U.S. patent application number 15/899463 was filed with the patent office on 2018-11-29 for injectable silk fibroin particles and uses thereof.
The applicant listed for this patent is Trustees of Tufts College, University of Pittsburgh - of the Commonwealth System of Higher Education, Wake Forest University Health Sciences. Invention is credited to Evangelia Bellas, David L. Kaplan, Kacey Marra, J. Peter Rubin, James J. Yoo.
Application Number | 20180339084 15/899463 |
Document ID | / |
Family ID | 48290608 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180339084 |
Kind Code |
A1 |
Bellas; Evangelia ; et
al. |
November 29, 2018 |
INJECTABLE SILK FIBROIN PARTICLES AND USES THEREOF
Abstract
The inventions provided herein relate to compositions, methods,
delivery devices and kits for repairing or augmenting a tissue in a
subject. The compositions described herein are injectable such that
they can be placed in a tissue to be treated with a
minimally-invasive procedure (e.g., by injection) and/or be molded
flexibly into a tissue void of any shape and/or size. In some
embodiments, the composition described herein comprises a plurality
of silk fibroin particles, which can retain their original volume
within the tissue for a period of time. The compositions can be
used as a filler to replace a tissue void, e.g., for tissue repair
and/or augmentation, or as a scaffold to support tissue
regeneration and/or reconstruction. In some embodiments, the
compositions described herein can be used for soft tissue repair or
augmentation.
Inventors: |
Bellas; Evangelia;
(Philadelphia, PA) ; Marra; Kacey; (Canonsburg,
PA) ; Rubin; J. Peter; (Pittsburgh, PA) ;
Kaplan; David L.; (Concord, MA) ; Yoo; James J.;
(Winston-Salem, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trustees of Tufts College
University of Pittsburgh - of the Commonwealth System of Higher
Education
Wake Forest University Health Sciences |
Medford
Pittsburgh
Winston-Salem |
MA
PA
NC |
US
US
US |
|
|
Family ID: |
48290608 |
Appl. No.: |
15/899463 |
Filed: |
February 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14357443 |
May 9, 2014 |
9931434 |
|
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PCT/US2012/064450 |
Nov 9, 2012 |
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15899463 |
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61557603 |
Nov 9, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/48 20130101;
A61L 27/58 20130101; A61P 17/00 20180101; Y10T 428/2982 20150115;
A61P 19/04 20180101; A61L 27/56 20130101; A61P 43/00 20180101; A61L
2400/06 20130101; A61L 27/227 20130101; A61L 27/54 20130101; A61L
2430/34 20130101; A61L 27/3604 20130101; A61P 21/00 20180101; A61P
15/08 20180101; A61L 27/3834 20130101; A61L 27/48 20130101; C08L
89/00 20130101 |
International
Class: |
A61L 27/22 20060101
A61L027/22; A61L 27/56 20060101 A61L027/56; A61L 27/54 20060101
A61L027/54; A61L 27/58 20060101 A61L027/58; A61L 27/48 20060101
A61L027/48; A61L 27/38 20060101 A61L027/38; A61L 27/36 20060101
A61L027/36; C08L 89/00 20060101 C08L089/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
No. EB002520 awarded by the National Institutes of Health and
W81XWH-08-2-0032 awarded by the US Army. The government has certain
rights in the invention.
Claims
1.-110. (canceled)
111. A composition comprising: dry porous silk fibroin particles,
wherein: (i) the porosity is at least 70%; (ii) the silk fibroin
constitutes at least 30% of the total composition; and (iii) the
particles have a size of about 1 .mu.m to about 1,000 .mu.m.
112. The composition of claim 111, wherein the composition is
formulated for injection.
113. The composition of claim 112, wherein the injection is
subcutaneous.
114. The composition of claim 112, wherein the injection is into a
tissue site.
115. The composition of claim 113, wherein the tissue site is a
soft tissue site.
116. The composition of claim 113, wherein the tissue site is a
hard tissue site.
117. The composition of claim 115, wherein the hard tissue site is
bone tissue.
118. The composition of claim 111, wherein the dry porous silk
fibroin particles have a porosity of at least about 80% or at least
about 90%.
119. A method of repairing or augmenting a tissue site in a subject
comprising injecting the composition of claim 111.
120. The composition of claim 111, wherein the composition further
comprises at least one active agent.
121. The composition of claim 120, wherein the at least one active
agent is or comprises at least one of a biologically active agent,
a cosmetically active agent, a cell attachment agent, a contrast
agent, or any combinations thereof.
