U.S. patent application number 17/169195 was filed with the patent office on 2021-06-17 for multi-layer biomaterial for tissue regeneration and wound healing.
The applicant listed for this patent is Children's Medical Center Corporation, Tufts University. Invention is credited to Carlos R. Estrada, Eun Seok Gil, David L. Kaplan, Joshua R. Mauney.
Application Number | 20210178017 17/169195 |
Document ID | / |
Family ID | 1000005429970 |
Filed Date | 2021-06-17 |
United States Patent
Application |
20210178017 |
Kind Code |
A1 |
Mauney; Joshua R. ; et
al. |
June 17, 2021 |
MULTI-LAYER BIOMATERIAL FOR TISSUE REGENERATION AND WOUND
HEALING
Abstract
The technology described herein is directed to compositions
comprising at least a first porous biomaterial layer and a second
impermeable biomaterial layer and methods relating thereto. In some
embodiments, the compositions and methods described herein relate
to wound healing, e.g. repair of wounds and/or tissue defects.
Inventors: |
Mauney; Joshua R.;
(Watertown, MA) ; Estrada; Carlos R.; (Brookline,
MA) ; Kaplan; David L.; (Concord, MA) ; Gil;
Eun Seok; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Medical Center Corporation
Tufts University |
Boston
Medford |
MA
MA |
US
US |
|
|
Family ID: |
1000005429970 |
Appl. No.: |
17/169195 |
Filed: |
February 5, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14377128 |
Aug 6, 2014 |
10912862 |
|
|
PCT/US2013/024744 |
Feb 5, 2013 |
|
|
|
17169195 |
|
|
|
|
61595233 |
Feb 6, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B29L 2031/7532 20130101;
A61L 27/3604 20130101; A61L 2300/254 20130101; A61L 2300/608
20130101; A61L 2430/22 20130101; B29C 39/003 20130101; B29L
2031/755 20130101; A61L 27/56 20130101; A61L 2300/406 20130101;
A61L 2300/43 20130101; A61L 2300/256 20130101; A61L 2300/258
20130101; A61L 27/227 20130101; A61L 2300/414 20130101; A61L
2300/41 20130101; A61L 27/58 20130101; A61L 2300/412 20130101; A61L
27/54 20130101; A61L 2300/22 20130101; A61L 27/3834 20130101 |
International
Class: |
A61L 27/22 20060101
A61L027/22; A61L 27/54 20060101 A61L027/54; A61L 27/56 20060101
A61L027/56; A61L 27/38 20060101 A61L027/38; A61L 27/36 20060101
A61L027/36; A61L 27/58 20060101 A61L027/58; B29C 39/00 20060101
B29C039/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with federal funding under Grant No.
P41 EB002520 awarded by the National Institutes of Health and the
National Institutes of Biomedical Imaging and Bioengineering and
Grant Nos. P50 DK065298-06, T32-DK60442, and K99DK083616-01A2
awarded by the National Institutes of Health and the National
Institutes of Diabetes and Digestive and Kidney Diseases. The U.S.
government has certain rights in the invention.
Claims
1. A composition comprising a first and second layer; the first
layer comprising a porous biomaterial matrix; and the second layer
comprising a impermeable biomaterial.
2. The composition of claim 1, wherein the matrix is a structure
selected from the group consisting of: foams; hydrogels; electro
spun fibers; gels; fiber mats; sponges; 3-dimensional scaffolds;
non-woven mats; woven materials; knit materials; fiber bundles; and
fibers.
3. The composition of any of claims 1-2, wherein the biomaterial is
selected from the group consisting of: silk fibroin; PGA; collagen;
polyethylene oxide, collagen, fibronectin, keratin, polyaspartic
acid, polylysin, alginate, chitosan, chitin, and hyaluronic
acid.
4. The composition of any of claims 1-3, wherein the silk fibroin
comprises Bombyx mori silk fibroin.
5. The composition of any of claims 1-4, wherein the first and
second layers comprise the same biomaterial.
6. The composition of any of claims 1-4, wherein the first and
second layers comprise different biomaterials.
7. A composition comprising a first and second layer; the first
layer comprising a porous silk fibroin matrix; and the second layer
comprising a impermeable silk fibroin film.
8. The composition of any of claims 1-7, further comprising an
agent.
9. The composition of claim 8, wherein the agent is selected from
the group consisting of: an antibiotic; an agent to attract cells;
a cell; a stem cell; a ligand; a growth factor; a platelet; and a
component of extracellular matrix.
10. The composition of any of claims 1-9, wherein the average pore
size of the biomaterial matrix is at least 200 .mu.m.
11. The composition of any of claims 1-10, wherein the average pore
size of the biomaterial matrix is at least 300 .mu.m.
12. The composition of any of claims 1-11, wherein the average pore
size of the biomaterial matrix is at least 400 .mu.m.
13. The composition of any of claims 1-12, wherein the average pore
size of the biomaterial matrix is from about 200 .mu.m to about 600
.mu.m.
14. The composition of any of claims 1-13, wherein the average pore
size of the biomaterial matrix is from about 300 .mu.m to about 500
.mu.m.
15. The composition of any of claims 1-14, wherein the average pore
size of the biomaterial matrix is about 400 .mu.m.
16. The composition of any of claims 1-15, wherein the average pore
size of the biomaterial matrix is about 300 .mu.m.
17. The composition of any of claims 1-16, wherein the impermeable
layer is from about 10 .mu.m to about 600 .mu.m thick.
18. The composition of any of claims 1-17, wherein the impermeable
layer is from about 100 .mu.m to about 400 .mu.m thick.
19. The composition of any of claims 1-18, wherein the impermeable
layer is from about 150 .mu.m to about 250 .mu.m thick.
20. The composition of any of claims 1-19, wherein the impermeable
layer is from about 25 .mu.m to about 150 .mu.m thick.
21. The composition of any of claims 1-20, wherein the impermeable
layer is about 200 .mu.m thick.
22. The composition of any of claims 1-21, wherein the composition
is from about 0.01 cm to about 5 cm thick.
23. The composition of any of claims 1-22, wherein the composition
is from about 0.1 cm to about 3 cm thick.
24. The composition of any of claims 1-23, wherein the composition
is from about 0.1 cm to about 2 cm thick.
25. The composition of any of claims 1-24, wherein the composition
is about 1 cm thick.
26. The composition of any of claims 1-25, wherein the composition
has a shape selected from the group consisting of: a sheet; a tube;
and a contoured sheet.
27. A method of producing a composition of any of claims 1-26, the
method comprising; contacting a porous biomaterial matrix with a
impermeable biomaterial layer.
28. A method of producing a composition of any of claims 1-26, the
method comprising; (c) casting an admixture of aqueous biomaterial
solution and NaCl; (d) contacting the composition resulting from
step (.alpha.) with water.
29. The method of claim 28, wherein step (.alpha.) is performed
with the solution in contact with a pre-existing impermeable
biomaterial layer.
30. The method of any of claims 28-29, wherein the NaCl is
granular.
31. The method of any of claims 28-30, wherein the biomaterial
solution is a silk fibroin solution.
32. The method of any of claims 28-31, wherein the silk fibroin
solution has a concentration of from about 2% wt/vol to about 15%
wt/vol.
33. The method of any of claims 28-32, wherein the silk fibroin
solution has a concentration of from about 4% wt/vol to about 10%
wt/vol.
34. The method of any of claims 28-33, wherein the silk fibroin
solution has a concentration of about 6% wt/vol.
35. The method of any of claims 28-34, wherein the admixture is
cast for from about 12 hours to about 96 hours.
36. The method of any of claims 28-35, wherein the admixture is
cast for from about 24 hours to 72 hours.
37. The method of any of claims 28-36, wherein the admixture is
cast for about 48 hours.
38. The method of any of claims 28-37, wherein step (b) proceeds
for from about 12 hours to about 120 hours.
39. The method of any of claims 28-38, wherein step (b) proceeds
for from about 24 hours to about 96 hours.
40. The method of any of claims 28-39, wherein step (b) proceeds
for about 72 hours.
41. The method of any of claims 28-40, wherein the water is
distilled water.
42. The method of any of claims 28-41, wherein the water is removed
and replaced with a fresh volume of water at least once during step
(b).
43. The method of any of claims 28-42, wherein, during the casting
step, the admixture of biomaterial solution and NaCl is in contact
with an impermeable layer.
44. The method of any of claims 27-43, wherein the impermeable
biomaterial layer comprises silk fibroin.
45. The method of any of claims 27-44, wherein the method further
comprises a first step of casting silk fibroin solution to form an
impermeable silk fibroin layer.
46. The method of claim 45, wherein the silk fibroin solution has a
concentration of from about 2% wt/vol to about 15% wt/vol.
47. The method of claim 45, wherein the silk fibroin solution has a
concentration of from about 4% wt/vol to about 10% wt/vol.
48. The method of claim 45, wherein the silk fibroin solution has a
concentration of about 8% wt/vol.
49. The method of any of claims 27-48, wherein the composition is
produced under sterile conditions or sterilized after the rinsing
step is complete.
50. The method of any of claims 27-49, wherein the composition is
produced in a linear shape.
51. The method of any of claims 27-50, wherein the composition is
produced in a tubular shape.
52. The method of any of claims 27-51, wherein the method further
comprises adding an agent to at least one layer.
53. The method of claim 52, wherein the agent is a therapeutic
agent.
54. The method of any of claims 27-53, wherein the method further
comprises altering the .beta.-sheet content of a silk fibroin
layer.
55. The method of claim 54, wherein the .beta.-sheet content of a
silk fibroin layer is altered using a method selected from the
group consisting of: contacting the layer with water vapor; drying;
dehydration; water annealing; stretching; compression; solvent
immersion; immersion in methanol; immersion in ethanol; pH
adjustment; heat treatment; and sonication.
56. The method of any of claims 54-55, wherein the .beta.-sheet
content of a silk fibroin layer is altered after adding an agent to
the layer.
57. A method for wound healing or repair of a tissue defect, the
method comprising applying a composition of any of claims 1-26 to
the wound or tissue defect.
58. The method of claim 57, wherein the wound or tissue defect is
located in a hollow organ.
59. The method of claim 58, wherein the hollow organ is selected
from the group consisting of: the bladder; a portion of the
gastrointestinal tract; the stomach, and the intestines.
60. The method of claim 57, wherein the wound or tissue defect is
selected from the group consisting of: a skin wound or defect; a
diabetic ulcer; a bone wound or defect; a joint wound or defect; a
meniscus wound or defect; an articular cartilage wound or defect; a
soft tissue wound or defect; a lung tissue wound or defect; and a
kidney wound or defect.
61. The method of any of claims 57-60, wherein the impermeable
layer is oriented to prevent the movement of material into or out
of the wound or defect.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Application No. 61/595,233 filed Feb. 6,
2012, the contents of which are incorporated herein by reference in
their entirety.
TECHNICAL FIELD
[0003] The technology described herein relates to biomaterial
compositions and methods of making and using them.
BACKGROUND
[0004] A number of biomaterials, including acellular matrices,
tissue grafts, and polymeric substances (e.g. PGA) have been
explored for use in tissue regeneration and/or wound healing.
However, existing scaffolds, while permitting a certain level of
tissue regeneration lead to long-term graft failure due to implant
contracture, graft rupture, and/or fibrosis. These problems lead to
delays in wound healing and even morbidity.
SUMMARY
[0005] The technology described herein is directed to multi-layer
biomaterial compositions and methods relating thereto. The layers
of these compositions differ in their physical properties and
provide a complete composition that serves both as a scaffold for
tissue regeneration as well as a suturable substrate capable of
sealing a wound or tissue defect.
[0006] In one aspect, the technology described herein relates to a
composition comprising a first and second layer; the first layer
comprising a porous biomaterial matrix; and the second layer
comprising an impermeable biomaterial. In some embodiments, the
matrix can be a structure selected from the group consisting of:
foams; hydrogels; electrospun fibers; gels; fiber mats; sponges;
3-dimensional scaffolds; non-woven mats; woven materials; knit
materials; fiber bundles; and fibers. In some embodiments, the
biomaterial can be selected from the group consisting of silk
fibroin; PGA; collagen; polyethylene oxide, collagen, fibronectin,
keratin, polyaspartic acid, polylysin, alginate, chitosan, chitin,
and hyaluronic acid. In some embodiments, the silk fibroin can
comprise Bombyx mori silk fibroin. In some embodiments, the first
and second layers can comprise the same biomaterial. In some
embodiments, the first and second layers can comprise different
biomaterials. In one aspect, described herein is a composition
comprising a first and second layer; the first layer comprising a
porous silk fibroin matrix; and the second layer comprising a
impermeable silk fibroin film. In some embodiments, the composition
can further comprise an agent. In some embodiments, agent can be
selected from the group consisting of: an antibiotic; an agent to
attract cells; a cell; a stem cell; a ligand; a growth factor; a
platelet; and a component of extracellular matrix.
[0007] In some embodiments, the average pore size of the
biomaterial matrix can be at least 200 .mu.m. In some embodiments,
the average pore size of the biomaterial matrix can be at least 300
.mu.m. In some embodiments, the average pore size of the
biomaterial matrix can be at least 400 .mu.m. In some embodiments,
the average pore size of the biomaterial matrix can be from about
200 .mu.m to about 600 .mu.m. In some embodiments, the average pore
size of the biomaterial matrix can be from about 300 .mu.m to about
500 .mu.m. In some embodiments, the average pore size of the
biomaterial matrix can be about 400 .mu.m. In some embodiments, the
average pore size of the biomaterial matrix can be about 300
.mu.m.
[0008] In some embodiments, the impermeable layer can be from about
10 .mu.m to about 600 .mu.m thick. In some embodiments, the
impermeable layer can be from about 100 .mu.m to about 400 .mu.m
thick. In some embodiments, the impermeable layer can be from about
150 .mu.m to about 250 .mu.m thick. In some embodiments, the
impermeable layer can be from about 25 .mu.m to about 150 .mu.m
thick. In some embodiments, the impermeable layer can be about 200
.mu.m thick. In some embodiments, the composition can be from about
0.01 cm to about 5 cm thick. In some embodiments, the composition
can be from about 0.1 cm to about 3 cm thick. In some embodiments,
the composition can be from about 0.1 cm to about 2 cm thick. In
some embodiments, the composition can be about 1 cm thick.
[0009] In some embodiments, the composition can have a shape
selected from the group consisting of: a sheet; a tube; and a
contoured sheet.
[0010] In one aspect, the technology described herein relates to a
method of producing a composition as described herein, the method
comprising; contacting a porous biomaterial matrix with a
impermeable biomaterial layer. In one aspect, the technology
described herein relates to a method of producing a composition as
described herein, the method comprising; (a) casting an admixture
of aqueous biomaterial solution and NaCl; (b) contacting the
composition resulting from step (a) with water. In some
embodiments, step (a) can be performed with the solution in contact
with a pre-existing impermeable biomaterial layer. In some
embodiments, the NaCl can be granular.
[0011] In some embodiments, the biomaterial solution can be a silk
fibroin solution. In some embodiments, the silk fibroin solution
can have a concentration of from about 2% wt/vol to about 15%
wt/vol. In some embodiments, the silk fibroin solution can have a
concentration of from about 4% wt/vol to about 10% wt/vol. In some
embodiments, the silk fibroin solution can have a concentration of
about 6% wt/vol.
[0012] In some embodiments, the admixture can be cast for from
about 12 hours to about 96 hours. In some embodiments, the
admixture can be cast for from about 24 hours to 72 hours. In some
embodiments, the admixture can be cast for about 48 hours. In some
embodiments, step (b) can proceed for from about 12 hours to about
120 hours. In some embodiments, step (b) can proceed for from about
24 hours to about 96 hours. In some embodiments, step (b) can
proceed for about 72 hours.
