots of media from the
wells containing the polymer films were lyophilized prior to
measurements of this protein by ELISA. Three samples for each
polymer were tested and the ELISA was repeated twice.
[0146] Von Kossa Staining
[0147] Three samples for each polymer were stained for mineralized
tissue using Von Kossa silver nitrate staining.
[0148] Expression of Alkaline Phosphatase and Osteocalcin mRNAs
[0149] The expression of alkaline phosphatase and osteocalcin mRNAs
in cells was assessed by semi-quantitative reverse transcriptase
polymerase chain reaction (RT-PCR) using RNA extracted from cells
cultured for 21 d on the films. Seven days after seeding, the
culture media was replaced with mineralization media (3 mM
.beta.-glycerophosphate and 50 .mu.g/ml of ascorbic acid were
added). The cells were cultured for additional 2 weeks, for a total
period of 3 weeks. Total RNA was extracted from four samples per
group using a RNeasy mini kit provided by Qiagen. The quality and
amount of total RNA was measured for each polymer group. Total RNA
was reverse transcribed to obtain cDNA using a reverse
transcriptase reaction (Superscript II, Gibco). The cDNAs for
osteocalcin, alkaline phosphatase, and
Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were amplified
using a PCR protocol described previously (GIBCO BRL manufacturers
instruction). The primer sequences (Table I) for osteocalcin,
alkaline phosphatase, and GAPDH were obtained using the FASTA
program (Genetic Computer Group, Madison, Wis.). Preliminary
studies were also conducted to optimize the number of PCR cycles
for each primer (Table II), and to determine the range of RNA,
which exhibits proportionality to cDNA. The PCR products were
electrophoreses on 1% (wt) agarose gels containing ethidium
bromide. The gels were photographed under UV light and were
evaluated by densitometry for the expression of osteocalcin and
alkaline phosphatase mRNAs relative to GAPDH.
[0150] Statistical Anlysis
[0151] Analysis of variance (ANOVA) with Tukey post hoc comparisons
was used to assess levels of significance for all the assays.
2TABLE I Primers used in RT-PCR Size Gene Species Forward primer
Reverse primer (bp) Alkaline Rat 5' 5' 379 ATCGCCTATCA GCAAGAAGAA
phospha- GCTAATGCAC GCCTTTGGG tase Osteo- Rat/ 5' CAACCCCAATTG 5'
339 calcin Human TGACGAGC TGGTGCGATC CATCACAGAG GAPDH Mouse/ 5'
ACCACAGTCCAT 5' TCCACCACCCT 452 Human/ GCCATCAC GTTGCTGTA Rat
[0152]
3TABLE II PCR optimization cycles Gene cDNA (.mu.l) Cycles Alkaline
1 25 phosphatase Osteocalcin 1 35 GAPDH 1 23
[0153] Results
[0154] Cell Attachment and Proliferation on Bioresorbable
Polymers:
[0155] No observable difference in cell morphology was evident
between the various polymer films and as compared to TCPS. Cell
attachment to the various biopolymer films was equivalent to TCPS
following 24 h of incubation. At day 3, cells proliferated well on
all films with the exception of 40/60 PCL/PLA, where proliferation
was 60% relative to TCPS. Furthermore, 95/5 PLA/PGA and 90/10
PGA/PLA films supported a significantly (p<0.05) higher degree
of cell proliferation compared to TCPS and 40/60 PCL/PLA (FIG.
7A).