122. The composition of claim 121, wherein the biologically active
agent is selected from the group consisting of: a therapeutic
agent, an anesthetic, a cell growth factor, a peptide, a
peptidomimetic, an antibody or a portion thereof, an antibody-like
molecule, nucleic acid, a polysaccharide, and any combinations
thereof.
123. The composition of claim 121, wherein the cell attachment
agent is selected from the group consisting of: hyaluronic acid,
collagen, crosslinked hyaluronic acid/collagen, an integrin-binding
molecule, chitosan, elastin, fibronectin, vitronectin, laminin,
proteoglycans, any derivatives thereof, and any combinations
thereof.
124. The composition of claim 121, wherein the cosmetically active
agent is selected from the group consisting of: an anti-aging
agent, an anti-free radical agent, an anti-oxidant, a hydrating
agent, a whitening agent, a colorant, a depigmenting agent, a
sun-blocking agent, a muscle relaxant, and any combinations
thereof.
125. The composition of claim 111, wherein the dry porous silk
fibroin particles exclude an amphiphilic peptide.
126. The composition of claim 125, wherein the amphiphilic peptide
comprises a RGD motif.
127. The composition of claim 111, further comprising a dermal
filler material.
128. The composition of claim 127, wherein the dermal filler
material is selected from the group consisting of poly(methyl
methacrylate) microspheres, hydroxylapatite, poly(L-lactic acid),
hyaluronic acid, collagen, and any combinations thereof.
129. A delivery device comprising the composition of claim 111.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C .sctn.
119(e) of U.S. Provisional Application No. 61/557,603 filed Nov. 9,
2011, the content of which is incorporated herein by reference in
its entirety.
TECHNICAL FIELD OF DISCLOSURE
[0003] The inventions provided herein generally relate to silk
fibroin-based materials for biomedical applications, e.g., in soft
tissue repair, augmentation and/or reconstruction.
BACKGROUND
[0004] The restoration of soft tissue defects from trauma, surgical
excision or congenital defects should start with a strategy that
will maintain tissue size and shape to near normal dimensions for
extended time frames. Current clinical strategies include free fat
transfers and artificial fillers. In the case of breast cancer
patients receiving mastectomies, silicone shells filled with saline
or silicone are used to replace the void. This leaves the patient
with an unnatural look and feel, and the risk of capsular
contracture resulting in a revision surgery. The fat grafting and
artificial filler options fail to retain volume over time. Thus,
the fat grafting and artificial filler options can require a second
surgical site, have avascular necrosis and generally do not
regenerate the original tissue.
[0005] Bovine and human collagen have gained widespread use as
injectable materials for soft tissue augmentation and filling.
Collagen, the principal extracellular structural protein of the
animal body, has been used as an implant material to replace or
augment connective tissue, such as skin, tendon, cartilage and
bone. Additionally, collagen has been injected or implanted into
the human body for cosmetic purposes for a number of years.
However, the use of collagen in soft tissue augmentation and/or
filling could be costly and it does not have a long lasting effect,
e.g., the results often only last for about 3 months.
[0006] Hyaluronic acid (HA) is a glycosaminoglycan that is
naturally found in the human body and is widely distributed
throughout connective, epithelial, and neural tissues. Compositions
of non-crosslinked hyaluronic acid tend to degrade within a few
months after injection and thus require fairly frequent reinjection
to maintain their soft tissue augmenting effect. More recently,
compositions of cross-linked hyaluronic acid have been used for
soft tissue augmentation. However, such cross-linked compositions
contain fairly large particles, around approximately 2 mm each, of
hyaluronic acid suspended in a gel. While the larger particles
could have a longer lasting effect, the larger particle size can
make the injection more challenging and create an unpleasant
experience to a recipient.
[0007] In summary, the major disadvantages of the current
strategies for soft tissue regeneration, repair and/or augmentation
include a large amount of tissues required for grafting large
tissue defects; donor site morbidity, possibility of second
surgical site, avascular necrosis; loss of shape and/or size of the
scaffolds over time; material mismatch with native tissue; and
failure to regenerate tissue. Accordingly, there is a strong need
to develop a strategy or a scaffold that can be administered with a
minimally invasive procedure and will provide sustained retention
of volume restoration for at least 3 months or longer, e.g., for at
least 6 months or at least one year, while the body gradually
remodels and regenerates the site into near-normal tissue structure
and function.