[0013] In some embodiments, the water can be distilled water. In
some embodiments, the water can be removed and replaced with a
fresh volume of water at least once during step (b). In some
embodiments, during the casting step, the admixture of biomaterial
solution and NaCl can be in contact with an impermeable layer. In
some embodiments, the impermeable biomaterial layer can comprise
silk fibroin.
[0014] In some embodiments, the method can further comprises a
first step of casting silk fibroin solution to form an impermeable
silk fibroin layer. In some embodiments, the silk fibroin solution
can have a concentration of from about 2% wt/vol to about 15%
wt/vol. In some embodiments, the silk fibroin solution can have a
concentration of from about 4% wt/vol to about 10% wt/vol. In some
embodiments, the silk fibroin solution can have a concentration of
about 8% wt/vol.
[0015] In some embodiments, the composition can be produced under
sterile conditions or sterilized after the rinsing step is
complete. In some embodiments, the composition can be produced in a
linear shape. In some embodiments, the composition can be produced
in a tubular shape. In some embodiments, the method can further
comprise adding an agent to at least one layer. In some
embodiments, the agent can be a therapeutic agent. In some
embodiments, the method can further comprise altering the
.beta.-sheet content of a silk fibroin layer. In some embodiments,
the .beta.-sheet content of a silk fibroin layer can be altered
using a method selected from the group consisting of: contacting
the layer with water vapor; drying; dehydration; water annealing;
stretching; compression; solvent immersion; immersion in methanol;
immersion in ethanol; pH adjustment; heat treatment; and
sonication. In some embodiments, the .beta.-sheet content of a silk
fibroin layer can be altered after adding an agent to the
layer.
[0016] In one aspect, the technology described herein relates to a
method for wound healing or repair of a tissue defect, the method
comprising applying a composition as described herein to the wound
or tissue defect. In some embodiments, the wound or tissue defect
can be located in a hollow organ. In some embodiments, the hollow
organ can be selected from the group consisting of: the bladder; a
portion of the gastrointestinal tract; the stomach, and the
intestines. In some embodiments, the wound or tissue defect can be
selected from the group consisting of: a skin wound or defect; a
diabetic ulcer; a bone wound or defect; a joint wound or defect; a
meniscus wound or defect; an articular cartilage wound or defect; a
soft tissue wound or defect; a lung tissue wound or defect; and a
kidney wound or defect. In some embodiments, the impermeable layer
can be oriented to prevent the movement of material into or out of
the wound or defect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 depicts scanning electron microscopy analysis and
tensile testing of the structural and mechanical properties of
bilayer compositions produced according to Method 1 of Example
1.
[0018] FIG. 2 depicts scanning electron microscopy analysis and
tensile testing of the structural and mechanical properties of
bilayer compositions produced according to Method 2 of Example
1.
[0019] FIG. 3 depicts cystogram images of bladders at 1 and 3
months post-implatation.
[0020] FIG. 4 depicts urodynamic assessment of silk augmented
bladder at 3 months of implantation in comparison to non augmented
controls.
[0021] FIGS. 5A-5B depict the results of structural and mechanical
analyses of scaffold groups. FIG. 5A depicts photomicrographs of
representative SEM images demonstrating top and cross-sectional
views of matrix configurations. Inset: Bottom FF scaffold view.
[0022] FIG. 5B depicts the results of evaluation of ultimate
tensile strength (UTS), elastic modulus (EM), and % elongation to
failure (ETF) in matrix groups defined in FIG. 5A.
Means.+-.standard deviation per data point. (*) represents data
previously reported in Gomez et al., 2011.
[0023] FIGS. 6A-6B depict the characterization of urinary stone
incidence and size in regenerated bladders. FIG. 6A is a table of
the quantification of stone frequency and diameter in bladders
augmented with each matrix group and cystotomy controls. FIG. 6B
depicts photomicrographs of urinary stones in regenerated bladders
and controls. Arrows denote stones. Scale bar=2.5 mm.
[0024] FIGS. 7A-7B depict cystometric analysis and quantification
of urodynamic parameters in augmented bladders following 10 weeks
post-implantation. FIG. 7A depicts representative cystometric
tracings of voiding cycles displayed by cystotomy controls, GS1,
GS2, and FF augmented bladders. (*) denotes individual voids. FIG.
7B depicts a table of comparisons of urodynamic parameters between
experimental groups displayed in [A]. (*)=p<0.05 in comparison
to cystotomy controls.
[0025] FIGS. 8A-8C demonstrate that silk augmented bladders display
increases in capacity over time. FIGS. 8A (pre-op) and 8B (3 month
post-implant) depict cystograms of bladders. FIG. 8C depicts
cystometric analysis of bladder capacity (capacity at 20 cm
H.sub.2O) pre-op and 3 months after augmentation with silk
scaffold.
[0026] FIGS. 9A-9D depict the structural analysis of bi-layer silk
scaffold and porcine bladder integration. FIGS. 9A (side view) and
9B (top view) depict images of electron microscopy of a bi-layer
scaffold. FIG. 9C depicts an image of a bi-layer scaffold
composition. FIG. 9D depicts a view of bladder dome with implanted
scaffold.
[0027] FIGS. 10A-10B depict analysis of stone formation in
implants. FIG. 10A depicts a table of stone frequency and diameter
in each treatment group. FIG. 10B depicts photographs of stone
formation.
[0028] FIGS. 11A-11B depict representative cystometric tracings of
voiding cycles displayed by experimental groups (FIG. 11A) and
comparisons of urodynamic parameters (FIG. 11B) of the tracings
shown in FIG. 11B. (*)=p<0.05 in comparison to cystotomy
[0029] FIGS. 12A-12B depict graphs of the tensile (FIG. 12A) and
functional (FIG. 12B) properties of regenerated bladder tissues.
FIG. 12A depicts stress-strain profiles of regenerated and control
tissues. FIG. 12B depicts contraction responses in intact (+M) and
demucosalized smooth muscle (-M) in response to carbachol in organ
bath cultures. Dome-regenerated tissue. Body-normal bladder. Molar
concentrations of carbachol are shown on the x-axis.
[0030] FIG. 13 depicts a schematic of the ileoplasty model in rats
for Bi-layer silk implantation.
[0031] FIG. 14 depicts graphs of tensile testing analyses of
regenerated and native procine bladder tissues performed to assess
mechanical stress.
[0032] FIGS. 15A-15B depict schematics of exemplary embodiments of
the compositions described herein.
[0033] FIG. 16 depicts an electromicrography demonstrating the
presence of primary and secondary pores in a porous layer.
[0034] FIG. 17 depicts photographs of gross tissue observations of
rat ileum implanted with bi-layer silk scaffold at 10 weeks
post-implantation. Dashed boxes indicate regenerated tissue.
[0035] FIG. 18 depicts images of gross tissue obersvations (left
panel) and microCT (right panel) of gastrointestinal tract
implanted with bi-layer silk scaffold 10 weeks post-operative. The
images demonstrate GI tract continuity after installation of
contrast agent (ileoplasty was performed). Arrows indicate area of
regenerated tissue.
[0036] FIGS. 19A-19B demonstrate that silk scaffolds support
urethral continuity following onlay urethroplasty in rabbits. FIG.
19A depicts an image of the the model of surgical procedure and
silk implantation within rabbit urethra. FIG. 19B depicts
retrograph urethrograms demonstrating no reduction in urethral
caliber or stricture formation following 3 months of silk
implantation. Controls represent urethras which were surgically
incised, claosed, and maintained in parallel with silk implanted
animals. Results are representative of N=2 animals performed with
silk implants as well as controls. Arrows denote original
implantation site or sham injury.
DETAILED DESCRIPTION
[0037] Described herein are compositions relating to a multi-layer
biomaterial composition and methods relating thereto. As described
herein, the inventors have demonstrated that multi-layer
biomaterial compositions comprising at least a porous biomaterial
matrix layer and an impermeable biomaterial layer provide a
scaffold for tissue regeneration, elasticity properties that mimic
native tissues, a readily suturable substrate, and the ability to
effectively seal a wound or defect (e.g. prevent the passage of
fluids and/or cellular materials through the wound or defect). As
further demonstrated, these multilayer compositions provide
improvements in wound healing as compared to monolayer
compositions.
[0038] In one aspect, described herein is a composition comprising
a first and second layer; the first layer comprising a porous
biomaterial matrix; and the second layer comprising an impermeable
biomaterial.
[0039] As used herein, the term "matrix" refers to the physical
structure which contains the biomaterial. Non-limiting examples of
matrix structures include foams; hydrogels; electrospun fibers;
gels; fiber mats; sponges; 3-dimensional scaffolds; non-woven mats;
woven materials; knit materials; fiber bundles; and fibers and
other material formats (See, e.g. Rockwood et al. Nature Protocols
2011 6:1612-1631 and US Patent Publications 2011/0167602;
2011/0009960; 2012/0296352; and U.S. Pat. No. 8,172,901; each of
which is incorporated by reference herein in its entirety). The
structure of the matrix can be selected by one of skill in the art
depending upon the intended application of the composition, e.g.
electrospun matrices can have greater surface area than foams and
can thus be preferable in applications where, e.g. lung and/or
kidney tissue is to regenerated, as the greater surface area
encourages mass/gas exchange.
[0040] In some embodiments, the composition is a hydrogel. As used
herein, the term "hydrogel" refers to a three-dimensional polymeric
structure that is insoluble in water but which is capable of
absorbing and retaining large quantities of water to form a stable,
often soft and pliable, structure. In some embodiments, water can
penetrate in between the polymer chains of the polymer network,
subsequently causing swelling and the formation of a hydrogel. In
general, hydrogels are superabsorbent. Hydrogels have many
desirable properties for biomedical applications. For example, they
can be made nontoxic and compatible with tissue, and they are
highly permeable to water, ions, and small molecules. Hydrogels are
super-absorbent (they can contain over 99% water) and can be
comprised of natural (e.g., silk) or synthetic polymers, e.g.,
PEG.
[0041] In some embodiments, the impermeable layer can be in the
form of a film. Thickness of the film can range from nanometers to
millimeters. For example, film thickness can range from about 1 nm
to about 1000 mm. In some embodiments, the film thickness can be
from about 1 nm to 1000 nm, from about 1 .mu.m about 1000 .mu.m,
from about 1 mm to about 1000 mm. In some embodiments, the film
thickness can be from about 500 nm to about 750 .mu.m, from about
750 nm to about 500 .mu.m, from about 1000 nm to about 250 .mu.m,
from about 10 .mu.m to about 100 .mu.m, from about 25 .mu.m to
about 75 .mu.m. In some embodiments, film thickness ranges from
about 10 nm to about 1 mm.
[0042] In some embodiments, the porous matrix layer can be in the
form of a foam. Foams can be made from methods known in the art,
including, for example, freeze-drying and gas foaming in which
water is the solvent or nitrogen or other gas is the blowing agent,
respectively, or by, e.g. leaching with salt and/or water-soluble
particles.
[0043] The biomaterial matrix of the first layer is a porous
biomaterial matrix. i.e., the matrix has porosity. As used herein,
the term "porosity" means the fractional volume (dimension-less) of
the composition that is composed of open space, e.g., pores or
other openings. Thus, porosity measures void spaces in a material
and is a fraction of volume of voids over the total volume, as a
percentage between 0 and 100% (or between 0 and 1). See for
example, Coulson J. M., et. al., Chemical Engineering, 1978, volume
2, 3.sup.rd Edition, Pergamon Press, 1978, page 126). Determination
of matrix porosity is well known to a skilled artisan, e.g., using
standardized techniques, such as mercury porosimetry and gas
adsorption, e.g., nitrogen adsorption. Generally, porosity of the
composition can range from 0.5 to 0.99, from about 0.75 to about
0.99, or from about 0.8 to about 0.95. In some embodiments,
porosity of the composition can be at least 0.75. In some
embodiments, porosity of the composition can be at least 0.8. In
some embodiments, porosity of the composition can be at least
0.9.
[0044] The porous layer provides for, e.g. cell migration and
tissue regeneration. The pore size of the pourous layer can vary,
e.g. accordingly to the size and/or shape of the cells and/or
tissue which will be regenerated. As used herein, the term "pore
size" refers to a diameter or an effective diameter of the
cross-sections of the pores. The term "pore size" can also refer to
an average diameter or an average effective diameter of the
cross-sections of the pores, based on the measurements of a
plurality of pores. The effective diameter of a cross-section that
is not circular equals the diameter of a circular cross-section
that has the same cross-sectional area as that of the non-circular
cross-section. In some embodiments, a hydrogel can be swollen when
the hydrogel is hydrated. The sizes of the pores size can then
change depending on the water content in the hydrogel. The pores
can be filled with a fluid such as water or air. It will be
understood by one of ordinary skill in the art that pores can
exhibit a distribution of sizes around the indicated "size." Unless
otherwise stated, the term "size" as used herein refers to the mode
of a size distribution of pores, i.e., the value that occurs most
frequently in the size distribution.
[0045] The pores can be substantially round cross-section or
opening. What is meant by "substantially round" is that the ratio
of the lengths of the longest to the shortest perpendicular axes of
the pore cross-section is less than or equal to about 1.5.
Substantially round does not require a line of symmetry. In some
embodiments, the ratio of lengths between the longest and shortest
axes of the pore cross-section is less than or equal to about 1.5,
less than or equal to about 1.45, less than or equal to about 1.4,
less than or equal to about 1.35, less than or equal to about 1.30,
less than or equal to about 1.25, less than or equal to about 1.20,
less than or equal to about 1.15 less than or equal to about
1.1.
[0046] In some embodiments, the average pore size of the
biomaterial matrix can be at least 200 .mu.m. In some embodiments,
the average pore size of the biomaterial matrix can be at least 300
.mu.m. In some embodiments, the average pore size of the
biomaterial matrix can be at least 400 .mu.m. In some embodiments,
the average pore size of the biomaterial matrix can be from about
200 .mu.m to about 600 .mu.m. In some embodiments, the average pore
size of the biomaterial matrix can be from about 300 .mu.m to about
500 .mu.m. In some embodiments, the average pore size of the
biomaterial matrix can be about 400 .mu.m. In some embodiments, the
average pore size of the biomaterial matrix can be about 300 .mu.m.
The average pore size of the matrix can be the average size (e.g.
diameter) of all the pores in the matrix. In some embodiments, the
average pore size of the matrix can be the average pore size (e.g.
diameter) of all the primary pores in the matrix. In some
embodiments, the matrix can comprise primary and secondary pores
(see, e.g. FIG. 16; where a single primary pore 14 is contained
within the dashed line circle and a secondary pore 16 is
indicated). The primary pores are substantially larger in diameter
than the secondary pores and the secondary pores interconnected the
primary pores. In some embodiments, the primary pores are the
population of pores which have an average diameter at least
3.times. the average pore size of the remaining pores.
[0047] The second layer of the composition comprises an impermeable
biomaterial layer. As used herein, "impermeable" refers to material
being able to prevent the passage of an aqueous solution across the
material at a particular temperature, tension and fluid pressure.
In some embodiments, the impermeable layer can have no observable
pores. In some embodiments, the impermeable layer can have no
observable pores which transit the entire thickness of the
layer.