[0156] Differentiation Assay:
[0157] Alkaline Phosphatase Enzyme Activity:
[0158] The profile for alkaline phosphatase activity expressed by
osteoblasts cultured on 95/5 PLA/PGA, 90/10 PGA/PLA and 95/5
PLA/PCL films was similar to the profile observed on TCPS. is
Alkaline phosphatase specific activities were significantly
(p<0.05) elevated for osteoblasts cultured on 40/60 PCL/PLA and
75/25 PGA/PCL films at days 14 and 21, respectively, compared to
other films and TCPS (FIG. 7B)
[0159] Expression of Alkaline Phosphatase and Osteocalcin mRNA:
[0160] The expression of mRNAs for alkaline phosphatase,
osteocalcin, and GAPDH for osteoblasts cultured on the 95/5
PLA/PGA, 40/60 PLA/PCL, 95/5 PLA/PCL films, and TCPS were evaluated
by densitometry. The results are depicted in FIG. 7C. It should be
noted that the data in FIG. 7B is at best semi-quantitative.
Nevertheless, the data suggests that 40/60 PCL/PLA film supported
significantly (p<0.05) higher levels of osteocalcin expression
compared to TCPS. The rest of the polymer surfaces were equivalent
to TCPS for both osteocalcin and AP mRNAs expression.
[0161] Conclusions
[0162] No major differences were observed with respect to cell
attachment and proliferation between the different bioresorbable
films or meshes tested following 6 days of incubation. Furthermore,
the results indicate that differences between these materials were
more obvious with respect to their differentiation characteristics.
Cells cultured on 40/60 PCL/PLA film showed enhanced alkaline
phosphatase activity and osteocalcin mRNA expression compared to
other films and TCPS following 14 and 21 days of incubation,
respectively.
[0163] References that may be referred to for a more complete
understanding of this techniques include, M. A. Aronow, L. C.
Gerstenfeld, T. A. Owen, M. S. Tassinari, G. S. Stein and J. B.
Lian: "Factors that promote progressive development of the
osteoblast phenotype in cultured fetal rat calvaria cells: Journal
of Cellular Physiology, 143: 213-221 (1990) and Stein, G. S., Lian,
J. B., and Owen, t. A. "Relationship of cell growth to the
regulation of tissue-specific gene expression during osteoblast
differentiation" FASEB, 4, 3111-3123 (1990).
Example 7
[0164] In Vivo Study of Foam Blend in Swine Dermal Wound Healing
Model
[0165] This example describes the results of implanting a 1 mm, 0.5
mm thickness foam tissue scaffolding in a swine full thickness
excisional wound model. The foam tissue scaffold was made from a
blend of 40/60 .epsilon.-caprolactone-co-lactide made as described
in Example 8 and 35/65 .epsilon.-caprolactone-co-glycolide
described in Example 9. These polymers were blended together and
formed into 1 mm and 0.5 mm foams substantially as described in
Example 3 (except that the cooling rate was 2.5.degree. C. per
minute and it was cooled only to -5.degree. C). Scanning electron
micrographs of a 0.5 mm foam are presented in FIGS. 9A, 9B and 9C.
The two thickness (0.5 mm and 1 mm) of foams were then tested in
the wound excisional model with and without PDGF being provided.
The resulting four different samples were then evaluated.
[0166] A blinded histologic evaluation was performed on 48 full
thickness excisional wounds from four pigs (12 sites per animal)
explanted at 8 days following wounding. The assessment was
performed on H&E stained slides. During the histologic
assessment, the following parameters were ranked/evaluated across
the specimen set 1) cellular invasion of the matrix qualitative and
quantitative assessments 2) infiltration of polymorphonuclear
leukoctyes (PMNs) into the contact zone (ventral surface) of the
matrix, 3) inflammation in the granulation tissue bed below
(ventral to) the matrix, 4) reaction of the epidermis to the
matrix, and 5) degree of fragmentation of the matrix.
[0167] Animal Husbandry:
[0168] The pigs were housed individually in cages (with a minimum
floor area of 10-sq. ft.) and given identification. All pigs were
assigned an individual animal number. A tag was placed on each
individual animal cage listing the animal number, species/strain,
surgical date, surgical technique and duration of the experiment
and date of euthanasia. Each animal was clearly marked with an
animal number on the base of the neck using a permanent marker.