SUMMARY
[0008] Embodiments of various aspects described herein are based
on, at least in part, engineering silk fibroin scaffolds in an
injectable format, e.g., silk fibroin particles, which can retain
at least a portion of the original volume within a tissue to be
repaired or augmented for a period of time. For example, such silk
fibroin particles can be placed with a minimally invasive procedure
(e.g., injection) into a subject's tissue to be repaired or
augmented as a filler to replace a void, e.g., for tissue
augmentation or repair, or as a scaffold, e.g., for tissue
regeneration or reconstruction.
[0009] Accordingly, one aspect provided herein is an injectable
composition for use in repairing or augmenting a tissue in a
subject, comprising a plurality of silk fibroin particles, wherein
at least a portion of the silk fibroin particles retain at least a
portion (e.g., at least about 50%) of their original volume within
the tissue to be repaired or augmented for a period of time (e.g.,
at least about 6 weeks).
[0010] Another aspect provided herein relates to a method for
repairing or augmenting a tissue in a subject. The method includes
placing in the tissue to be repaired or augmented a composition
comprising a plurality of silk fibroin particles, wherein at least
a portion of the silk fibroin particles retain at least a portion
of its original volume (e.g., at least about 50% or more) within
the tissue for a period of time (e.g., at least about 6 weeks or
longer). In one embodiment, the composition is placed into the
tissue to be repaired or augmented by injection.
[0011] In certain embodiments of the compositions and methods
provided herein, the silk fibroin particles can exclude an
amphiphilic peptide. In other embodiments, the silk fibroin
particles can include an amphiphilic peptide. An exemplary
amphiphilic peptide, for example, can comprise a RGD motif.
[0012] In some embodiments of the compositions and methods provided
herein, at least a portion of the silk fibroin particles can retain
at least about 50% of their original volume, including at least
about 60%, at least about 70%, at least about 80% or more, of their
original volume within the tissue for a period of time.
[0013] In some embodiments of the composition and method provided
herein, at least a portion of the silk fibroin particles can retain
at least a portion of its original volume for at least about 6
weeks, at least about 3 months, at least about 6 months or
longer.
[0014] Volume retention of the silk fibroin particles can be, in
part, controlled by modulating the degradation and/or solubility
properties of the silk fibroin particles. In such embodiments, at
least a portion of the silk fibroin particles can be adapted to
degrade no more than 50% of their original volume, for example,
including no more than 30%, no more than 10%, of their original
volume, in at least about 6 weeks, including at least about 3
months, 6 months or longer.
[0015] Depending on the defect size of the tissue and/or desired
properties of the silk fibroin particles, the silk fibroin
particles can be adapted to be any size. In some embodiments, the
silk fibroin particles can have a size suitable for injection into
a tissue. For example, the silk fibroin particles provided herein
can have a size of about 500 nm to about 5000 .mu.m. In some
embodiments, the silk fibroin particles can have a size of about 1
.mu.m to about 2000 .mu.m. In some embodiments, the silk fibroin
particles can have a size of about 10 .mu.m to about 1500 .mu.m. In
some embodiments, the silk fibroin particles can have a size of
about 1 .mu.m to about 1000 .mu.m. In some embodiments, the silk
fibroin particles can have a size of about 1 .mu.m to about 500
.mu.m. In some embodiments, the silk fibroin particles can have a
size of about 3 .mu.m to about 425 .mu.m. In some embodiments, the
silk fibroin particles can have a size of about 500 .mu.m to about
1200 .mu.m. In some embodiments, the silk fibroin particles can
have a size of about 800 .mu.m to about 1000 .mu.m.
[0016] The silk fibroin particles can be adapted to mimic the
structural morphology of native tissues and/or to deliver an active
agent to a local area of a tissue. For example, the silk fibroin
particles can be porous. In some embodiments, the porosity of the
silk fibroin particles can be adapted to mimic the structural
morphology and/or gradient of cellular densities found in native
tissue. In some embodiments, the porosity of the silk fibroin
particles can be adapted to deliver an active agent to a tissue in
a pre-determined release profile. In some embodiments, the porosity
of the silk fibroin particles can be adapted to retain at least a
portion of their original volume for a period of time. For example,
the silk fibroin porous particles can have a porosity of at least
about 1%, e.g., including at least about 3%, at least about 5%, at
least about 10%, at least about 15%, at least about 20%, at least
about 30%, at least about 50%, at least about 70%, at least about
80%, at least about 90% or higher. The pore size of such porous
silk fibroin particles can range from about 10 nm to about 2000
.mu.m, from about 50 nm to about 1500 .mu.m, from about 0.5 .mu.m
to about 1500 .mu.m, from about 1 .mu.m to about 1000 .mu.m, or
from about 1 .mu.m to about 500 .mu.m. In some embodiments, the
pore size of the porous silk fibroin particles can range from about
3 .mu.m to about 500 .mu.m. In some embodiments, the pore size of
the porous silk fibroin particles can range from about 8 .mu.m to
about 1000 .mu.m. In some embodiments, the silk fibroin particles
need not be porous.