[0048] The compositions described herein can be elastic. For
example, the composition can have an extensibility of about 500%,
about 400%, 300%, 200%, 100%, 50%, or about 25%. In some
embodiments, the composition can have an elastic modulus in the
range about 10-2 kPa to about 103 kPa. As used herein, the term
"elastic modulus" refers to an object or substance's tendency to be
deformed elastically (i.e., non-permanently) when a force is
applied to it. Generally, the elastic modulus of an object is
defined as the slope of its stress-strain curve in the elastic
deformation region. Specifying how stress and strain are to be
measured, including directions, allows for many types of elastic
moduli to be defined. Young's modulus (E) describes tensile
elasticity, or the tendency of an object to deform along an axis
when opposing forces are applied along that axis; it is defined as
the ratio of tensile stress to tensile strain. It is often referred
to simply as the elastic modulus. The bulk modulus (K) describes
volumetric elasticity, or the tendency of an object to deform in
all directions when uniformly loaded in all directions; it is
defined as volumetric stress over volumetric strain, and is the
inverse of compressibility. The bulk modulus is an extension of
Young's modulus to three dimensions. Three other elastic moduli are
Poisson's ratio, Lame's first parameter, and P-wave modulus. In
some embodiments, the composition can have an elastic modulus in
the range from about 1 kPa to about 25 kPa. In some embodiments,
the composition can have an elastic module of about 5 kPa, about 10
kPa, about 15 kPa, or about 20 kPa. In some embodiments, the
composition can have an Young's modulus in the range of from about
15 kPa to about 35 kPa.
[0049] The first and second layers are arranged such that the
second layer coats and/or covers no more than 70% of the surface
area of the first layer, e.g. 70% or less, 60% or less, 50% or
less, 40% or less, or 30% or less of the surface area of the first
layer. In some embodiments, wherein the entire composition has a
shape approximating a cuboid, the first layer can coat and/or cover
one face of the cuboid. In some embodiments, wherein the entire
composition has a shape approximating a cuboid, the first layer can
coat and/or cover the entirety of one face of the cuboid. In some
embodiments, where the entire composition has a shape approximating
a sheet or plane, the first layer can coat and/or cover one face of
the sheet or plane. In some embodiments, where the entire
composition has a shape approximating a sheet or plane, the first
layer can coat and/or cover the entirety of one face of the sheet
or plane.
[0050] Each of the layers of the composition can comprise one or
more biomaterials. As used herein, "biomaterial" refers to a
material that is biocompatible and biodegradable. As used herein,
the term "biocompatible" refers to substances that are not toxic to
cells. In some embodiments, a substance is considered to be
"biocompatible" if its addition to cells in vitro results in less
than or equal to approximately 20% cell death. In some embodiments,
a substance is considered to be "biocompatible" if its addition to
cells in vivo does not induce inflammation and/or other adverse
effects in vivo. As used herein, the term "biodegradable" refers to
substances that are degraded under physiological conditions. In
some embodiments, a biodegradable substance is a substance that is
broken down by cellular machinery. In some embodiments, a
biodegradable substance is a substance that is broken down by
chemical processes.
[0051] Non-limiting examples of biomaterials include, silk fibroin;
PGA; collagen; fibronectin, keratin, polyaspartic acid, polylysin,
alginate, chitosan, chitin, hyaluronic acid, glycosaminoglycan,
silk, fibrin, MATRIGEL.RTM., alginic acid, pectinic acid, carboxy
methyl cellulose, hyaluronic acid, heparin, heparin sulfate,
carboxymethyl chitosan, pullulan, gellan, xanthan, gelatin,
carboxymethyl starch, carboxymethyl dextran, chondroitin sulfate,
cationic guar, cationic starch, esters of alginic, or pectinic and
the like. In some embodiments, a layer of the composition can
comprise one or more biomaterials, e.g. one biomaterial, two
biomaterials, three biomaterials, four biomaterials, or more
biomaterials. In some embodiments, the first and second layer can
comprise the same biomaterial and/or the same combination of
biomaterials. In some embodiments, the first and second layer can
comprise different biomaterials and/or different combinations of
biomaterials.
[0052] In some embodiments, either layer can comprise silk fibroin.
In some embodiments, both layers can comprise silk fibroin. In some
embodiments, either layer can consist of silk fibroin. In some
embodiments, both layers can consist of silk fibroin. In some
embodiments, the silk fibroin comprises Bombyx mori silk fibroin.
As used herein, the term "silk fibroin" includes silkworm fibroin
and insect or spider silk protein. See e.g., Lucas et al., 13 Adv.
Protein Chem. 107 (1958). Any type of silk fibroin can be used.
Silk fibroin produced by silkworms, such as Bombyx mori, is the
most common and represents an earth-friendly, renewable resource.
For instance, silk fibroin can be attained by extracting sericin
from the cocoons of B. mori. Organic silkworm cocoons are also
commercially available. There are many different silks, however,
including spider silk (e.g., obtained from Nephila clavipes),
transgenic silks, genetically engineered silks, such as silks from
bacteria, yeast, mammalian cells, transgenic animals, or transgenic
plants (see, e.g., WO 97/08315; U.S. Pat. No. 5,245,012), and
variants thereof, that can be used. An aqueous silk fibroin
solution can be prepared using techniques known in the art.
Suitable processes for preparing silk fibroin solution are
disclosed, for example, in U.S. patent application Ser. No.
11/247,358; WO/2005/012606; and WO/2008/127401. The silk fibroin
solution can be diluted to a lower concentration with deionized
water, or can be concentrated, for example, to about 30% (w/v), if
desired. Additionally, silk fibroin can be chemically modified with
active agents in the solution, for example through diazonium or
carbodiimide coupling reactions, avidin-biodin interaction, or gene
modification and the like, to alter the physical properties and
functionalities of the silk protein. See, e.g., PCT/US09/64673;
PCT/US10/41615; PCT/US10/42502; U.S. application Ser. No.
12/192,588. In some embodiments, silk fibroin used herein can be
depleted of sericin by any methods known in the art.
[0053] Alternatively, the silk fibroin solution can be produced
using organic solvents. Such methods have been described, for
example, in Li, M., et al., J. Appl. Poly Sci. 2001, 79, 2192-2199;
Min, S., et al. Sen'I Gakkaishi 1997, 54, 85-92; Nazarov, R. et
al., Biomacromolecules 2004 May-June; 5(3):718-26.
[0054] Various methods of producing silk matrix are known in the
art. In some embodiments, a silk hydrogel can be produced by
sonicating a silk solution containing silk or silk fibroin. See,
e.g., U.S. Pat. App. No. U.S. 2010/0178304 and International App.
No.: WO 2008/150861, the contents of which are incorporated herein
by reference in their entirety for methods of silk fibroin gelation
using sonication.
[0055] In alternative embodiments, the silk matrix can be produced
by applying a shear stress to a silk solution. See, e.g.,
International App. No.: WO 2011/005381, the content of which is
incorporated herein by reference in its entirety for methods of
producing vortex-induced silk fibroin gelation for encapsulation
and delivery.
[0056] In other embodiments, the silk hydrogel can be produced by
modulating the pH of a silk solution. The pH of the silk solution
can be altered by subjecting the silk solution to an electric field
and/or reducing the pH of the silk solution with an acid. See,
e.g., U.S. App. No.: US 2011/0171239, the content of which is
incorporated herein by reference in its entirety, for details on
methods of producing pH-induced silk gels.
[0057] Either and/or both layers of the compositions described
herein can further comprise microparticles and/or nanoparticles,
e.g. biomaterial microparticles and/or nanoparticles, optionally
comprising a therapeutic agent. By way of non-limiting example,
various methods of producing silk microparticles or nanoparticles
are known in the art. In some embodiments, the silk microparticles
or nanoparticles can be produced by a polyvinyl alcohol (PVA) phase
separation method as described in, e.g., International App. No. WO
2011/041395, the content of which is incorporated herein by
reference in their entirety for methods of silk fibroin gelation
using sonication. Other methods for producing silk microparticles
or nanoparticles, e.g., described in U.S. App. No. U.S.
2010/0028451 and International App. No.: WO 2008/118133 (using
lipid as a template for making silk microspheres or nanospheres),
and in Wenk et al. J Control Release 2008; 132: 26-34 (using
spraying method to produce silk microspheres or nanospheres) can be
used for the purpose of making silk microparticles or nanoparticles
encapsulating a therapeutic agent. In some embodiments, the silk
microparticles or nanoparticles can be further embedded in a
biopolymer, e.g., to prolong the release of a therapeutic agent
over a period of time. In some embodiments, the biopolymer can be a
silk hydrogel to encapsulate the therapeutic agent-loaded silk
microparticles or nanoparticles. See, e.g., International App. No.:
WO 2010/141133 for methods of producing silk fibroin scaffolds for
antibiotic delivery.
[0058] The layers of the present composition can further comprise
at least one agent, e.g. 1 agent, 2 agents, 3 agents, 4 agents, 5
agents, or more agents. As used herein, an "agent" refers to any
molecule or combination of molecules added to a composition during
construction which is either a) not present in the biomaterial used
to construct the composition. In some embodiments, the agent can be
a bioactive agent or bioactive material. As used herein, "bioactive
agents" or "bioactive materials" refer to naturally occurring
biological materials, for example, extracellular matrix materials
such as fibronectin, vitronection, and laminin; cytokins; and
growth factors and differentiation factors. "Bioactive agents" also
refer to artificially synthesized materials, molecules or compounds
that have a biological effect on a biological cell, tissue or
organ. The molecular weights of the bioactive agent can vary from
very low (e.g. small molecules, 200-500 Daltons) to very high (e.g.
plasmid DNA, .about.2,000,000 Daltons). In some embodiments, the
bioactive agent is a small molecule. As used herein, the term
"small molecule" can refer to compounds that are "natural
product-like," however, the term "small molecule" is not limited to
"natural product-like" compounds. Rather, a small molecule is
typically characterized in that it contains several carbon-carbon
bonds, and has a molecular weight of less than 5000 Daltons (5 kD).
In some embodiments, a small molecule can have a molecular weight
of less than 3 kD. In some embodiments, a small molecule can have a
molecular weight of less than 2 kD. In some embodiments, a small
molecule can have a molecular weight of less than 1 kD. In some
embodiments, a small molecule can have a molecular weight of less
than 700 D.
[0059] In some embodiments, bioactive agent is a therapeutic agent.
As used herein, the term "therapeutic agent" refers to a substance
used in the diagnosis, treatment, or prevention of a disease. Any
therapeutic agent known to those of ordinary skill in the art to be
of benefit in the diagnosis, treatment or prevention of a disease
is contemplated as a therapeutic agent in the context of the
present invention. Therapeutic agents include pharmaceutically
active compounds, hormones, growth factors, enzymes, DNA, plasmid
DNA, RNA, siRNA, viruses, proteins, lipids, pro-inflammatory
molecules, antibodies, antibiotics, anti-inflammatory agents,
anti-sense nucleotides and transforming nucleic acids or
combinations thereof. Any of the therapeutic agents may be combined
to the extent such combination is biologically compatible.
[0060] Exemplary therapeutic agents include, but are not limited
to, those found in Harrison's Principles of Internal Medicine,
13.sup.th Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY;
Physicians' Desk Reference, 50.sup.th Edition, 1997, Oradell New
Jersey, Medical Economics Co.; Pharmacological Basis of
Therapeutics, 8.sup.th Edition, Goodman and Gilman, 1990; United
States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990;
current edition of Goodman and Oilman's The Pharmacological Basis
of Therapeutics; and current edition of The Merck Index, the
complete contents of all of which are incorporated herein by
reference.
[0061] Non-limiting examples of agents can include an antibiotic;
an agent to attract cells; a cell; a stem cell; a ligand; a growth
factor; a platelet; an antinflammatory; a component of
extracellular matrix; enzymes, proteins, nucleic acids, antibodies,
antibiotics, hemostatic agents and the like, as described herein.
See, e.g., WO2004/062697 and WO2005/012606. The agent can represent
any material capable of being embedded in the biomaterials. For
example, the active agent can be a therapeutic agent, or a
biological material, such as peptides, nucleic acids (e.g., DNA,
RNA, siRNA), nucleic acid analogs, nucleotides, oligonucleotides,
peptide nucleic acids (PNA), aptamers, antibodies or fragments or
portions thereof (e.g., paratopes or complementarity-determining
regions), antibody-like molecules, antigens or epitopes, hormones,
hormone antagonists, growth factors or recombinant growth factors
and fragments and variants thereof, cell attachment mediators (such
as RGD), cytokines, small molecules, drugs, dyes, amino acids,
vitamins, antioxidants, antifungals, antivirals, prodrugs, or
combinations thereof. See, e.g., PCT/US09/44117; U.S. Patent
Application Ser. No. 61/224,618). The active agent can also be a
combination of any of the above-mentioned agents. A cell, e.g. a
platelet, can be autologous to the subject or obtained from a
donor.
[0062] Exemplary antibiotic agents include, but are not limited to,
actinomycin; aminoglycosides (e.g., neomycin, gentamicin,
tobramycin); .beta.-lactamase inhibitors (e.g., clavulanic acid,
sulbactam); glycopeptides (e.g., vancomycin, teicoplanin,
polymixin); ansamycins; bacitracin; carbacephem; carbapenems;
cephalosporins (e.g., cefazolin, cefaclor, cefditoren,
ceftobiprole, cefuroxime, cefotaxime, cefipeme, cefadroxil,
cefoxitin, cefprozil, cefdinir); gramicidin; isoniazid; linezolid;
macrolides (e.g., erythromycin, clarithromycin, azithromycin);
mupirocin; penicillins (e.g., amoxicillin, ampicillin, cloxacillin,
dicloxacillin, flucloxacillin, oxacillin, piperacillin); oxolinic
acid; polypeptides (e.g., bacitracin, polymyxin B); quinolones
(e.g., ciprofloxacin, nalidixic acid, enoxacin, gatifloxacin,
levaquin, ofloxacin, etc.); sulfonamides (e.g., sulfasalazine,
trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole),
sulfadiazine); tetracyclines (e.g., doxycyline, minocycline,
tetracycline, etc.); monobactams such as aztreonam;
chloramphenicol; lincomycin; clindamycin; ethambutol; mupirocin;
metronidazole; pefloxacin; pyrazinamide; thiamphenicol; rifampicin;
thiamphenicl; dapsone; clofazimine; quinupristin; metronidazole;
linezolid; isoniazid; piracil; novobiocin; trimethoprim;
fosfomycin; fusidic acid; or other topical antibiotics. Optionally,
the antibiotic agents can also be antimicrobial peptides such as
defensins, magainin and nisin; or lytic bacteriophage. The
antibiotic agents can also be the combinations of any of the agents
listed above. See also PCT/US2010/026190.
[0063] In some embodiments, the bioactive agent can be a growth
factor or a cytokine. Suitable growth factors and cytokines
include, but are not limited, to stem cell factor (SCF),
granulocyte-colony stimulating factor (G-CSF),
granulocyte-macrophage stimulating factor (GM-CSF), stromal
cell-derived factor-1, steel factor, VEGF, TGF.beta., platelet
derived growth factor (PDGF), angiopoeitins (Ang), epidermal growth
factor (EGF), bFGF, HNF, NGF, bone morphogenic protein (BMP),
fibroblast growth factor (FGF), hepatocye growth factor,
insulin-like growth factor (IGF-1), interleukin (IL)-3,
IL-1.alpha., IL-1.beta., IL-6, IL-7, IL-8, IL-11, and IL-13,
colony-stimulating factors, thrombopoietin, erythropoietin,
fit3-ligand, and tumor necrosis factor .alpha. (TNF.alpha.). Other
examples are described in Barrientos et al. Wound Repair Regen 2008
16:585-601; Peplow and Baxter Photomed Laser Surg 2012 30:617-36;
Demidova-Rice et al. Adv Skin Wound Care 2012 25:349-370; Kiwanuka
et al. Clin Plast Surg 2012 39:239-248; each of which is
incorporated by reference herein in its entirety.
[0064] The composition can further comprise hemostatic agents since
hemostatic agents typically act to stop bleeding and tissue sealant
can bind to and close defects in the tissues. Combining the
hemostatic agents into the biomaterial can therefore present
desirable features during surgical repair to prevent or stop
bleeding as well as promote tissue reconstruction. Exemplary
hemostatic agents suitable for use herein include, but are not
limited to, thrombin, fibrin, fibrinogen, gelatin, collagen,
polysaccharide, cellulose, blood factors, and combinations
thereof.