[0169] The animal rooms were maintained at the range of 40 to 70%
R.H. and 15 to 24.degree. C. (59.0 to 75.2.degree. F.). The animals
were fed with a standard pig chow once per day, but were fasted
overnight prior to any experimental procedure requiring anesthesia.
Water was available ad libitum. A daily light:dark cycle of 12:12
hours was adopted.
[0170] Anesthesia:
[0171] On the initial day of the study, days of evaluation and the
day of necropsy, the animals were restrained and anesthetized with
either an intramuscular injection of Tiletamine HCl plus Zolazepam
HCl (Telazol.RTM., Fort Dodge Animal Health, Fort Dodge, Iowa 4
mg/ml) and Xylazine (Rompun.RTM., Bayer Corporation, Agriculture
Division, Animal Health, Shawnee Mission, Kansas, 4 mg/ml) or
Isoflurane (AErrane.RTM. Fort Dodge Animal Health, Fort Dodge,
Iowa) inhalatory anesthesia (5% vol.) administered via a nose cone.
When the animal was in the surgical suite, it was maintained on
Isoflurane (Aerrane.RTM.) inhalatory anesthesia (2% vol.)
administered via a nose cone. Food was available after recovery
from each procedure.
[0172] Preparation of the Surgical Site:
[0173] One day prior to the surgical procedure, body weights were
measured and the dorsal region of four pigs were clipped with an
electric clipper equipped with a #40 surgical shaving blade. The
shaved skin was then re-shaved closely with shaving cream and a
razor and then rinsed. The shaved skin and entire animal (excluding
the head) was then scrubbed with a surgical scrub brush-sponge with
PCMX cleansing solution (Pharmaseal.RTM. Scrub Care.RTM. Baxter
Healthcare Corporation, Pharmaseal Division, Valencia, Calif.) and
then with HIBICLENS.RTM. chlorhexidine gluconate (available from
COE Laboratories, Incorporated, Chicago, Ill.). The animal was
wiped dry with a sterile towel. Sterile NU-GAUZE* gauze (from
Johnson & Johnson Medical Incorporated, Arlington, Tex.) was
placed over the dorsal surface of each animal and secured with
WATERPROOF* tape (available from Johnson & Johnson Medical
Incorporated, Arlington, Texas). The entire torso region of the
animal was then wrapped with Spandage.TM. elastic stretch bandage
(available from Medi-Tech International Corporation, Brooklyn,
N.Y.) to maintain a clean surface overnight.
[0174] On the day of surgery, immediately prior to delivering the
animal to the surgical suite, the dorsal skin was again scrubbed
using a surgical scrub brush-sponge with PCMX cleansing solution
(Pharmaseal.RTM. Scrub Care.RTM.), rinsed and wiped dry using a
sterile towel, as performed on the previous day. The animals were
placed prone on the surgical table and wiped with 70% alcohol and
dried with sterile gauze. Using a sterile surgical marker (availabe
from Codman.COPYRGT. a division of Johnson & Johnson
Professional Incorporated, Raynham, Mass.) and an acetate template,
marks were made on the dorsal skin according to the desired
placement of each full-thickness wound.
[0175] Surgical Procedure:
[0176] Following anesthesia, under sterile conditions, twelve (12)
full-thickness excisions (1.5.times.1.5 cm) per animal were made in
two rows parallel to the spinal column on the left and right dorsal
regions using a scalpel blade. A pair of scissors and/or scalpel
blade was used to aid in the removal of skin and subcutaneous
tissue. Bleeding was controlled by use of a sponge tamponade.
Sufficient space was left between wounds to avoid wound-to-wound
interference. The excised tissue was measured for thickness using a
digital caliper.