[0017] The silk fibroin particles, in one embodiment, can be
fabricated by reducing a solid-state silk fibroin into particles.
For example, a solid-state silk fibroin can be reduced into
particles by a mechanical means, for example, but not limited to,
micronizing, milling, pulverizing, crushing, grinding, cutting, and
any combinations thereof. A solid-state silk fibroin can be made by
any methods known in the art. To produce a porous silk fibroin
structure, porogen leaching method or any other art-recognized can
be used. In some embodiments, the silk fibroin particles can have
no optical properties, for example, but not limited to,
diffraction. In such embodiments, the silk fibroin particles can be
produced from a solid-state silk fibroin without any optical
elements imprinted on or added thereto.
[0018] The injectable composition described herein comprising the
silk fibroin particles can further comprise at least one active
agent. In some embodiments, the silk fibroin particles of the
composition described herein can further comprise at least one
active agent. Non-limiting examples of the active agents can
include biologically active agents, cosmetically active agents,
cell attachment agents, a dermal filler material, and any
combinations thereof. In some embodiments, the active agent can be
a therapeutic agent. In some embodiments, the active agent can be a
cosmetically active agent. In some embodiments, the active agent
can be a dermal filler material.
[0019] In some embodiments, the injectable composition or the
composition comprising a plurality of silk fibroin particles can
further comprise at least one cell, e.g., a stem cell. In some
embodiments, the cell can be obtained from a biological fluid or
concentrate, such as lipoaspirate, bone marrow aspirate or any
combinations thereof.
[0020] Accordingly, in some embodiments, the injectable composition
or the composition comprising a plurality of silk fibroin particles
can further comprise a biological fluid or concentrate, such as
lipoaspirate, bone marrow aspirate or any combinations thereof. In
one embodiment, the injectable composition or the composition
comprising a plurality of silk fibroin particles can further
comprise a lipoaspirate. In these embodiments, the composition or
the injectable composition can comprise silk fibroin particles and
a biological fluid or concentrate (e.g., a lipoaspirate or a
bone-marrow aspirate) in a volume ratio of about 1:38 to about
12:19, or about 1:19 to about 10:19, or about 2:19 to about 8:19.
In one embodiment, the composition or the injectable composition
can comprise silk fibroin particles and a biological fluid or
concentrate (e.g., a lipoaspirate or a bone-marrow aspirate) in a
volume ratio of about 3:19. In another embodiment, the composition
or the injectable composition can comprise silk fibroin particles
and a biological fluid or concentrate (e.g., a lipoaspirate or a
bone-marrow aspirate) in a volume ratio of about 6:19.
[0021] In some embodiments, the composition described herein can
further comprise a hydrogel, e.g., in a form of separate hydrogel
particles, and/or distributed within silk fibroin particles.
[0022] Various embodiments of the composition described herein can
be injected into a tissue to be repaired or augmented by any known
methods in the art, e.g., subcutaneously, submuscularly, or
intramuscularly. When injected in a tissue, some embodiments of the
composition can be at least partially dry. Alternatively, the
composition can be at least partially hydrated, e.g., the
composition can further comprise a pharmaceutically-acceptable
carrier, e.g., a buffered solution, when injected in a tissue. In
some embodiments, the silk fibroin particles in the composition are
small enough such that the silk fibroin particles can be delivered
through a needle or a catheter without any prior compression before
being introduced into a tissue to be repaired or augment.
[0023] The tissue to be repaired or augmented by the composition
and/or the method described herein can be a soft tissue. Exemplary
examples of a soft tissue include, but are not limited to, a
tendon, a ligament, skin, a breast tissue, a fibrous tissue, a
connective tissue, a muscle, and any combinations thereof. In
certain embodiments, the soft tissue is skin. In other embodiments,
the soft tissue is a breast tissue.
[0024] A delivery device comprising one embodiment of an injectable
composition and/or silk fibroin particles is also provided herein.
A delivery device can include any conventional injection device
(e.g., a syringe) and/or any administration device that is
minimally invasive. Accordingly, in some embodiments, provided
herein relate to syringes comprising one embodiment of an
injectable composition. The syringe can further comprise a needle,
a cannula, and/or a catheter. In some embodiments, the delivery
device (e.g., a syringe) can further comprise an injection carrier,
e.g., a buffered solution. In some embodiments, the delivery device
(e.g., a syringe) can further comprise a local anesthetic.