[0065] Additional materials can also be blended into the
biomaterial. Such materials include, but are not limited to,
polyaspartic acid, polylysine, alginate, polycaprolactone,
polylactic acid, polyglycolic acid, polyhydroxyalkanoates,
dextrans, polyanhydrides, PEO, PEG, glycerol (see
PCT/US2009/060135), and other biocompatible polymers, see WO
2004/0000915.
[0066] The biomaterial can be modified if desired. One of skill in
the art can select appropriate methods to modify biomaterials,
e.g., depending on the side groups of the biomaterial, desired
reactivity of the biomaterial and/or desired charge density on the
biomaterial. In one embodiment, modification of a biomaterial can
use the amino acid side chain chemistry, such as chemical
modifications through covalent bonding, or modifications through
charge-charge interaction. Exemplary chemical modification methods
include, but are not limited to, carbodiimide coupling reaction
(see, e.g. U.S. Patent Application. No. US 2007/0212730), diazonium
coupling reaction (see, e.g., U.S. Patent Application No. US
2009/0232963), avidin-biotin interaction (see, e.g., International
Application No.: WO 2011/011347) and pegylation with a chemically
active or activated derivatives of the PEG polymer (see, e.g.,
International Application No. WO 2010/057142). As a further
non-limiting example, biomaterials, e.g. silk fibroin can also be
modified through gene modification to alter functionalities of the
biomaterial (see, e.g., International Application No. WO
2011/006133). For instance, the silk fibroin can be genetically
modified, which can provide for further modification of the silk
such as the inclusion of a fusion polypeptide comprising a fibrous
protein domain and a mineralization domain, which can be used to
form an organic-inorganic composite. See WO 2006/076711.
Additionally, the biomaterial can be combined with a chemical, such
as glycerol, that, e.g., affects flexibility of the composition.
See, e.g., WO 2010/042798, Modified Silk films Containing
Glycerol.
[0067] In some embodiments, the biomaterial provides controlled
release of the delivery of the embedded active agents (e.g.,
therapeutic agents or biological materials). Controlled release
permits dosages to be administered over time, with controlled
release kinetics. In some instances, delivery of the therapeutic
agent or biological material is continuous to the site where
treatment is needed, for example, over several weeks. Controlled
release over time, for example, over several days or weeks, or
longer, permits continuous delivery of the therapeutic agent or
biological material to obtain preferred treatments. The controlled
delivery vehicle is advantageous because it protects the
therapeutic agent or biological material from degradation in vivo
in body fluids and tissue, for example, by proteases. See, e.g.,
PCT/US09/44117. Controlled release of the agent from the
biomaterial can be designed to occur over time, for example, for
greater than about 12 hours or 24 hours, inclusive; greater than 1
month or 2 months or 5 months, inclusive. The time of release can
be selected, for example, to occur over a time period of about 12
hours to 24 hours, or about 12 hours to 1 week. In another
embodiment, release can occur for example on the order of about 1
month to 2 months, inclusive. The controlled release time can be
selected based on the condition treated. For example, a particular
release profile can be more effective where consistent release and
high local dosage are desired.
[0068] In an alternative embodiment, the biomaterials can include
plasmonic nanoparticles to form photothermal elements. Thermal
therapy has been shown to aid in the delivery of various agents,
see Park et al., Effect of Heat on Skin Permeability, 359 Intl. J.
Pharm. 94 (2008). In one embodiment, short bursts of heat on very
limited areas can be used to maximize permeability with minimal
harmful effects on surrounding tissues. Thus, plasmonic
particle-doped biomaterial can add specificity to thermal therapy
by focusing light to locally generate heat only via the
biomaterials. In some embodiments, the biomaterials can include
photothermal agents such as gold nanoparticles.
[0069] When referring to the "thickness" of the composition
described herein, reference is being made the distance from one
surface of the compostion to a second surface of the composition as
measured along the shortest line which will cross both layers (see,
e.g. FIG. 14B). In some embodiments, the impermeable layer can be
from about 10 .mu.m to about 600 .mu.m thick. In some embodiments,
the impermeable layer can be from about 100 .mu.m to about 400
.mu.m thick. In some embodiments, the impermeable layer can be from
about 150 .mu.m to about 250 .mu.m thick. In some embodiments, the
impermeable layer can be from about 25 .mu.m to about 150 .mu.m
thick. In some embodiments, the impermeable layer can be about 200
.mu.m thick. In some embodiments, the composition can be from about
0.01 cm to about 5 cm thick. In some embodiments, the composition
can be from about 0.1 cm to about 3 cm thick.
[0070] In some embodiments, the composition can be from about 0.1
cm to about 2 cm thick. In some embodiments, the composition can be
about 1 cm thick.
[0071] The compositions described herein can be formed in any
desired shape and size. The shape of the composition can be, by way
of non-limiting example, a sheet; a tube; and a contoured sheet. In
some embodiments, the compositions can be formed in substantially
flat sheets. In some embodiments, the compositions can be formed in
countoured sheets, e.g. sheet which have at least one curve, arc,
or angle. In some embodiments, the contour of the sheet can be such
that it matches the desired contour of the tissue to be
regenerated. In some embodiments, the composition can be formed in
a tube or a portion of a tube, e.g. a sheet which is curved, along
one dimension, at least 45 degrees, e.g. 45 degrees or more, 90
degrees or more, 180 degrees or more, 200 degrees or more, 240
degrees or more, 300 degrees or more, or 360 degrees or more.
[0072] In some embodiments, the impermeable layer can extend beyond
the porous layer, e.g. if the porous layer forms a 2''.times.2''
cube, the impermeable layer can form a 3''.times.3'' sheet on one
face of the cube. This can provide a more complete seal around the
wound and/or tissue defect and/or provide a substrate for, e.g.
suturing.
[0073] In one aspect, described herein is a method of producing a
composition as described herein, the method comprising contacting a
porous biomaterial matrix with an impermeable biomaterial layer.
Protein-protein interactions between the two layers are often
sufficient to maintain the contact between the two layers,
particularly when the porous biomaterial matrix is formed while in
contact with the impermeable layer. In further embodiments, the
layers can be annealed using methods known in the art, e.g. water
annealing or exposing the layers to a methanol to reduce water
solubility (see, e.g. Hu et al. Biomacromolecules 2011
12:1686-1696; which is incorporated by reference herein in its
entirety). In further embodiments, the layers can comprise agents
which cause the layers to maintain contact, e.g. one layer can
comprise an antibody and the other layer can comprise the ligand
for that antibody.
[0074] In one aspect, described herein is a method of producing a
composition as described herein, the method comprising; (a) casting
an admixture of aqueous biomaterial solution and a salt or
water-soluble particle; (b) contacting the composition resulting
from step (a) with water. In some embodiments, the salt can be
NaCl.
[0075] Casting can comprise the solidification and/or gelation of
an aqueous biomaterial over time as it gradually dehydrates.
Methods for casting biomaterials, e.g. silk fibroin, are known in
the art and described herein. See, e.g. descriptions of the
generation of hydrogels (WO2005/012606; PCTUS08/65076;
PCT/US08/65076), ultrathin films (WO2007/016524), thick films,
conformal coatings (WO2005/000483; WO2005/123114), microspheres
(PCT/US2007/020789), 3D porous matrices (WO2004/062697),
combinations of the films, microspheres and porous matrices
(PCT/US09144117), solid blocks (WO2003/056297), and fibers with
diameters ranging from the nanoscale (WO2004/0000915) to several
centimeters (U.S. Pat. No. 6,902,932) have been explored with
implications in biomaterials and regenerative medicine
(WO2006/042287; U.S. patent application Ser. No. 11/407,373;
PCT/US08/55072). Each of the foregoing references is incorporated
by reference herein in its entirety.
[0076] The thickness of a material produced by casting can be
controlled, e.g. by altering the amount and concentration of the
biomaterial solution and the rate of drying or by physical
manipulation, e.g. cutting of the cast product. The shape and size
of a cast biomaterial can be controlled by the selection of a
casting vessel, e.g. the vessel can have substantially the shape
and/or size and/or contour that it is desired that the cast
material assume.
[0077] In some embodiments, the impermeable layer forms
spontaneously on the bottom of the composition. A schematic of an
exemplary embodiment of such a composition is depicted in FIG. 15A,
wherein the impermeable layer 10 is composed of the same
biomaterial (at substantially the same concentration) as the porous
matrix layer 12. In some embodiments, the spontaneously formed
impermeable layer can be comprised, substantially, of the same
biomaterial as the porous matrix. An exemplary embodiment of a
protocol for producing such compositions is set forth in Example
9.
[0078] In some embodiments, step (a), as described above herein,
can be performed with the solution in contact with a pre-existing
impermeable biomaterial layer. The pre-existing impermeable layer
can have been cast in the same casting mold, or cast in a different
mold, and/or obtained by other methods (e.g. electrospinning). The
pre-existing impermeable layer can be oriented in the casting mold
in any desired configuration and/or location, e.g. in the bottom or
on the side of the mold.
[0079] In some embodiments, the impermeable layer can comprise a
different biomaterial, mix of biomaterials (and, e.g. agents), or
different concentrations of biomaterial as the porous matrix layer.
A schematic of an exemplary embodiment of such a composition is
depicted in FIG. 15B, wherein the impermeable layer 10 is composed
of a different biomaterial as the porous matrix layer 12.
[0080] In some embodiments, the impermeable biomaterial layer can
comprise silk fibroin. In some embodiments, the method further can
comprise a first step of casting silk fibroin solution to form an
impermeable silk fibroin layer. In some embodiments, the silk
fibroin solution can have a concentration of from about 2% wt/vol
to about 15% wt/vol. In some embodiments, the silk fibroin solution
can have a concentration of from about 4% wt/vol to about 10%
wt/vol. In some embodiments, the silk fibroin solution can have a
concentration of about 8% wt/vol. Films, e.g. silk fibroin films
suitable for use as impermeable biolayers can be formed by casting
of purified silk fibroin solution which crystallizes upon exposure
to air, humidity or dry nitrogen gas, as some examples, without the
need for exogenous crosslinking reactions or post processing
crosslinking for stabilization. See, e.g., PCT/US07/83600;
PCT/US07/83620: PCT/US07/83605: each of which is incorporated by
reference herein in its entirety.
[0081] In some embodiments, the pores of the porous matrix are
formed by the salt or water-soluble particle via salt-leaching. In
some embodiments, the salt or water-soluble particle can be
granular. In some embodiments, the salt or water-soluble particle
can comprise crystals and/or grains having an average size of from
about 300 .mu.m to about 1000 .mu.m. In some embodiments, the salt
or water-soluble particle can comprise crystals and/or grains
having an average size of from about 400 .mu.m to about 800 .mu.m.
In some embodiments, the salt or water-soluble particle can
comprise crystals and/or grains having an average size of from
about 500 .mu.m to about 600 .mu.m.
[0082] In some embodiments, the biomaterial solution used to form
the biomaterial matrix can be a silk fibroin solution. In some
embodiments, the silk fibroin solution can have a concentration of
from about 2% wt/vol to about 15% wt/vol. In some embodiments, the
silk fibroin solution can have a concentration of from about 4%
wt/vol to about 10% wt/vol. In some embodiments, the silk fibroin
solution can have a concentration of about 6% wt/vol.
[0083] The admixture can be cast for until the desired thickness of
the porous layer is produced. The necessary time will vary, as
described elsewhere herein, and is readily determined and adjusted
by one of ordinary skill in the art. In some embodiments, the
admixture can be cast for from about 12 hours to about 96 hours. In
some embodiments, the admixture can be cast for from about 24 hours
to 72 hours. In some embodiments, the admixture can be cast for
about 48 hours.
[0084] Step (b) of the methods described above herein removes the
salt or water-soluble particle, leaving pores in the biomaterial
matrix. Step (b) can proceed, e.g. until the salt or water-soluble
particle has been substantially removed from the composition, e.g.
until less than 10% of the salt or water-soluble particle remains
and/or until 90% of the pores (e.g. primary pores) no longer
contain detectable grains and/or crystals of salt or water-soluble
particle. The amount of time necessary for step (b) can therefore
vary according with the thickness of the compositon, the volume of
water used, the temperature of the water, and/or whether the water
is caused to flow and/or be agitated, as is understood by one of
skill in the art. In some embodiments, step (b) can proceed for
from about 1 hour to about 2 weeks. In some embodiments, step (b)
can proceed for from about 12 hours to about 120 hours. In some
embodiments, step (b) can proceed for from about 24 hours to about
96 hours. In some embodiments, step (b) can proceed for about 72
hours. The water used in step (b) can be, e.g. distilled water,
deionized water, and/or sterile water. In some embodiments, the
water can be distilled water. In some embodiments, the water can
further comprise, e.g. a buffer, a preservative, and/or an agent.
In some embodiments, the water can be removed and replaced with a
fresh volume of water at least once during step (b). In some
embodiments, the water can be continually removed and replaced with
fresh volumes of water during step (b), e.g. a continuous flow of
water over, around, and/or through the composition can be
provided.
[0085] The compositions described herein can be used to increase
wound healing and/or tissue regeneration. Accordingly, the
compositions described herein can be provided in a sterile
condition prior to implantation in a subject. In some embodiments,
the composition can be produced under steril conditions. In some
embodiments, the composition can be sterilized after it is formed.
Any method of sterilization known in the art can be used, e.g.
autoclaving, gamma irradiation or e-beam sterilization.
[0086] In some embodiments, the composition can be produced in a
linear shape. In some embodiments, the composition can be produced
in a planar shape. In some embodiments, the composition can be
produced in a contoured shape. In some embodiments, the composition
can be produced in a tubular chape.
[0087] The compositions can be provided in a multitude of sizes and
shapes to suit particular applications, e.g. a size and shape
appropriate for treating a bladder defect, and/or a size and shape
appropriate for treating a bone defect. Alternatively, the
compositions described herein can be cut and/or formed by a medical
professional for individual applications, e.g. a 4''.times.4''
sheet of a composition as described herein can be cut to fit a 2''
diameter circular wound. In some embodiments, the porous layer can
also be reduced in thickness, e.g. by cutting or slicing, to
achieve the total desired thickness, e.g. a thickness which is
approximately the same as the tissue into which the composition is
to be implanted. The size and shape necessary for particular wound
healing and/or tissue regeneration applications can be readily
determined by one of skill in the art.
[0088] In some embodiments, the methods described herein can
comprise adding an agent to at least one layer. In some
embodiments, the agent can be a therapeutic agent. Non-limiting
examples of agents are described above herein.
[0089] In some embodiments, the matrix and/or impermeable layer
comprise silk fibroin. Silk fibroin can be modified after a matrix
and/or impermeable layer is formed and/or after an agent is added
to the layer. In some embodiments, after formation of the silk
matrix and/or impermeable layer, the method can further comprise
exposing the silk matrix and/or impermeable layer to a
post-treatment that will affect at least one silk fibroin property.
For example, post-treatment of a silk material can affect silk
fibroin properties including beta-sheet content, solubility, active
agent loading capacity, degradation time, active agent permeability
or any combinations thereof. In some embodiments, a layer
comprising silk fibroin can be caused to form beta-sheets, e.g.
after an agent has been added to the layer. Silk post-processing
options, e.g., to increase beta-sheet content, include, but not
limited to, controlled slow drying (Hu et al. Biomacromolecules
2011 12:1686-1696; Lu et al., 10 Biomacromolecules 1032 (2009)),
water annealing (Jin et al., Water-Stable Silk Films with Reduced
.beta.-Sheet Content, 15 Adv. Funct. Mats. 1241 (2005); and Hu et
al. Biomacromolecules 2011 12:1686-1696), stretching (Demura &
Asakura, Immobilization of glucose oxidase with Bombyx mori silk
fibroin by only stretching treatment and its application to glucose
sensor, 33 Biotech & Bioengin. 598 (1989)), compressing, and
solvent immersion, including methanol (Hofmann et al., 2006),
ethanol (Miyairi et al., 1978), glutaraldehyde (Acharya et al.,
2008) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)
(Bayraktar et al., 2005); pH adjustment (see, e.g., U.S. Patent
App. No. US2011/0171239, the content of which is incorporated
herein by reference), heat treatment, shear stress (see, e.g.,
International App. No.: WO 2011/005381, the content of which is
incorporated herein by reference), sonication (see, e.g., U.S. Pat.