[0177] Application of the Treatment and Dressing:
[0178] Each wound was submitted to a prepared, coded treatment
regimen (study participants were blinded to all treatments). The
primary dressing consisting of the sterile individual test article
(1.5.times.1.5 cm soaked in sterile saline for 24 hours) was placed
into the wound deficit in a predetermined scheme. The secondary
dressing, a non-adherent, saline soaked, square of RELEASE*
dressing (manufactured by Johnson & Johnson Medical
Incorporated, Arlington, Texas) was placed on top of the test
article. A layer of BIOCLUSIVE* dressing (available from Johnson
& Johnson Medical Incorporated, Arlington, Tex.) was sealed
over the wounds to keep the wound moist and the dressing in place.
Strips of Reston.TM. (3M Medical-Surgical Division, St. Paul,
Minn.) polyurethane self-adhering foam were placed between the
wounds to avoid cross-contamination due to wound fluid leakage, and
to protect the wounds from damage and the dressing from
displacement. A layer of NU-GAUZE* gauze was placed on top of the
BIOCLUSIVE* dressing and Reston.TM. foam, and was secured with
WATERPROOF* tape to protect the dressings. The animals were then
dressed with Spandage.TM. elastic net to help keep the dressings in
place.
[0179] The secondary dressings were removed and replaced daily with
a fresh piece of saline soaked RELEASE* secondary dressing. The
primary dressings (test articles) were not disturbed unless the
unit was displaced or pushed out of the wound deficit.
[0180] Post-operative Care and Clinical Observations:
[0181] After performing the procedures under anesthesia, the
animals were returned to their cages and allowed to recover. The
animals were given analgesics (buprenorphine hydrochloride
[Buprenex Injectable, 0.01 mg/kg, im] sold by Reckitt & Colman
Products, Hull, England) immediately post-surgery and the following
day. After recovering from anesthesia, the pigs were observed for
behavioral signs of discomfort or pain. No signs of pain were
observed.
[0182] Each pig was observed twice daily after the day of surgery
to determine its health status on the basis of general attitude and
appearance, food consumption, fecal and urinary excretion and
presence of abnormal discharges.
[0183] Euthanasia:
[0184] At the end of the study (8 days post-wounding), each animal
was euthanized under anesthesia, with an intravenous injection of
(1 ml/10 pounds body weight) Socumb.TM. pentobarbital sodium and
phenytoin sodium euthanasia solution (sold by The Butler Company,
Columbus, Ohio) via the marginal ear vein. Following euthanasia,
the animals were observed to ensure that respiratory function had
ceased and there was no palpable cardiac function. A stethoscope
facilitated the assessment for the lack of cardiac function.
[0185] Tissue Harvesting:
[0186] Immediately following euthanasia, each wound, together with
the underlying fat and a small portion of surrounding skin was
excised. The tissue was placed in 10% neutral buffered
formalin.
[0187] Evaluations:
[0188] Visual Wound Assessment:
[0189] General observations were recorded for days 1-3, including
displacement, wound reaction and physical characteristics of the
scaffold. Detailed clinical evaluations were performed on days 4-8
post-wounding. Assessments were recorded as to the presence/absence
(yes=1/no=0) and/or degree (given a score) of the following
parameters:
[0190] Dressing Conditions: air exposed, displacement of test
article, channeling, communication and moisture content of the
RELEASE* secondary dressing (scored as: 4=moist, 3=moist/dry,
2=dry/moist, 1=dry).
[0191] Wound Bed Conditions: moisture content of test article
(scored as: 4=moist, 3=moist/dry, 2=dry/moist, 1=dry), inflammation
(scored as: 3=severe, 2=moderate, 1=slight, 0=none), reinjury
(scored as: 3=severe, 2=moderate, 1=slight, 0=none), clots,
folliculitis, infection, level of test article (scored as: 4=super
raised, 3=raised, 2=even, 1=depressed), fibrin (scored as:
3=severe, 2=moderate, 1=slight, 0=none), and erythema. Color of the
test article was also observed.
[0192] Tissue Processing:
[0193] Excised tissue samples were taken at day eight. The entire
wound was harvested and placed into 10% neutral buffered formalin.