[0025] In some embodiments of any aspects described herein, the
compositions and/or delivery devices can be stored or transported
at a temperature about 0.degree. C. and about 60.degree. C., e.g.,
between about 10.degree. C. and about 60.degree. C. or between
about 15.degree. C. and about 60.degree. C. At such temperatures,
the bioactivity of active agents embedded or distributed inside the
silk fibroin particles can be stabilized for a period of time.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows an exemplary method of using one or more
embodiments of the injectable compositions described herein. The
porous silk fibroin scaffold bits (e.g., formed by reducing a
solid-state porous silk fibroin into particles or bits) can be
mixed with lipoaspirate as a carrier, optionally containing
adipose-derived stem cells (ASCs), to form an exemplary injectable
composition. The injectable compositions can then be injected into
a subject, e.g., an animal model.
[0027] FIG. 2 shows images of one or more embodiments of the
injectable compositions described herein. The silk fibroin scaffold
particles can be of any size, for example, from submicrons to about
2 mm. The left panel shows that the silk fibroin scaffold particles
can have a size of about 3 .mu.m to about 425 .mu.m, while the
right panel shows that the silk fibroin scaffold particles can have
a size of about 0.8 mm to about 1 mm.
[0028] FIG. 3 shows a set of hematoxylin and eosin images of
injectable silk fibroin porous particles mixed with various ratios
of lipoaspirate at the 6-week post-injection. The silk fibroin
porous particles (with a pore size of about 3 .mu.m to about 500
.mu.m) as shown in the first row of the images were produced by
micronizing a porous silk fibroin scaffold with a pore size of
about 300 microns to about 500 microns, while the ones shown in the
second and third rows of the images (silk fibroin particles with a
pore size of about 8 .mu.m to about 1000 .mu.m) were produced from
a silk fibroin scaffold with a pore size of about 850 microns to
about 1000 microns. The term "dose" as used in FIG. 3 refers to the
amount of the silk fibroin particles injected in a subject, e.g.,
an animal model. In some embodiments, the dose can be indicated by
the final volume ratio of silk fibroin particles to lipoaspirate.
For example, the final volume ratios can range from about 3:19 to
about 6:19.
DETAILED DESCRIPTION OF THE INVENTION
[0029] Described herein are methods, compositions, delivery
devices, and kits for repairing or augmenting a tissue in a
subject. In accordance with embodiments of various aspects
described herein, an injectable format of silk fibroin scaffolds
(e.g., silk fibroin particles) can be placed (e.g., by injection)
into a tissue to be repaired or augmented and retain at least a
portion of their original volume (e.g., at least about 50% of their
original volume) within the tissue to be repaired or augmented for
a period of time (e.g., at least about 6 weeks). Such injectable
silk fibroin particles can be introduced into a defect site with a
minimally-invasive procedure, while enabling a skilled practitioner
to flexibly mold the injectable silk fibroin particles to fit into
a defect of any shape and/or size.
Silk Fibroin Particles
[0030] Silk fibroin particles described herein can retain their
original volume upon administration to a tissue (e.g., by
injection) to be repaired or augmented for a period of time.
[0031] By "original volume" in reference to the silk fibroin
particles described herein is generally meant the volume of silk
fibroin particles as measured immediately before the silk fibroin
particles are placed into a tissue to be repaired or augmented, or
the corresponding increase in tissue volume as measured immediately
after the silk fibroin particles are placed in a tissue to be
repaired or augmented. For example, the original volume of silk
fibroin particles can be measured, for example, within about 20
minutes, before or after the silk fibroin particles are placed into
a tissue to be repaired or augmented. In some instances, the
original volume of the silk fibroin particles can be measured, for
example, about 10 seconds, about 15 seconds, about 20 seconds,
about 25 seconds, about 30 seconds, about 1 minute, about 2
minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6
minutes, about 7 minutes, about 8 minutes, about 9 minutes, about
10 minutes, about 11 minutes, about 12 minutes, about 13 minutes,
about 14 minutes, about 15 minutes, about 16 minutes, about 17
minutes, about 18 minutes, about 19 minutes, or about 20 minutes,
before or after the silk fibroin particles are placed into a tissue
to be repaired or augmented. In some embodiments, the volume of the
silk fibroin particles prior to placing into a tissue can refer to
the volume of the silk fibroin particles in a dried state. In
alternative embodiments, the volume of the silk fibroin particles
prior to placing into a tissue can refer to the volume of silk
fibroin particles in a hydrated state. In other embodiments, the
volume of the silk fibroin particles prior to placing in a tissue
can refer to the volume of silk fibroin particles suspended in a
fluid or a carrier. In some embodiments, the volume of the silk
fibroin particles prior to placing in a tissue can refer to the
injection volume of the mixture comprising the silk fibroin
particles.