App. No. U.S. 2010/0178304; PCT/US2010/036841; and International
App. No.: WO 2008/150861, the contents of which are incorporated
herein by reference), and any combinations thereof. See, e.g.,
WO/2004/062697; WO 2008/127404; Wang et al., 36 Intl. J. Biol.
Macromol. 66-70 (2005); and Kim et al., 5 Biomacromol. 786-92
(2004); Matsumoto et al., 110 J. Phys. Chem. B 21630-38 (2006).
Each of the foregoing references is incorporated by reference
herein in its entirety.
[0090] In one aspect, described herein is a method for wound
healing or repair of a tissue defect, the method comprising
applying a composition as described herein to the wound or tissue
defect. Non-limiting examples of wounds and/or tissue defects can
include a skin wound or defect; a diabetic ulcer; a bone wound or
defect; a joint wound or defect; a meniscus wound or defect; an
articular cartilage wound or defect; a soft tissue wound or defect;
a lung tissue wound or defect; a burn; a liver wound or defect; a
pancrease wound or defect; a gallbladder wound or defect; a kidney
wound or defect; a heart wall wound or defect; and/or a vascular
tissue wound or defect. In some embodiments, the wound or defect
can be the result of injury or disease (e.g. trauma or degenerative
disease). In some embodiments, the wound or defect can be
surgically created, e.g. an incision or surgical removal of excess,
damaged, or pathological tissue or material (e.g. removal of a
tumor).
[0091] In some embodiments, the compositions described herein can
be used to treat spinal wound or defects, e.g. intravertebral disc
wounds or defects. In some emboidments, the composition described
herein can be provided with in a flexible sheet or tube and
positioned to be positioned around the spinal column, e.g. with the
impermeable layer oriented next to the spine and the matrix layer
oriented on the outer surface of the composition when placed on the
spinal column. Without wishing to be bound by theory, the matrix
layer can provide load bearing properties whiel the impermeable
layer does not stick or or become tethered to the spinal
column.
[0092] Wounds to be treated include open or closed, or as either
acute or chronic in origin. In one embodiment, the compositions can
be used to treat an open wound. Open wounds include, but are not
limited to, incisions or incised wounds; lacerations or irregular
tear-like wounds caused by some blunt trauma; avulsion; abrasions
(grazes) such as superficial wounds in which the topmost layer of
the skin (the epidermis) is scraped off; puncture wounds such as
those caused by an object puncturing the skin; penetration wounds
such as those caused by an object entering and coming out from the
skin; and gunshot wounds. The wounds to be treated here can also
include closed wounds such as contusions, hematomas, crush injury,
chronic or acute wounds.
[0093] In some embodiments, the impermeable layer can be oriented
to prevent the movement of material into or out of the wound or
defect. By way of non-limiting example, the impermeable layer can
prevent fluids and/or contaminants from entering a wound (e.g.
preventing dirt from entering the skin or preventing fluids from
entering the lung), and/or the impermeable layer can prevent fluids
and/or materials from exiting a tissue via the wound or defect
(e.g. preventing fluids from leaking out of the bladder or
intestines via a defect or wound).
[0094] In some embodiments, the compositions can be placed on a
wound or defect. In some embodiments, the compositions can be
attached to the site of a wound or defect, e.g. by suturing,
stapling, and/or the use of adhesives. One of skill in the art can
readily determine how to secure a composition as described herein
to a particular wound and/or tissue defect.
[0095] The compositions described herein, by virtue of the
differing properties of the multiple layers, can provide a scaffold
for tissue regeneration while at the same time, sealing the
contents of a hollow organ in and/or preventing entry of fluids
and/or particles (e.g. debris) into the region the composition is
covering. Accordingly, in some embodiments, the wound or tissue
defect is located in a hollow organ. Non-limiting examples of
hollow organs include the bladder; a portion of the
gastrointestinal tract; the stomach, and the intestines.
[0096] In some embodiments, the composition can be applied to a
wound and/or defect in a bone. The bilayer compositions described
herein mimic the differences and orientation of the compact and
spony bone tissues, thereby promoting tissue regeneration of the
bone tissue.
[0097] In some embodiments, the compositions described herein can
be used to promote wound healing or repair of a tissue defect at a
load-bearing joint. The qualitatively different degrees of surface
roughness of the compositions described herein (e.g. a silk fibroin
film is smoother than a silk fibroin foam) can provide a smooth
surface for lubrication in joints while the rougher surface can
cushion impacts and provide for host tissue integration. The same
advantages are applicable to meniscus and articular cartilage
repair.
[0098] The invention also provides kits and device containing the
biomaterials and/or compositions described herein and instructions
to carry out any of the methods described herein.
[0099] For convenience, the meaning of some terms and phrases used
in the specification, examples, and appended claims, are provided
below. Unless stated otherwise, or implicit from context, the
following terms and phrases include the meanings provided below.
The definitions are provided to aid in describing particular
embodiments, and are not intended to limit the claimed invention,
because the scope of the invention is limited only by the claims.
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. If there
is an apparent discrepancy between the usage of a term in the art
and its definition provided herein, the definition provided within
the specification shall prevail.
[0100] For convenience, certain terms employed herein, in the
specification, examples and appended claims are collected here.
[0101] The terms "increased", "increase", or "enhance" are all used
herein to mean an increase by a statically significant amount. In
some embodiments, the terms "increased", "increase", or "enhance"
can mean an increase of at least 10% as compared to a reference
level, for example an increase of at least about 20%, or at least
about 30%, or at least about 40%, or at least about 50%, or at
least about 60%, or at least about 70%, or at least about 80%, or
at least about 90% or up to and including a 100% increase or any
increase between 10-100% as compared to a reference level, or at
least about a 2-fold, or at least about a 3-fold, or at least about
a 4-fold, or at least about a 5-fold or at least about a 10-fold
increase, or any increase between 2-fold and 10-fold or greater as
compared to a reference level. In the context of a marker or
symptom, a "increase" is a statistically significant increase in
such level.
[0102] As used herein, a "subject" means a human or animal. Usually
the animal is a vertebrate such as a primate, rodent, domestic
animal or game animal. Primates include chimpanzees, cynomologous
monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents
include mice, rats, woodchucks, ferrets, rabbits and hamsters.
Domestic and game animals include cows, horses, pigs, deer, bison,
buffalo, feline species, e.g., domestic cat, canine species, e.g.,
dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and
fish, e.g., trout, catfish and salmon. In some embodiments, the
subject is a mammal, e.g., a primate, e.g., a human. The terms,
"individual," "patient" and "subject" are used interchangeably
herein.
[0103] Preferably, the subject is a mammal. The mammal can be a
human, non-human primate, mouse, rat, dog, cat, horse, or cow, but
is not limited to these examples. Mammals other than humans can be
advantageously used as subjects that represent animal models of
wound healing. A subject can be male or female.
[0104] A "subject in need" of treatment for a particular condition
can be a subject having that condition, diagnosed as having that
condition, or at risk of developing that condition.
[0105] As used herein, the terms "treat," "treatment," "treating,"
or "amelioration" refer to therapeutic treatments, wherein the
object is to reverse, alleviate, ameliorate, inhibit, slow down or
stop the progression or severity of a condition associated with a
disease or disorder, e.g. a wound or tissue defect. The term
"treating" includes reducing or alleviating at least one adverse
effect or symptom of a condition, disease or disorder associated
with a wound or tissue defect. Treatment is generally "effective"
if one or more symptoms or clinical markers are reduced.
Alternatively, treatment is "effective" if the progression of a
disease is reduced or halted. That is, "treatment" includes not
just the improvement of symptoms or markers, but also a cessation
of, or at least slowing of, progress or worsening of symptoms
compared to what would be expected in the absence of treatment.
Beneficial or desired clinical results include, but are not limited
to, alleviation of one or more symptom(s), diminishment of extent
of disease, stabilized (i.e., not worsening) state of disease,
delay or slowing of disease progression, amelioration or palliation
of the disease state, remission (whether partial or total), and/or
decreased mortality, whether detectable or undetectable. The term
"treatment" of a disease also includes providing relief from the
symptoms or side-effects of the disease (including palliative
treatment).
[0106] The term "statistically significant" or "significantly"
refers to statistical significance and generally means a two
standard deviation (2SD) or greater difference.
[0107] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages can mean.+-.1%.
[0108] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the method or composition, yet open
to the inclusion of unspecified elements, whether essential or
not.
[0109] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0110] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of elements that do not materially affect the basic
and novel or functional characteristic(s) of that embodiment.
[0111] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. Although methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of this disclosure, suitable methods and materials are
described below. The abbreviation, "e.g." is derived from the Latin
exempli gratia, and is used herein to indicate a non-limiting
example. Thus, the abbreviation "e.g." is synonymous with the term
"for example."
[0112] Definitions of common terms in cell biology and molecular
biology can be found in "The Merck Manual of Diagnosis and
Therapy", 19th Edition, published by Merck Research Laboratories,
2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell Science
Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published
by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321);
Kendrew et al. (eds.), Molecular Biology and Biotechnology: a
Comprehensive Desk Reference, published by VCH Publishers, Inc.,
1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences
2009, Wiley Intersciences, Coligan et al., eds.
[0113] Unless otherwise stated, the present invention was performed
using standard procedures, as described, for example in Sambrook et
al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001);
Davis et al., Basic Methods in Molecular Biology, Elsevier Science
Publishing, Inc., New York, USA (1995); Current Protocols in
Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley
and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic
Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition
(2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol.
57, Jennie P. Mather and David Barnes editors, Academic Press, 1st
edition, 1998) which are all incorporated by reference herein in
their entireties.
[0114] Other terms are defined herein within the description of the
various aspects of the invention.
[0115] All patents and other publications; including literature
references, issued patents, published patent applications, and
co-pending patent applications; cited throughout this application
are expressly incorporated herein by reference for the purpose of
describing and disclosing, for example, the methodologies described
in such publications that might be used in connection with the
technology described herein. These publications are provided solely
for their disclosure prior to the filing date of the present
application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention or for any other reason.
All statements as to the date or representation as to the contents
of these documents is based on the information available to the
applicants and does not constitute any admission as to the
correctness of the dates or contents of these documents.
[0116] The description of embodiments of the disclosure is not
intended to be exhaustive or to limit the disclosure to the precise
form disclosed. While specific embodiments of, and examples for,
the disclosure are described herein for illustrative purposes,
various equivalent modifications are possible within the scope of
the disclosure, as those skilled in the relevant art will
recognize. For example, while method steps or functions are
presented in a given order, alternative embodiments may perform
functions in a different order, or functions may be performed
substantially concurrently. The teachings of the disclosure
provided herein can be applied to other procedures or methods as
appropriate. The various embodiments described herein can be
combined to provide further embodiments. Aspects of the disclosure
can be modified, if necessary, to employ the compositions,
functions and concepts of the above references and application to
provide yet further embodiments of the disclosure. Moreover, due to
biological functional equivalency considerations, some changes can
be made in protein structure without affecting the biological or
chemical action in kind or amount. These and other changes can be
made to the disclosure in light of the detailed description. All
such modifications are intended to be included within the scope of
the appended claims.
[0117] Specific elements of any of the foregoing embodiments can be
combined or substituted for elements in other embodiments.
Furthermore, while advantages associated with certain embodiments
of the disclosure have been described in the context of these
embodiments, other embodiments may also exhibit such advantages,
and not all embodiments need necessarily exhibit such advantages to
fall within the scope of the disclosure.
[0118] The technology described herein is further illustrated by
the following examples which in no way should be construed as being
further limiting.
[0119] Some embodiments of the technology described herein can be
defined according to any of the following numbered paragraphs:
[0120] 1. A composition comprising a first and second layer; [0121]
the first layer comprising a porous biomaterial matrix; and [0122]
the second layer comprising a impermeable biomaterial. [0123] 2.
The composition of paragraph 1, wherein the matrix is a structure
selected from the group consisting of: [0124] foams; hydrogels;
electrospun fibers; gels; fiber mats; sponges; 3-dimensional
scaffolds; non-woven mats; woven materials; knit materials; fiber
bundles; [0125] and fibers. [0126] 3. The composition of any of
paragraphs 1-2, wherein the biomaterial is selected from the group
consisting of: [0127] silk fibroin; PGA; collagen; polyethylene
oxide, collagen, fibronectin, keratin, polyaspartic acid,
polylysin, alginate, chitosan, chitin, and hyaluronic acid. [0128]
4. The composition of any of paragraphs 1-3, wherein the silk
fibroin comprises Bombyx mori silk fibroin. [0129] 5. The
composition of any of paragraphs 1-4, wherein the first and second
layers comprise the same biomaterial. [0130] 6. The composition of
any of paragraphs 1-4, wherein the first and second layers comprise
different biomaterials. [0131] 7. A composition comprising a first
and second layer; [0132] the first layer comprising a porous silk
fibroin matrix; and [0133] the second layer comprising a
impermeable silk fibroin film. [0134] 8. The composition of any of
paragraphs 1-7, further comprising an agent. [0135] 9. The
composition of paragraph 8, wherein the agent is selected from the
group consisting of: [0136] an antibiotic; an agent to attract
cells; a cell; a stem cell; a ligand; a growth factor; a platelet;
and a component of extracellular matrix. [0137] 10. The composition
of any of paragraphs 1-9, wherein the average pore size of the
biomaterial matrix is at least 200 .mu.m. [0138] 11. The
composition of any of paragraphs 1-10, wherein the average pore
size of the biomaterial matrix is at least 300 .mu.m. [0139] 12.
The composition of any of paragraphs 1-11, wherein the average pore
size of the biomaterial matrix is at least 400 .mu.m. [0140] 13.
The composition of any of paragraphs 1-12, wherein the average pore
size of the biomaterial matrix is from about 200 .mu.m to about 600
.mu.m. [0141] 14. The composition of any of paragraphs 1-13,
wherein the average pore size of the biomaterial matrix is from
about 300 .mu.m to about 500 .mu.m. [0142] 15. The composition of
any of paragraphs 1-14, wherein the average pore size of the
biomaterial matrix is about 400 .mu.m. [0143] 16. The composition
of any of paragraphs 1-15, wherein the average pore size of the
biomaterial matrix is about 300 .mu.m. [0144] 17. The composition
of any of paragraphs 1-16, wherein the impermeable layer is from
about 10 .mu.m to about 600 .mu.m thick. [0145] 18. The composition
of any of paragraphs 1-17, wherein the impermeable layer is from
about 100 .mu.m to about 400 .mu.m thick. [0146] 19. The
composition of any of paragraphs 1-18, wherein the impermeable
layer is from about 150 .mu.m to about 250 .mu.m thick. [0147] 20.
The composition of any of paragraphs 1-19, wherein the impermeable
layer is from about 25 .mu.m to about 150 .mu.m thick. [0148] 21.