The tissue was prepared for frozen sections. The tissue was trimmed
and mounted onto the object holder with Tissue-Tek.RTM. OCT
Compound (sold by Sakura Finetechnical Company, Limited, Tokyo,
Japan) and quickly frozen. The specimens were sectioned on the
cryostat at 10 .mu.m and stained with a frozen H&E stain.
[0194] Histological Assessments (Day 8 Post-wounding)
[0195] Histological evaluations for granulation tissue (area and
length) and epithelialization were assessed using H&E stained
specimens using a magnification of 20-40.times.. Granulation tissue
height was determined by dividing the area by the length.
[0196] Histopathological evaluation of the tissue samples was
assessed using the H&E stained specimens, they were first
assessed under 100.times. to 400.times. magnification.
[0197] Results
[0198] There was cellular invasion into the interstices of the
matrix in the majority of all test sites. In the majority of sites
this invasion was true tissue ingrowth comprised of varying
populations of fibroblasts, macrophages, macrophage giant cells,
and endothelial-like cells, there also appeared to be capillary
formation. Significant formation of dense fibrous connective tissue
layer dorsal to the matrices essentially embedding the matrices in
the tissue, was seen at several sites for the 0.5 mm foams with and
without PDGF. The 1 mm matrices were either at the surface of the
tissue bed or sloughed. Macrophage giant cell formation seemed to
be greater in the 0.5 mm versus the 1 mm foam-scaffolds. In sites
where the 1 mm foam was being sloughed or partially separated from
the underlying granulation tissue there was death of the invading
cells forming masses of pyknotic cell debris.
[0199] Complete incorporation of the matrix into the granulation
tissue bed was seen at several sites for the 0.5 mm foam
scaffoldings. FIGS. 10 and 11 illustrate the incorporation of these
matrices into the granulation tissue bed. FIG. 10 is a dark filed
40.times. pictomicrograph of a trichrome stained tissue sample.
FIG. 11 is a 100.times. composite photomicrograph of a trichrome
stained sample illustrating the cellular invasion of a foam
containing PDGF. Complete incorporation of the matrices into the
granulation tissue bed is evident in both pictures. The dense
fibrous tissue above the foam scaffolding is evident in both
pictures. These results indicate the 0.5 mm foams will provide a
suitable substrate for the growth of epidermal tissue.
Example 8
[0200] Synthesis of a Random
Poly(.epsilon.-caprolactone-co-glycolide)
[0201] A random copolymer of .epsilon.-caprolactone-glycolide with
a 35/65 molar composition was synthesized by ring opening
polymerization reaction. The method of synthesis was essentially
the method described in U.S. Pat. No. 5,468,253 in Example 6 (which
is hereby incorporated herein by reference). The amount of
diethylene glycol initiator added was adjusted to 1.15 mmole/mole
of monomer to obtain the following characteristics of the dried
polymer: The inherent viscosity (I.V.) of the copolymer was 1.59
dL/g in hexafluoroisopropanol at 25.degree. C. The molar ratio of
PCL/PGA was found to be 35.5/64.5 by proton NMR with about 0.5%
residual monomer. The glass transition (Tg) and the melting points
(Tm) of the copolymer were found to be -1.degree. C., 60.degree. C.
and 126.degree. C. respectively, by DSC.