[0032] As used herein, the term "retain" refers to maintaining the
volume (e.g., size and/or shape) of at least a portion of the silk
fibroin particles described herein over a period of time. In some
embodiments, at least a portion of the silk fibroin particles can
retain over a period of time at least about 20% of their original
volume, including, for example, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90% of their original volume or
higher. In some embodiments, at least a portion of the silk fibroin
particles can retain over a period of time at least about 1%, at
least about 3%, at least about 5%, at least about 10%, at least
about 15%, at least about 20% of their original volume or higher.
In some embodiments, at least a portion of the silk fibroin
particles can retain 100% of their original volume, e.g., no
detectable changes in the volume, within the tissue to be repaired
or augmented for a period of time. In one embodiment, at least a
portion of the silk fibroin particles can retain at least about 1%
of their original volume within the tissue to be repaired or
augmented for a period of time. In one embodiment, at least a
portion of the silk fibroin particles can retain at least about 50%
of their original volume within the tissue to be repaired or
augmented for a period of time. In one embodiment, at least a
portion of the silk fibroin particles can retain at least about 60%
of their original volume within the tissue to be repaired or
augmented for a period of time. In one embodiment, at least a
portion of the silk fibroin particles can retain at least about 70%
of their original volume within the tissue to be repaired or
augmented for a period of time. In one embodiment, at least a
portion of the silk fibroin particles can retain at least about 80%
of their original volume within the tissue to be repaired or
augmented for a period of time. The volume of the silk fibroin
particles placed into a tissue can be determined or indicated by a
change in at least one of the tissue properties, e.g., tissue
volume, tissue elasticity, and/or tissue hardness. In some
embodiments, the volume of the silk fibroin particles placed into a
tissue can be determined from explants, e.g., weight measurements
and/or volume displacement. In one embodiment, the volume of the
silk fibroin particles placed into a tissue can be monitored and/or
measured by imaging.
[0033] The silk fibroin particles can retain at least a portion of
their original volume for any period of time, e.g., weeks, months,
or years. In some embodiments, the silk fibroin particles can
retain, e.g., at least about 50% of their original volume
(including e.g., at least about 60%, at least about 70%, at least
about 80%, or higher, of their original volume) for at least about
6 weeks, at least about 7 weeks, at least about 8 weeks, at least
about 3 months, at least about 4 months, at least about 5 months,
at least about 6 months, at least about 7 months, at least about 8
months, at least about 9 months, at least about 10 months, at least
about 11 months, at least about 1 year, at least about 2 years, at
least 3 years, at least about 4 years, at least 5 years or longer.
In certain embodiments, the silk fibroin particles can retain,
e.g., at least about 70% of their original volume or higher, for at
least about 3 months or longer. In other embodiments, there can be
no significant changes in the volume of the silk fibroin particles
after placed into a tissue to be repaired or augmented for at least
about 3 months or longer. In some embodiments, the silk fibroin
particles can retain, e.g., at least about 70% of their original
volume or higher, for at least about 6 months or longer (including,
e.g., at least about 9 months, at least about 12 months, at least
about 18 months or longer). In other embodiments, there can be no
significant changes in the volume of the silk fibroin particles
after placed into a tissue to be repaired or augmented for at least
about 6 months or longer. In particular embodiments, the silk
fibroin particles can retain at least about 20% of their original
volume or higher for at least about 1 year or longer (including,
e.g., at least about 2 years, at least about 3 years, at least
about 4 years, at least about 5 years or longer). In some
embodiments, the silk fibroin particles can retain at least about
50% of their original volume or higher for at least about 1 year or
longer (including, e.g., at least about 2 years, at least about 3
years, at least about 4 years, at least about 5 years or longer).
In one embodiment, at least a portion of the silk fibroin particles
can retain at least about 1%, at least about 3%, at least about 5%,
at least about 10%, at least about 15%, at least about 20%, at
least about 30%, at least about 40% of their original volume or
more, within the tissue to be repaired or augmented for at least
about 6 weeks (including, e.g., at least about 3 months, at least
about 6 months, or longer).