The composition of any of paragraphs 1-20, wherein the impermeable
layer is about 200 .mu.m thick. [0149] 22. The composition of any
of paragraphs 1-21, wherein the composition is from about 0.01 cm
to about 5 cm thick. [0150] 23. The composition of any of
paragraphs 1-22, wherein the composition is from about 0.1 cm to
about 3 cm thick. [0151] 24. The composition of any of paragraphs
1-23, wherein the composition is from about 0.1 cm to about 2 cm
thick. [0152] 25. The composition of any of paragraphs 1-24,
wherein the composition is about 1 cm thick. [0153] 26. The
composition of any of paragraphs 1-25, wherein the composition has
a shape selected from the group consisting of: [0154] a sheet; a
tube; and a contoured sheet. [0155] 27. A method of producing a
composition of any of paragraphs 1-26, the method comprising;
[0156] contacting a porous biomaterial matrix with a impermeable
biomaterial layer. [0157] 28. A method of producing a composition
of any of paragraphs 1-26, the method comprising; [0158] (a)
casting an admixture of aqueous biomaterial solution and NaCl;
[0159] (b) contacting the composition resulting from step (a) with
water. [0160] 29. The method of paragraph 28, wherein step (a) is
performed with the solution in contact with a pre-existing
impermeable biomaterial layer. [0161] 30. The method of any of
paragraphs 28-29, wherein the NaCl is granular. [0162] 31. The
method of any of paragraphs 28-30, wherein the biomaterial solution
is a silk fibroin solution. [0163] 32. The method of any of
paragraphs 28-31, wherein the silk fibroin solution has a
concentration of from about 2% wt/vol to about 15% wt/vol. [0164]
33. The method of any of paragraphs 28-32, wherein the silk fibroin
solution has a concentration of from about 4% wt/vol to about 10%
wt/vol. [0165] 34. The method of any of paragraphs 28-33, wherein
the silk fibroin solution has a concentration of about 6% wt/vol.
[0166] 35. The method of any of paragraphs 28-34, wherein the
admixture is cast for from about 12 hours to about 96 hours. [0167]
36. The method of any of paragraphs 28-35, wherein the admixture is
cast for from about 24 hours to 72 hours. [0168] 37. The method of
any of paragraphs 28-36, wherein the admixture is cast for about 48
hours. [0169] 38. The method of any of paragraphs 28-37, wherein
step (b) proceeds for from about 12 hours to about 120 hours.
[0170] 39. The method of any of paragraphs 28-38, wherein step (b)
proceeds for from about 24 hours to about 96 hours. [0171] 40. The
method of any of paragraphs 28-39, wherein step (b) proceeds for
about 72 hours. [0172] 41. The method of any of paragraphs 28-40,
wherein the water is distilled water. [0173] 42. The method of any
of paragraphs 28-41, wherein the water is removed and replaced with
a fresh volume of water at least once during step (b). [0174] 43.
The method of any of paragraphs 28-42, wherein, during the casting
step, the admixture of biomaterial solution and NaCl is in contact
with an impermeable layer. [0175] 44. The method of any of
paragraphs 27-43, wherein the impermeable biomaterial layer
comprises silk fibroin. [0176] 45. The method of any of paragraphs
27-44, wherein the method further comprises a first step of casting
silk fibroin solution to form an impermeable silk fibroin layer.
[0177] 46. The method of paragraph 45, wherein the silk fibroin
solution has a concentration of from about 2% wt/vol to about 15%
wt/vol. [0178] 47. The method of paragraph 45, wherein the silk
fibroin solution has a concentration of from about 4% wt/vol to
about 10% wt/vol. [0179] 48. The method of paragraph 45, wherein
the silk fibroin solution has a concentration of about 8% wt/vol.
[0180] 49. The method of any of paragraphs 27-48, wherein the
composition is produced under sterile conditions or sterilized
after the rinsing step is complete. [0181] 50. The method of any of
paragraphs 27-49, wherein the composition is produced in a linear
shape. [0182] 51. The method of any of paragraphs 27-50, wherein
the composition is produced in a tubular shape. [0183] 52. The
method of any of paragraphs 27-51, wherein the method further
comprises adding an agent to at least one layer. [0184] 53. The
method of paragraph 52, wherein the agent is a therapeutic agent.
[0185] 54. The method of any of paragraphs 27-53, wherein the
method further comprises altering the .beta.-sheet content of a
silk fibroin layer. [0186] 55. The method of paragraph 54, wherein
the .beta.-sheet content of a silk fibroin layer is altered using a
method selected from the group consisting of: [0187] contacting the
layer with water vapor; drying; dehydration; water annealing;
[0188] stretching; compression; solvent immersion; immersion in
methanol; [0189] immersion in ethanol; pH adjustment; heat
treatment; and sonication. [0190] 56. The method of any of
paragraphs 54-55, wherein the .beta.-sheet content of a silk
fibroin layer is altered after adding an agent to the layer. [0191]
57. A method for wound healing or repair of a tissue defect, the
method comprising applying a composition of any of paragraphs 1-26
to the wound or tissue defect. [0192] 58. The method of paragraph
57, wherein the wound or tissue defect is located in a hollow
organ. [0193] 59. The method of paragraph 58, wherein the hollow
organ is selected from the group consisting of: [0194] the bladder;
a portion of the gastrointestinal tract; the stomach, and the
intestines. [0195] 60. The method of paragraph 57, wherein the
wound or tissue defect is selected from the group consisting of:
[0196] a skin wound or defect; a diabetic ulcer; a bone wound or
defect; a joint wound or defect; a meniscus wound or defect; an
articular cartilage wound or defect; a soft tissue wound or defect;
a lung tissue wound or defect; and a kidney wound or defect. [0197]
61. The method of any of paragraphs 57-60, wherein the impermeable
layer is oriented to prevent the movement of material into or out
of the wound or defect.
EXAMPLES
Example 1: Bi-Layer Silk Sheet for Hollow Organ Repair
[0198] Described herein is a 3-D biomaterial composed of silk
fibroin derived from Bombyx mori silkworm cocoons. This scaffold is
composed of a bi-layer structure consisting of one porous layer
buttressed by a thin non-permeable, barrier layer. This scaffold is
intended for use, for example, in the repair of hollow organ
defects such as the urinary bladder in procedures such as
augmentation cystoplasty. The composition's bi-layer structure
permits, e.g. retention of hollow organ contents (i.e. urine)
following initial implantation into defect sites while the porous
layer allows for host tissue ingrowth during defect
consolidation.
[0199] Preparation of silk solutions: B. mori silkworm cocoons were
boiled for 20 min in an aqueous solution of 0.02M Na2CO3 and then
rinsed thoroughly with distilled water to extract the glue-like
sericin proteins and wax. The extracted silk fibroin was then
dissolved in 9.3M LiBr solution at 60 C for 6 h. This solution was
dialyzed in distilled water using a Slide-a-lyzer dialysis cassette
(MWCO, 3500) for 4 d yielding an 8% (wt/vol) aqueous silk fibroin
solution (See e.g., Kim et al., 2004; which is incorporated by
reference herein in its entirety). Two methods of casting the
resultant silk matrix are described below.
[0200] Preparation of Matrix:
[0201] Method 1: A variant of a previously published solvent
casting/salt leaching technique was used (Kim et al., 2004). 75 ml
of an aqueous silk fibroin solution (6% wt/vol) was poured into a
rectangular casting vessel (12 cm.times.10 cm) and granular NaCl
(150 g, 500-600 .mu.M average crystal size) was mixed with the silk
solution. The resultant solution was allowed to cast for 2 d at
room temperature and then NaCl was removed by washing in distilled
water for 2 d. Spontaneous formation of the bi-layer scaffold was
achieved. Scanning electron microscopy and tensile testing was used
to determine structural and mechanical properties (FIG. 1).
[0202] Method 2: 20 ml of an aqueous silk fibroin solution (8%
wt/vol) was poured into a rectangular casting vessel (12
cm.times.10 cm) and dried in a laminar flow hood to achieve
formation of a silk film. Next as described above, 75 ml of an
aqueous silk fibroin solution (6% wt/vol) was poured into the
rectangular casting vessel (12 cm.times.10 cm) and granular NaCl
(150 g, 500-600 .mu.M average crystal size) was mixed with the silk
solution on top of the silk film. The resultant solution was
allowed to cast for 2 d at room temperature and then NaCl was
removed by washing in distilled water for 2 d. Fusion of the silk
film and bulk matrix occurred to generate bi-layer scaffold.
Scanning electron microscopy and tensile testing was used to
determine structural and mechanical properties (FIG. 2).
[0203] Surgical Evaluations: Robotic augmentation cystoplasty
procedure used to validate the performance of silk matrix produced
in Method 1. Scaffold was implantation for 3 months in which animal
was catheterized for 1 week post-op and then allowed to
spontaneously void. The structure of augmented porcine bladder
structure and kidneys following 3 months of silk matrix
implantation was examined. No evidence of hydronephrosis was
observed. Histological and immunohistochemical analyses of
regenerated bladder tissue supported by silk matrix following 3
months of implantation were performed, comparing the regenerated
tissue to non augmented regions of bladder tissue. Regenerated
tissue displayed morphology mimicking that of the non-augmented
regions, as well as displaying .alpha.-actin, uroplakin, p63,
cytokeratin, and SM22.alpha. expression, both at the periphery and
center of the regenerated tissue (data not shown). Furthermore, the
silk matrix was observed to degrade over the course of implantation
(data not shown and FIG. 3). FIG. 4 depicts urodynamic assessment
of silk augmented bladder at 3 months of implantation in comparison
to non augmented controls.
Example 2: The Effect of Matrix Processing Techniques on the
Performance of Silk Scaffolds in a Rat Model of Augmentation
Cystoplasty
[0204] Congenital and acquired urinary tract pathologies such as
neurogenic bladder, bladder exstrophy, and posterior urethral
valves routinely require enterocystoplasty in order to reduce
urinary storage and voiding pressures and mitigate the risk of
renal damage and incontinence. Although the use of autologous
gastrointestinal segments represents the gold standard of care for
bladder reconstruction, this strategy is associated with
significant complications including chronic urinary tract
infection, metabolic abnormalities, and secondary malignancies.
Biomaterials including acellular bladder matrix, small intestinal
submucosa (SIS), and poly-glycolic acid (PGA), either alone or
seeded with primary or progenitor bladder cell sources, have been
previously explored as alternatives for bladder defect repair in a
variety of animal models as well as short-term clinical studies.
Despite the ability of these matrices to support bladder tissue
regeneration, restoration of organ function is often hindered by
suboptimal scaffold properties which frequently lead to long-term
graft failure due to implant contracture, graft rupture, and/or
fibrosis. Therefore, there is a major clinical need to develop
novel scaffold configurations which can overcome these limitations
and thus serve as viable options for bladder tissue
engineering.
[0205] Silk fibroin-based biomaterials encompass a unique array of
properties including high structural strength and elasticity,
diverse processing plasticity, and tunable biodegradability which
make them well suited for the consolidation of hollow organ
defects. As described herein, the performance of silk biomaterials
was evaluated in a rat model of augmentation cystoplasty. The
ability of various 3-D scaffold configurations produced from
different processing techniques, including gel spinning or solvent
casting/salt leaching, were compared for their potential to support
bladder tissue repair and functional voiding responses. It was
hypothesized that NaCl-leaching of aqueous-based silk fibroin
solutions may offer particular advantages over gel spinning for the
construction of biomaterials for bladder tissue engineering.
[0206] By manipulating the NaCl crystal diameter, the solvent
casting/salt leaching method allows for greater scalability of
scaffold pore size (470-940 .mu.m) [Kim et al., 2005] in relation
to lyophilization of gel spun matrices which routinely generates
pore diameters below 200 .mu.m. In addition, lower concentrations
of silk fibroin solutions are utilized for the formation of
NaCl-leached scaffolds (<10% wt/vol) in comparison to the gel
spun spinning technique which requires solutions containing 20-30%
wt/vol for scaffold fabrication. A systematic analysis of the
impact of processing methods on the ability of silk scaffolds to
support bladder tissue regeneration is a crucial step in the
development of biomaterial configurations for clinical organ
repair.
[0207] Materials and Methods
[0208] Biomaterials. Aqueous silk fibroin solutions were prepared
from Bombyx mori silkworm cocoons using previously described
procedures [Kim et al., 2005]. Three distinct groups of silk-based
matrices were fabricated from these solutions by either a gel
spinning technique [Lovett et al., 2008] or a
solvent-casting/salt-leaching method [Kim et al., 2005]. Gel spun
scaffolds were produced by spinning concentrated silk solutions
[25-30% (wt/vol), 0.5 ml/scaffold] onto a rotating (200 rpm) and
axially reciprocating mandrel (6 mm in diameter) using a custom gel
spinning platform and program. Two groups of matrices previously
shown to support murine bladder augmentation, but with different
structural and mechanical properties, were generated with various
winding and post-winding conditions using this method [Mauney et
al., 2011; Gomez et al., 2011]. GS1 matrices were spun with an
axial slew rate (ASR) of 2 mm/sec followed by treatment with
methanol. GS2 scaffolds were composed of .about.0.4 ml of silk
solution spun at an ASR of 40 mm/sec followed by .about.0.1 ml spun
at 2 mm/sec in order to consolidate gaps between the resultant silk
fibers. This matrix group was then subjected to lyophilization and
subsequent methanol treatment. The FF scaffold group was composed
of 3-D porous silk foams which were annealed to silk films on their
top external face. Briefly, a silk fibroin solution (8% wt/vol) was
poured into a rectangular casting vessel and dried in a laminar
flow hood for 48 h to achieve formation of a silk film. A 6% wt/vol
silk fibroin solution was then mixed with sieved granular NaCl
(500-600 .mu.M average crystal size) and layered on to the surface
of the silk film. The resultant solution was allowed to cast for 48
h and NaCl was subsequently removed by washing the scaffold for 72
h in distilled water with regular volume changes. Fusion of the
silk film with the bulk foam matrix occurred to generate a bi-layer
scaffold configuration. Silk matrix groups were then sterilized in
70% ethanol, rinsed in phosphate buffered saline (PBS) overnight,
and subjected to analytical or surgical procedures described below.
Small intestinal submucosa (SIS) (Cook, Bloomington, Ind.)
scaffolds were evaluated in parallel as standard points of
comparison since this biomaterial has been previously deployed in
bladder augmentation approaches in both animal and human models
[Kropp et al., 1998; Caione et al., 2012].
[0209] Scanning electron microscopy (SEM). Structural analysis of
matrix groups was performed in order to assess differences in
scaffold morphology generated by various fabrication techniques.
Matrix samples were sputter coated with gold and imaged using a
Hitachi S-520 Scanning Electron Microscope.
[0210] Mechanical testing. Uniaxial tensile tests were performed as
previously described [Gomez et al., 2011] on an Instron 3366
testing frame (Norwood, Mass.) equipped with a 100 N capacity load
cell and Biopuls pneumatic clamps. Matrix groups (N=3-4 per group)
were hydrated in PBS for at least 24 h to reach a swelling
equilibrium prior to testing. Test samples were submerged in a
temperature-controlled testing container (Biopuls) filled with PBS
(37.degree. C.). A displacement control mode with a crosshead
displacement rate of 5 mm/min was used, and the gauge length was 15
mm. The initial elastic modulus (EM), ultimate tensile strength
(UTS) and % elongation to failure were calculated from
stress/strain plots. EM was calculated by using a least-squares
(LS) fitting between 0.02 N load and 5% strain past this initial
load point. UTS was determined as the highest stress value attained
during the test and the % elongation to failure was the last data
point before a >10% decrease in the load.