Example 9
[0202] Synthesis of 40:60 Poly(.epsilon.-caprolactone-co-L-lactide)
by Sequential Addition
[0203] In the glove box, 100 .mu.L (33 .mu.mol) of a 0.33 M
stannous octoate solution in toluene, 115 .mu.L (1.2 mmol) of
diethylene glycol, 24.6 grams (170 mmol) of L-lactide, and 45.7
grams (400 mmol) of .epsilon.-caprolactone were transferred into a
silanized, flame dried, two neck, 250 mL round bottom flask
equipped with a stainless steel mechanical stirrer and a nitrogen
gas blanket. The reaction flask was placed in an oil bath already
set at 190.degree. C. and held there. Meanwhile, in the glove box,
62.0 grams (430 mmol) L-lactide were transferred into a flame
dried, pressure equalizing addition funnel. The funnel was wrapped
with heat tape and attached to the second neck of the reaction
flask. After 6 hours at 190.degree. C., the molten L-lactide was
added to the reaction flask over 5 minutes. The reaction was
continued overnight for a total reaction time of 24 hours at
190.degree. C. The reaction was allowed to cool to room temperature
overnight. The copolymer was isolated from the reaction flask by
freezing in liquid nitrogen and breaking the glass. Any remaining
glass fragments were removed from the copolymer using a bench
grinder. The copolymer was again frozen with liquid nitrogen and
broken off the mechanical stirring paddle. The copolymer was ground
into a tared glass jar using a Wiley Mill and allowed to warm to
room temperature in a vacuum oven overnight. 103.13 grams of 40:60
poly(.epsilon.-caprolactone-co-L-lactide) were added to a tared
aluminum pan and then devolitilized under vacuum at 110.degree. C.
for 54 hours. 98.7 grams (95.7% by weight) of copolymer were
recovered after devolitilization. The inherent viscosity was
measured and found to be 1.1 dL/g in CHCl.sub.3 at 25.degree. C.
(c=0.1 g/dL). FTIR (cast film from CHCl.sub.3 onto KBr window,
cm.sup.-1): 2993, 2944, 2868, 1759, 1456, 1383, 1362, 1184, 1132,
1094, 870, and 756. .sup.1H NMR (400 MHz, HFAD/Benzene, ppm):
.delta. 1.25, 2 broad lines (e); 1.35, 2 lines (f); 1.42, 3 lines;
1.55, 2 lines; 2.22, 3 lines; 2.35, 4 broad lines; 4.01, 3 lines;
4.05, 3 lines; 4.2, quartet; 5.05, 3 broad lines; 5.15, 4 lines.
Polymer composition by .sup.1H NMR: 41.8% PCL, 57.5% PLA, 0.8%
L-lactide, <0.1% .epsilon.-caprolactone. DSC (20.degree. C./min,
first heat): T.sub.m=154.8.degree. C., .DELTA.H.sub.m=18.3 J/g. GPC
(molecular weights determined in THF using poly(methyl
methacrylate) standards, daltons): M.sub.w=160,000,
M.sub.n=101,000, PDI=1.6.
Example 10
[0204] Synthesis of 95/5 PLA/PCL Copolymer
[0205] In the glove box, 170 .mu.L (1.8 mmol) of diethylene glycol,
350 .mu.L (115 .mu.mol) of a 0.33 M stannous octoate solution in
toluene, 17.1 grams (150 mmol) of .epsilon.-caprolactone, and 410.4
grams (2.85 mol) of L-lactide were placed into a silanized, flame
dried, 1000 mL round bottom equipped with a stainless steel
mechanical stirrer and vacuum take off connector in order to
maintain a dry nitrogen gas blanket. The reaction flask was placed
in an oil bath already heated to 185.degree. C. and then held there
for 3 hours. The flask was removed from the oil bath and allowed to
cool down to room temperature. The polymer was isolated by wrapping
the flask with aluminum foil, freezing it in liquid nitrogen, and
then grinding away any adhered glass to the polymer. The copolymer
was then ground in a Wiley mill. The ground polymer was vacuum
dried at 80.degree. C. for 24 hours. 302 grams of copolymer were
collected. The inherent viscosity was 2.1 dL/g in chloroform
[25.degree. C., c=0.l g/dL]. The copolymer composition was measured
by proton NMR spectroscopy and found to be 97.2 mole percent PLA
and 2.8 mole percent PCL. No residual monomer was detected.
* * * * *