[0034] The volume retention of the silk fibroin particles can also
be characterized by, e.g., degradation of the silk fibroin
particles. Generally, the slower the silk fibroin particles
degrade, the longer the silk fibroin particles can retain their
original volume in a tissue. Accordingly, some embodiments provided
herein are directed to injectable compositions for use in repairing
or augmenting a tissue in a subject, the compositions comprising a
plurality of silk fibroin particles, wherein at least a portion of
the silk fibroin particles are adapted to degrade within the tissue
to be repaired or augmented over a period of time.
[0035] As used in reference to the silk fibroin particles described
herein, the term "degrade" or "degradation" refers to a decrease in
volume or size of the silk fibroin particles. The degradation of
the silk fibroin particles can occur via cleavage of the silk
fibroin particles into smaller fragments and/or dissolution of the
silk fibroin particles or fragments thereof. In some embodiments,
at least a portion of the silk fibroin particles can be adapted to
degrade no more than 80% of their original volume, including, for
example, no more than 70%, no more than 60%, no more than 50%, no
more than 40%, no more than 30%, no more than 20%, no more than 10%
of their original volume or lower. In some embodiments, at least a
portion of the silk fibroin particles can exhibit no significant
degradation (e.g., no detectable changes in the volume) within the
tissue to be repaired or augmented. In one embodiment, at least a
portion of the silk fibroin particles can be adapted to degrade no
more than 50% of their original volume within the tissue to be
repaired or augmented for a period of time. In one embodiment, at
least a portion of the silk fibroin particles can be adapted to
degrade no more than 40% of their original volume within the tissue
to be repaired or augmented for a period of time. In one
embodiment, at least a portion of the silk fibroin particles can be
adapted to degrade no more than 30% of their original volume within
the tissue to be repaired or augmented for a period of time. In one
embodiment, at least a portion of the silk fibroin particles can be
adapted to degrade no more than 20% of their original volume within
the tissue to be repaired or augmented for a period of time. In one
embodiment, at least a portion of the silk fibroin particles can be
adapted to degrade no more than 10% of their original volume within
the tissue to be repaired or augmented for a period of time.
[0036] The silk fibroin particles can be adapted to degrade at any
rate. In some embodiments, the silk fibroin particles can be
adapted to degrade at least a portion of their original volume over
any period of time, e.g., weeks, months, or years. In some
embodiments, the silk fibroin particles can be adapted to degrade,
e.g., no more than 50% of their original volume (including e.g., no
more than 40%, no more than 30%, no more than 20% or lower, of
their original volume) in at least about 6 weeks, at least about 7
weeks, at least about 8 weeks, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, at
least about 1 year, at least about 2 years, at least about 3 years,
at least about 4 years, at least about 5 years or longer. In
certain embodiments, the silk fibroin particles can be adapted to
degrade, e.g., no more than 30% of their original volume or lower,
in at least about 3 months or longer. In other embodiments, there
can be no significant degradation (i.e., no detectable changes in
the volume of the silk fibroin particles) after placed into a
tissue to be repaired or augmented for at least about 3 months or
longer. In some embodiments, the silk fibroin particles can be
adapted to degrade, e.g., no more than 30% of their original volume
or lower, in at least about 6 months or longer (including, e.g., at
least about 9 months, at least about 12 months, at least about 18
months or longer). In other embodiments, there can be no
significant degradation (i.e., no detectable changes in the volume
of the silk fibroin particles) after placed into a tissue to be
repaired or augmented for at least about 6 months or longer. In
particular embodiments, the silk fibroin particles can be adapted
to degrade no more than 80% of their original volume or lower in at
least about 1 year or longer (including, for example, at least
about 2 years, at least about 3 years, at least about 4 years, at
least about 5 years or longer). In some embodiments, the silk
fibroin particles can be adapted to degrade no more than 50% of
their original volume or lower in at least about 1 year or
longer.
[0037] The same or similar formulation of the silk fibroin
particles or injectable compositions can manifest different
responses in a subject. By way of example only, the volume
retention or degradation rate of the silk fibroin particles in a
tissue can vary from one subject to another, e.g., because of
different tissue microenvironment such as species and/or levels of
various proteins or enzymes (e.g., proteolytic enzymes) present in
the tissue.