[0211] Rat bladder augmentation. Biomaterial groups were evaluated
in a bladder augmentation model using adult female immunocompetent
Sprague Dawley rats (6 weeks old, Charles River Laboratories,
Wilmington, Mass.) following IACUC approved protocols as previously
described [Tu et al., 2012]. Briefly, animals were anesthetized
using isoflurane inhalation and then shaved to expose the surgical
site. A low midline laparotomy incision was then made and the
underlying tissue (rectus muscle and peritoneum) was dissected free
to expose the bladder. The anterior portion (immediately distal to
the dome) of the bladder was marked with 7-0 polypropylene
(Prolene) sutures in a square configuration. A longitudinal
cystotomy incision was then made in the anterior bladder wall in
the middle of these holding sutures using fine scissors to create a
bladder defect. A square piece of biomaterial (7.times.7 mm.sup.2)
was then anastamosed to this site using a 7-0 vicryl continuous
suture. In addition, a control group of animals receiving a
cystotomy alone were treated similarly. A watertight seal was
confirmed by filling the bladder with sterile saline via
instillation through a 30 gauge hypodermic needle. Matrix and
control groups were assessed independently for 10 weeks of
implantation with animals subsequently subjected to cystometric,
histological and immunohistochemical analyses described below.
[0212] Cystometric analyses. Bladder urodynamics were evaluated in
all rodents using conscious unrestrained cystometry at 10 weeks
post implantation as previously described [Tu et al., 2012]. A
suprapubic catheter was surgically inserted in the bladder prior to
this. After induction with isoflurane anesthesia, the animals were
prepped and draped in a sterile fashion. A dorsal midline incision
was made in between the scapulae of the rats. A laparotomy was
created using a ventral midline incision. Polyethylene-50 tubing
with a flared tip was tunneled from the dorsal incision into the
peritoneal cavity. A purse-string 6-0 prolene stitch was used to
secure the flared tip of the polyethylene-50 tubing into the dome
of the bladder. The exteriorized polyethylene-50 tubing on the
dorsal aspect was attached to a luer lock adapter and secured to
the skin with a 3-0 silk suture. Cystometry was conducted 1-3 d
after suprapubic catheter placement. The suprapubic catheter was
attached to a physiological pressure transducer (model MLT844,
ADInstruments, Colorado Springs, CO) to allow measurement of
intravesical pressure, while the bladder was continuously infused
with sterile PBS at 100 .mu.l/min. Post void residual volume was
measured by aspirating the suprapubic catheter at the conclusion of
cystometry. After establishment of a regular voiding pattern,
multiple other variables were extrapolated from the cystometric
tracings, such as compliance, voided volume, peak voiding pressure,
intercontraction interval and spontaneous non voiding contractions
(SNVC). A total of 6 animals per group with 4 voids per animal were
analyzed to determine urodynamic parameters.
[0213] Histological and immunohistochemical analyses. Following 10
weeks of implantation, animals were euthanized by CO.sub.2
asphyxiation and bladders were excised for standard histological
processing. Briefly, organs were fixed in 10% neutral-buffered
formalin, dehydrated in graded alcohols, and then embedded in
paraffin in an axial orientation to capture the entire
circumferential surface of the bladder within each section. Correct
orientation (anterior vs posterior) within the paraffin block was
determined by suture placement on the specimen. Sections (10 .mu.m)
were cut and then stained with hematoxylin and eosin (H&E) or
Masson's trichrome (MTS) as previously described [Gomez et al.,
2011]. For immunohistochemical (IHC) analysis, contractile smooth
muscle markers such as .alpha.-smooth muscle actin (.alpha.-SMA)
and SM22a; urothelial-associated proteins, uroplakins (UP) and p63;
neuronal and endothelial markers, Fox3 and CD31, respectively were
detected using the following primary antibodies: anti-.alpha.-SMA
[Sigma-Aldrich, St. Louis, Mo., cat. #A2457, 1:200 dilution],
anti-SM22.alpha. [Abcam, Cambridge, Mass., cat. #ab14106, 1:200
dilution], anti-pan-UP [rabbit antisera raised against total bovine
UP extracts, 1:100 dilution], anti-p63 [Santa Cruz Biotechnology,
Santa Cruz, Calif., cat. #sc-8431, 1:200 dilution], anti-Fox3
[Abcam, cat. #ab104225, 1:200 dilution], anti-CD31 [Abcam, cat.
#ab228364, 1:100 dilution]. Sections were then incubated with
species-matched Cy3-conjugated secondary antibodies (Millipore,
Billerica, Mass.) and nuclei were counterstained with 4',
6-diamidino-2-phenyllindole (DAPI). Specimens were visualized using
an Axioplan-2 microscope (Carl Zeiss MicroImaging, Thornwood, N.Y.)
and representative images were acquired using Axiovision software
(version 4.8).
[0214] Statistical analysis. Urodynamic measurements were analyzed
by generalized estimating equations with post-hoc Bonferroni
testing using commercially available statistical software (SAS9.3
software, www.sas.com). Statistically significant values were
defined as p<0.05. Urodynamic parameters are displayed as
means.+-.standard deviation.
[0215] Results and Discussion
[0216] SEM analyses of silk matrix groups revealed that scaffold
processing techniques as well as distinct fabrication parameters
led to selective differences in overall biomaterial structural
architecture (FIG. 5A). As observed in previous studies [Mauney et
al., 2011; Gomez et al., 2011; Franck et al., 2013], GS1 scaffolds
consisted of compact multi-laminates of parallel-oriented silk
fibers while GS2 matrices were composed of porous (pore size range,
5-50 .mu.m) lamellar-like sheets buttressed by a dense outer layer.
In contrast, FF scaffolds consisted of a bi-layer structure with
compartments dictated by the mode of fabrication. The
solvent-casting/NaCl-leached layer comprised the bulk of the matrix
and resembled a foam configuration with large pores (pore size,
.about.400 .mu.m) interconnected by a network of smaller pores
dispersed along their periphery. This layer was fused on the
external face with a homogeneous, nonporous silk film (200 .mu.m
thick) generated by the annealing of dehydrated silk solutions
during matrix casting. Pore occlusion of the solvent
cast/NaCl-layer was also observed in the bulk matrix along the
plane adjacent to the casting vessels in scaffolds produced in the
absence of silk films (data not shown); however continuity of this
feature was heterogeneous along the surface area as well as highly
variable between matrix replicates. Tensile testing of FF scaffolds
prior to implantation (FIG. 5B) demonstrated a lower degree of UTS
and EM compared to the previously published properties of both gel
spun matrix configurations [Gomez et al., 2011]. SIS scaffolds
exhibited substantially higher UTS and EM in comparison to all silk
groups. Elongation to failure measurements revealed that the FF
group was .about.4 fold more elastic than any other matrix
configuration tested suggesting that although these scaffolds had
lower respective UTS they could achieve larger degrees of
deformation; a potential advantage for the consolidation of highly
distensible bladder defects.
[0217] The porous foam compartment of the FF scaffold configuration
independent of the annealed silk film was first analyzed for its
potential to support bladder defect integration within the rat
model of augmentation cystoplasty. During initial implantation,
poor degrees of scaffold suture retention were noted leading to
dehiscence at the suture line between the matrix and the bladder
wall. In addition, ex vivo assessments of initial defect closure
following surgical integration demonstrated prominent fluid leaks
within center of the scaffold coupled with a failure of the implant
to support bladder distension during saline instillation. The
inability of silk foams to restore the integrity of defect sites is
presumably due to the interconnected porous nature of this scaffold
type which was insufficient to mimic the barrier function of the
native bladder wall following a rise in intravesical pressure. In
contrast, FF scaffolds consisting of silk films annealed to the
exterior face of the porous silk foams were observed to support
organ integrity and distension following surgical integration
similar to the performance of gel spun silk scaffold configurations
and SIS. These results demonstrate that the annealed silk film
compartment is essential for the bi-layer FF group to maintain
initial defect consolidation within this model system.
[0218] Over the course of the 10 week implantation period, survival
rates of the augmented animals in 3 out of 4 scaffold groups were
similar to cystotomy controls prior to scheduled euthanasia.
Bladder reconstruction with GS1 and FF groups displayed survival
rates of 100% (10/10 for GS1 and 8/8 for FF) while animals
implanted with SIS exhibited an 88% survival rate (8/9); both
values were comparable to the 90% rate observed following cystotomy
alone. In contrast, animals implanted with GS2 scaffolds displayed
a reduced rate of survival at 60% (9/15). All spontaneous animal
deaths in each group occurred within the first post-operative week
and post-mortem analyses revealed urinary ascites as the probable
cause in all cases due to scaffold perforation. Previous reports
deploying GS2 matrices for murine bladder augmentation demonstrated
a 100% survival rate (6/6), however the greater tendency for animal
death from scaffold urine leaks observed in this study is
presumably related to the increase in scaffold area used between
the rat and murine models. The lyophilization process utilized
during GS2 construction is prone to generating microfractures in
the nonporous outer layer of this scaffold configuration (FIG. 5A)
[Gomez et al., 2011] thereby increasing the potential for the
integrity of the matrix to become compromised with increased
surface area.
[0219] Following 10 weeks post-op, gross tissue evaluations of the
lower urinary tract revealed no signs of bladder mucus or
hydronephrosis in any of the sacrificed animals, however the
presence of lumenal bladder stones was evident in all experimental
groups examined (FIGS. 6A-6B). These data are consistent with other
previous studies which have shown that the formation of bladder
calculi is a common occurrence in rat models of bladder
reconstruction with biodegradable acellular materials [Kropp et
al., 1995; Vaught et al., 1996; Iijima et al., 2007]. The incidence
of urinary calculi was the highest in the gel spun silk cohorts
with animals augmented with GS1 and GS2 matrices exhibiting a
frequency of 75% and 71%, respectively. SIS implants elicited
similar extents of stone formation by comparison with a frequency
of 57%. In contrast, rats subjected to cystotomy alone or FF
scaffold implantation demonstrated the lowest respective incidence
of stone formation at 13% and 20%. In addition, stone diameters in
these groups were also found to be substantially lower (2 mm) in
comparison to all other experimental conditions (3-4 mm). These
results show that the frequency and extent of bladder stone
formation within the rat augmentation cystoplasty model is
dependent on biomaterial composition (silk versus SIS) as well as
the processing method utilized for silk scaffold construction.
[0220] Histological examination of whole bladder sections (H&E
and MTS) demonstrated that each scaffold group supported robust
degrees of connective tissue ingrowth which traversed from the
periphery of the native bladder wall to the interior of the
original defect site (data not shown). Residual GS1 and GS2
matrices were localized to the bladder lumen and were primarily
intact exhibiting minimal extents of degradation. These results are
consistent with the gross tissue observations wherein scaffold
remnants from both groups were found encapsulated within lumenal
bladder stones. In contrast to our previous study [Gomez et al.,
2011], the rate of in vivo scaffold degradation was not
substantially elevated by the porous architecture of GS2 matrices
in comparison to the non porous GS1 group. An increase in GS2 area
utilized in the rat (7.times.7 mm.sup.2) versus the murine
(4.times.4 mm.sup.2) model may have allowed for enhanced structural
stability and therefore less fragmentation of the bulk matrix
following 10 weeks of implantation. Indeed, similar relationships
between degradation profiles and scale-up of scaffold dimensions
have been reported for a variety of biomaterial formulations [Wu et
al., 2004].
[0221] The degradation pattern of the FF scaffolds was found to be
dependent on the structural architecture of the bi-layer matrix
with the porous region exhibiting extensive degrees of
fragmentation within the bladder wall while the non porous silk
films remained largely intact. Higher initial levels of silk
fibroin utilized for film construction in respect to the porous
compartment may have also contributed to the observed differences
in degradation profile. In addition, the enhanced rate of
degradation demonstrated by the porous regions of the FF scaffolds
in comparison to the GS2 matrices was presumably related to the
increase in pore size as well as the lower content of silk fibroin
which would have allowed for more efficient exposure of the matrix
interior to proteolytic enzymes and subsequent polymer hydrolysis.
Analysis of the degradation profile of collagenous SIS scaffolds
revealed more extensive fragmentation in respect to all silk groups
with minimal degrees of residual matrix detected within the bladder
lumen. Taken together, these data demonstrate that the silk
scaffolds investigated in this study are more structurally stable
than SIS matrices in vivo, however alterations in the rate of silk
scaffold degradation can be achieved through different scaffold
processing techniques with the solvent casting/salt-leaching method
encouraging increased degrees of matrix degradation in comparison
to gel spinning protocols.
[0222] For each biomaterial group, H&E and MTS analyses
demonstrated the presence of robust smooth muscle bundles (H&E:
pink; MTS: red) localized throughout the periphery of the
regenerated bladder wall (data not shown). IHC assessments revealed
that the reconstituted smooth muscle layers of all scaffold groups
stained positive for both .alpha.-SMA and SM22.alpha. contractile
protein expression to similar extents as those observed with the
cystotomy controls indicating prominent smooth muscle maturation
(data not shown). No evidence of severe fibrotic events was
detected in these regions in any of the experimental groups
examined. In addition, an ECM-rich lamina propria populated with
fibroblastic populations was also evident in the regenerated
tissues supported by all matrix configurations. In comparison to
cystotomy controls, histological features within the lamina propria
indicative of minimally acute inflammatory reactions were noted at
each implantation site characterized by the presence of disperse
eosinophil granulocytes, however evidence of substantial chronic
inflammatory events was not observed in response to any
experimental condition.
[0223] Histological evaluations also demonstrated that each implant
group supported the formation of a multi-layer urothelium covering
the entire lumenal surface of the original defect site (data not
shown). The transitional nature of the urothelium was confirmed in
all regenerated tissues by IHC analysis wherein p63-positive basal
and intermediate cell layers were lined with lumenal p63-negative
superficial cells. Varying degrees of hyperplasia were observed in
the basal and intermediate cell compartments supported by all
implant groups in comparison to cystotomy controls. This feature
may reflect incomplete urothelial maturation since normalization of
basal/intermediate cell proliferation is required during wound
healing for native tissue stratification to be achieved [de Boer et
al., 1994; Gomez et al., 2011]. However across all matrix groups,
robust pan-UP protein expression was noted in both regenerated
superficial and intermediate cell layers to levels similar to those
observed in control tissues. Expression and assembly of UP proteins
into heterodimers which form asymmetrical unit membranes is
essential for maintaining the integrity of the urothelial
permeability barrier [Kong et al., 2004].
[0224] IHC analyses revealed evidence of de novo vascularization
and innervation processes in the regenerated tissues supported by
each implant group (data not shown). Vessels containing prominent
CD31 positive endothelial cells were present throughout the
original defect sites while neuronal lineages displaying Fox3
peri-nuclear protein expression were found localized to the
sub-urothelial region of the regenerated bladder walls similar to
cystotomy controls. These results demonstrate that silk scaffold
configurations described in this study are capable of supporting
regeneration of innervated, vascularized smooth muscle and
urothelial tissues to levels comparable to conventional SIS
matrices in a rat model of bladder defect repair.
[0225] The functionality of reconstructed bladders was assessed by
conscious unrestrained cystometry following 10 weeks of biomaterial
implantation. Voided volume was used as a surrogate marker for
bladder capacity given that post-void residual volumes were similar
in each experimental group (data not shown) [Chang et al., 2009].
Rats augmented with GS1 and FF scaffolds had significantly higher
mean voided volumes compared to cystotomy controls and in contrast
the other scaffold groups, GS2 and SIS. In accordance with larger
voided volumes, animals with GS1 and FF implants also had
significantly longer intercontraction intervals compared to control
levels, indicating greater functional bladder capacity since more
time was required for bladder filling between voiding cycles. SNVC
were used as a measure of detrusor overactivity as previously
described [Abrams et al., 2003]. Rats implanted with SIS, GS1 and
GS2 matrices had significantly higher numbers of SNVC compared to
the cystotomy group. In contrast, animals augmented with FF
scaffolds displayed SNVC levels similar to controls. The difference
in SNVC is likely due to the increase in frequency and size of
intra-lumenal calculi observed in the SIS, GS1, and GS2 in respect
to control and FF groups since bladder stones are known to elicit
organ irritation resulting in detrusor overactivity and urge
incontinence [Blaivas et al., 2009]. Compliance is a measure of the
ability of the urinary bladder to store large volumes of urine at
low intravesical pressures. Significant gains in bladder compliance
were observed in rats implanted with FF scaffolds in comparison to
cystotomy controls and in contrast to all other scaffold
configurations. Peak intravesical pressures however were similar
between all experimental groups. These results are consistent with
the ability of FF scaffolds to support bladder tissue regeneration
with increased functional capacity relative to controls and
distensible mechanical properties (FIGS. 7A-7B).
REFERENCES
[0226] Mauney J R, Cannon G M, Lovett M L, Gong E M, Di Vizio D, et
al. (2011) Evaluation of gel spun silk-based biomaterials in a
murine model of bladder augmentation. Biomaterials 32: 808-818.
[0227] Gomez P, Gil E S, Lovett M L, Rockwood D N, Di Vizio D, et
al. (2011) The effect of manipulation of silk scaffold fabrication
parameters on matrix performance in a murine model of bladder
augmentation. Biomaterials 32: 7562-7570. [0228] Chang S J, Yang S
S. Variability, related factors and normal reference value of
post-void residual urine in healthy kindergarteners. J Urol. 2009
October; 182(4 Suppl):1933-8. doi: 10.1016/j.juro.2009.02.086.
[0229] Abrams P. Describing bladder storage function: overactive
bladder syndrome and detrusor overactivity. Urology. 2003 November;
62(5 Suppl 2):28-37; discussion 40-2. [0230] Hofmann S, Hagenmuller
H, Koch A M, Muller R, Vunjak-Novakovic G, Kaplan D L, Merkle H P,
Meinel L. Control of in vitro tissue-engineered bone-like
structures using human mesenchymal stem cells and porous
silkscaffolds. Biomaterials. 2007 February; 28(6):1152-62. Epub
2006 Nov. 7. [0231] Wu L, Ding J. In vitro degradation of
three-dimensional porous poly(D,L-lactide-co-glycolide) scaffolds
for tissue engineering. Biomaterials. 2004 December;
25(27):5821-30. Kropp B P, Eppley B L, Prevel C D, Rippy M K,
Harruff R C, Badylak S F, Adams M C, Rink R C, Keating M A.
Experimental assessment of small intestinal submucosa as a bladder
wall substitute. Urology. 1995 September; 46(3):396-400.
Example 3
[0232] Currently, autologous gastrointestinal segments are utilized
as the primary option for bladder reconstructive procedures despite
their inherent morbidity and significant complication rate.
Biomaterials derived from Bombyx mori silk fibroin represent
attractive alternatives for bladder tissue engineering given their
mechanical robustness, processing plasticity, and biodegradability.
As described herein, it was hypothesized that acellular silk
matrices would effectively mediate tissue regeneration in a large
animal model of bladder augmentation.
[0233] Methods. Scaffolds (6.times.6 cm.sup.2) were generated from
aqueous solutions of 6% silk fibroin by a previously reported
solvent casting/sodium chloride-leaching process. Scanning electron
microscopy (SEM) and tensile testing were performed to ascertain
structural and mechanical properties of scaffolds prior to
implantation. Matrices were anastomosed to the bladder dome of
Yorkshire pigs (N=6) either through open or robot-assisted
augmentation cystoplasty. After an initial catheter drainage period
of 7 days, pigs were allowed to void spontaneously and housed for 3
months. Cystometric analysis was used to determine bladder capacity
both pre-operatively and 3 months post-op. Following euthanasia,
histological (H&E and Masson's trichrome) and
immunohistochemical (IHC) analyses of smooth muscle contractile
protein expression (a-actin and SM22a), urothelial-associated
markers (cytokeratins, p63, and uroplakins), and markers of
innervation (Fox3) and vascularization (von willebrand factor
(wWF)) were assessed at the periphery and center of the original
implantation site as well as a nonsurgical control region. The
results demonstrate that silk scaffolds support smooth muscle and
urothelial regeneration as well as innervation and vascularization
of the defect site. The implant periphery anc center displayed
robust smooth muscle (.alpha.-actin, SM22a), urothelial
(cytokeratins, p63, and uroplakins), neuronal (Fox3) and vascular
regeneration (vonWBF, von willebrand factor) marker expression 3
mos post-op (data not shown).
[0234] Silk augmented bladders displayed increased in capacity over
time (FIGS. 8A-8B) and exhibited tensile and functional properties
similar to native controls (FIGS. 12A-12B).
[0235] Acellular bi-layer silk scaffolds represent an effective
biomaterial system for mediating bladder tissue regeneration and
functional outcomes in a large animal model. These matrices may
offer advantages over conventional gastrointestinal segments and
other cellularized biomaterials for augmentation cystoplasty.
Example 4
[0236] Scaffolds (6.times.6 cm.sup.2) were generated from aqueous
solutions of 6% silk fibroin by a previously reported solvent
casting/sodium chloride-leaching process. Scanning electron
microscopy (SEM) and tensile testing were performed to ascertain
structural and mechanical properties of scaffolds prior to
implantation. Matrices were anastomosed to the bladder dome of
non-diseased Yorkshire pigs (N=2) either through open or
robot-assisted augmentation cystoplasty and maintained for 3
months. Cystometric analysis was used to determine bladder capacity
both pre-operatively and 3 months post-op. Following euthanasia,
histological (H&E and Masson's trichrome) and
immunohlstochemical (IHC) analyses of smooth muscle contractile
protein expression (.alpha.-actin and SM22a) and
urothelial-associated markers (cytokeratins and uroplakins) were
assessed at the periphery and center of the original Implantation
site as well as a nonsurgical control region.
[0237] SEM characterization of silk matrices demonstrated the
formation of a bl-layer structure consisting of an internal porous
network (pore size .about.300 .mu.m) buttressed on one side by a
thin layer of amorphous silk (-50 pm thick) which resulted in
surface pore occlusion. Tensile testing of silk scaffolds revealed
an average ultimate tensile strength of 430 kPa, tensile modulus of
70 kPa, and elongation to failure of 28%. Following 1 week of
Initial catheterization, animals were capable of voluntary voiding
throughout the entire implantation period. Cystometric analyses of
augmented bladders at 3 months post-op revealed substantial
increases (>2-fold) in organ capacity in comparison to
pre-operative values and weight-matched unoperated controls.
Histological and IHC evaluations of both the periphery and central
regions of the regenerated tissues demonstrated robust smooth
muscle bundle formation displaying a-actin and SM22.alpha.
expression as well as the presence of a multi-layered urothelium
exhibiting both prominent uroplakin and cytokeratin positivity
similar to control regions. In addition, substantial degradation of
the silk matrix was noted with only discrete scaffold remnants
present within the interior of the implantation site with no areas
of fibrosis or stone formation observed.
[0238] Acellular bi-layer silk scaffolds represent an effective
biomaterial system for mediating bladder tissue regeneration and
functional outcomes in a large animal model and may offer
advantages over conventional gastrointestinal segments and
previously described cellularized biomaterials for augmentation
cystoplasty.
Example 5
[0239] Scaffolds (6.times.6 cm.sup.2) were generated from 6% silk
fibroin solutions using a solvent casting/sodium chloride-leaching
process. Matrices were anastomosed to the bladder dome of Yorkshire
pigs (N=6) and maintained for 3 months. Cystometric analysis was
used to determine bladder capacity both pre-operatively and at 3
months. Regenerated tissues were evaluated by histological (H&E
and Masson's trichrome) and immunohistochemical (INC) analyses of
smooth muscle contractile protein expression (a-actin and SM22a),
urothelial-associated markers (cytokeratins, p63, uroplakins), and
markers of innervation (Fox3) and vascularization (von willebrand
factor (vWF)). Nonsurgical control regions served as negative
controls.
[0240] Following scaffold implantation, 5/6 swine survived to the
scheduled 3 month euthanasia while 1 animal was sacrificed within
the first week due to urinary ascites. Following 1 week of initial
catheterization, 4/5 animals were capable of voluntary voiding
throughout the entire implantation period. Cystometric analyses of
augmented bladders (4/5 animals) at 3 months post-op revealed
substantial increases (>2-fold) in organ capacity in comparison
to pre-operative values and non surgical controls. In 4/5 animals,
histological and IHC evaluations of the regenerated tissues
demonstrated robust smooth muscle bundles displaying a-actin and
SM22a as well as the presence of a transitional urothelium
exhibiting uroplakin, p63, and cytokeratin positivity similar to
control regions. Fox3 and vWF-positive cells were also observed
throughout the implant site indicating de novo innervation and
vascularization.
[0241] Following 1 week of initial urinary diversion via urethral
catheter, animals were capable of voluntary voiding. Cystometry at
3 months revealed substantial increases (>2-fold) in bladder
capacity in 5/6 augmented pigs in comparison to pre-op values and
weight-matched unoperated controls. Histological and IHC
evaluations of both the periphery and central regions of the
regenerated tissues demonstrated robust smooth muscle formation as
well as a multi-layered urothelium, similar to nonsurgical control
regions. Innervation and vascularization markers were also apparent
in the regenerated tissue. Regenerated tissues were evaluated by
histological (H&E and Masson's trichrome) and
immunohistochemical (IHC) analyses for smooth muscle contractile
proteins (.alpha.-actin and SM22a), urothelial (cytokeratins, p63,
uroplakins), innervation (Fox3) and vascularization (CD31)
markers.
[0242] Acellular silk scaffolds represent an effective biomaterial
system for mediating bladder tissue regeneration and functional
outcomes in a large animal model and offer advantages over GI
segments and previously described cellularized biomaterials for
augmentation cystoplasty.
Example 6
[0243] Two groups of silk scaffolds were produced by a gel spinning
process and consisted of either smooth, compact multi-laminates
(GS1) or rough, porous lamellar-like sheets (GS2). FF silk
scaffolds were produced with a solvent-casting/salt-leaching method
and consisted of porous foams annealed to compact films. Bladder
augmentations were performed in Sprague Dawley rats with acellular
scaffolds (7.times.7 mm.sup.2) and animals were maintained for 10
weeks. Small intestinal submucosa (SIS) scaffolds were assessed in
parallel while control animals received a cystotomy alone. A total
of 8-10 animals per group were evaluated using cystometry,
histological (H&E and Masson's trichrome), and
immunohistochemical (IHC) assays at 10 weeks post-op.
[0244] Regenerated tissues were analyzed for smooth muscle
contractile protein expression (a-actin and SM22a),
urothelial-associated markers (p63 and uroplakins), and markers of
innervation (Fox3) and vascularization (von willebrand factor
(wWF)). Robust expression of these markers within the integration
site, demonstrating that the scaffold groups support innvervated,
vascularized smooth muscle and urothelium. Silk scaffolds support
defect consolidation and the configuration influences stone
formation, as shown in FIGS. 10A-10B. Additionally, silk scaffold
configurations differently influence voiding function, as shown in
FIGS. 11A-11B.
[0245] Variations in silk scaffold structure were demonstrated to
influence the frequency of urinary stone formation with the FF
group displaying a 20% incidence of stones compared to 71 and 75%
rates observed in the GS1 and GS2 groups. Histological and IHC
analyses demonstrated comparable extents of smooth muscle
regeneration and contractile protein (a-actin and SM22a) expression
within the original defect sites of all groups. The regenerated
tissues in each group also contained a transitional urothelium with
positive uroplakin and p63 protein expression. De novo innervation
and vascularization of the regenerated tissues was also confirmed
in each group by robust Fox3 and CD31 protein expression.
Cystometric analysis revealed significant increases in voided
volumes in all groups compared to cystotomy controls. Frequency of
non voiding contractions was similar in FF rats compared to
controls, however the GS1 and GS2 cohorts exhibited significant
overactivity, suggesting an increase in detrusor dysfunction with
these matrices.
[0246] Silk scaffolds promote bladder tissue regeneration with
functional voiding performance dependent on initial biomaterial
fabrication techniques.
Example 7
[0247] The silk bilayer compositions described above herein were
used to repair a surgically-created ileum defect (FIG. 13). Ileum
tissue at the site of the bilayer composition implantation
regenerated (FIG. 17) and demonstrated GI tract continuity by 10
weeks post-op, as determined by gross examination and microCT scan
(FIG. 18). Histological and immunohistological analyses of proximal
regenerated region of the ileum augmented with the bi-layer silk
scaffold was performed 10 weeks post operative. Expression of
smooth muscle contractile markers (.alpha.-SMA and SM22a) as well
as epithelial markers (pancytokeratins) were observed in the
regenerated tissue (data not shown).
Example 8
[0248] Silk bi-layer compositions were made according to Method 2
of Example 1, e.g. a silk fibroin foam was cast while in contact
with a pre-existing silk fibroin film, and used to regenerate
porcine bladder tissue.
[0249] Methods: Matrices were anastomosed to the bladder dome of
Yorkshire pigs (N=4) either through open augmentation cystoplasty,
and after an initial catheter drainage period of 7 days, were
maintained for 3 months. Cystometric analysis was used to determine
bladder capacity both pre-operatively and 3 months post-op (Table
1). Following euthanasia, histological (H&E) and
immunohistochemical (IHC) analyses of smooth muscle contractile
protein expression (a-actin and SM22a), urothelial-associated
markers (cytokeratins, p63, and uroplakins), and markers of
innervation (synaptophysin) and vascularization (CD31) were
assessed at the periphery and center of the original implantation
site as well as a nonsurgical control region. Marker expression
indicated smooth muscle and urothelial regeneration as well as
vascularization and innervation in the implant area. Tensile
testing analyses of regenerated and native porcine bladder tissue
was performed to assess mechanical properties (FIG. 14).
Regenerated tissue performed, on average, similarly to native
tissue.
TABLE-US-00001 TABLE 1 Bladder capacity (capacity at 20 cm H2O)
pre-operatively and at 3 months after augmentation with silk
scaffold. Animal Pre-operative bladder 3 month bladder Replicates
capacity (ml) capacity (ml) Pig 1 358 1000 Pig 2 1000 1600 Pig 3
450 1600 Pig 4 850 1600
Example 9
[0250] B. mori silkworm cocoons were boiled for 20 min in an
aqueous solution of 0.02M Na2CO3 and then rinsed thoroughly with
distilled water to extract the glue-like sericin proteins and wax.
The extracted silk fibroin was then dissolved in 9.3M LiBr solution
at 60 C for 6 h. This solution was dialyzed in distilled water
using a Slide-a-lyzer dialysis cassette (MWCO, 3500) for 4 d
yielding an 8% (wt/vol) aqueous silk fibroin solution. 75 ml of an
aqueous silk fibroin solution (6% wt/vol) was poured into a
rectangular casting vessel (12 cm.times.10 cm) and granular NaCl
(150 g, 500-600 .mu.M average crystal size) mixed with the silk
solution. The resultant solution was allowed to cast for 2 d at
room temperature and then NaCl was removed by washing in distilled
water for 2 d. Spontaneous formation of the bi-layer scaffold was
achieved with random pore occlusion occurring at the bottom of the
matrix plane adjacent to the face of the casting vessel. For
implantation, the porous compartment can be trimmed in thickness to
allow for the total bi-layer scaffold thickness to approximate the
thickness of the surrounding host tissue. The area of the scaffold
was also trimmed to accommodate the defect area.
Example 10
[0251] FIGS. 19A-19B demonstrate that silk scaffolds support
urethral continuity following onlay urethroplasty in rabbits. FIG.
19A depicts an image of the the model of surgical procedure and
silk implantation within rabbit urethra. FIG. 19B depicts
retrograph urethrograms demonstrating no reduction in urethral
caliber or stricture formation following 3 months of silk
implantation. Controls represent urethras which were surgically
incised, claosed, and maintained in parallel with silk implanted
animals. Results are representative of N=2 animals performed with
silk implants as well as controls. Arrows denote original
implantation site or sham injury.
* * * * *
References