[0038] In some embodiments, the silk fibroin particles can be
adapted to maintain a constant volume retention rate and/or
degradation rate over a period of time. In some embodiments, the
silk fibroin particles can be adapted to have a volume retention
rate or degradation rate varying with time. For example, the silk
fibroin particles can be coated with a polymeric material, e.g.,
silk fibroin of a different concentration and/or a different
biodegradable and biocompatible polymer. Such coating can possess a
different function and/or a different degradation rate from that of
the silk fibroin particle core. By way of example only, the coating
of the silk fibroin particle can contain at least one active agent
and be adapted to degrade at a different rate (e.g., at a faster
rate) from that of the silk fibroin particle core. Thus, upon
placing the silk fibroin particles in a tissue, the coating of the
silk fibroin particles can be adapted to degrade faster, e.g., to
release the active agent for relieving the pain and/or promoting
the wound healing, while the core of the silk fibroin particles can
retain their volume for a longer period of time.
[0039] Silk fibroin is a particularly appealing biopolymer
candidate to be used for various embodiments described herein,
e.g., because of its versatile processing e.g., all-aqueous
processing (Sofia et al., 54 J. Biomed. Mater. Res. 139 (2001);
Perry et al., 20 Adv. Mater. 3070-72 (2008)), relatively easy
functionalization (Murphy et al., 29 Biomat. 2829-38 (2008)), and
biocompatibility (Santin et al., 46 J. Biomed. Mater. Res. 382-9
(1999)). For example, silk has been approved by U.S. Food and Drug
Administration as a tissue engineering scaffold in human implants.
See Altman et al., 24 Biomaterials: 401 (2003).
[0040] 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 in different embodiments described herein. Silk fibroin
produced by silkworms, such as Bombyx mori, is the most common and
represents an earth-friendly, renewable resource. For instance,
silk fibroin used in a silk film may 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.
[0041] In various embodiments, the silk fibroin can be modified for
different applications and/or desired mechanical or chemical
properties (e.g., to facilitate formation of a gradient of active
agent in silk fibroin particles). One of skill in the art can
select appropriate methods to modify silk fibroins, e.g., depending
on the side groups of the silk fibroins, desired reactivity of the
silk fibroin and/or desired charge density on the silk fibroin. In
one embodiment, modification of silk fibroin 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). Silk fibroin can also be modified
through gene modification to alter functionalities of the silk
protein (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
silk fibroin matrix can be combined with a chemical, such as
glycerol, that, e.g., affects flexibility of the matrix. See, e.g.,
WO 2010/042798, Modified Silk films Containing Glycerol.
[0042] As used herein, the phrase "silk fibroin particles"
generally refer to particles comprising silk fibroin. In some
embodiments, the phrase "silk fibroin particles" refers to
particles in which silk fibroin constitutes at least about 30% of
the total composition, including at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95% or higher, of the total
composition. In certain embodiments, the silk fibroin particles can
be substantially formed from silk fibroin. In various embodiments,
the silk fibroin particles can be substantially formed from silk
fibroin comprising at least one active agent.
[0043] The silk fibroin particles described herein can be adapted
to be any shape, e.g., a spherical shape, polygonal-shaped,
elliptical-shaped. As used in reference to silk fibroin particles,
the term "particle" as used herein refers to a particle of any
shape, e.g., but not limited to, a spherical shape, a polygonal
shape, or an elliptical shape. The particle size can vary with a
number of factors including, without limitations, the size of the
tissue to be repaired or augmented and/or desired properties of the
silk fibroin particles, e.g., volume retention or degradation
profile. In some embodiments, the particle size can range from
about 500 nm to about 5000 .mu.m, about 1 .mu.m to about 2000
.mu.m, about 10 .mu.m to about 1500 .mu.m, about 20 .mu.m to about
1000 .mu.m, about 50 .mu.m to about 750 .mu.m, or about 100 .mu.m
to about 500 .mu.m. In certain embodiments, the silk fibroin
particles provided herein can have a size of about 500 nm to about
5000 .mu.m. In some embodiments, the silk fibroin particles can
have a size of about 1 .mu.m to about 2000 .mu.m. In some
embodiments, the silk fibroin particles can have a size of about 10
.mu.m to about 1500 .mu.m. In some embodiments, the silk fibroin
particles can have a size of about 1 .mu.m to about 1000 .mu.m. In
some embodiments, the silk fibroin particles can have a size of
about 1 .mu.m to about 500 .mu.m. In some embodiments, the silk
fibroin particles can have a size of about 3 .mu.m to about 425
.mu.m. In some embodiments, the silk fibroin particles can have a
size of about 500 .mu.m to about 1200 .mu.m. In some embodiments,
the silk fibroin particles can have a size of about 800 .mu.m to
about 1200 .mu.m. In some embodiments, the silk fibroin particles
can have a size of about 1 .mu.m to about 5 .mu.m. In some
embodiments, the silk fibroin particles can have a size of about 5
.mu.m to about 20 .mu.m. In some embodiments, the silk fibroin
particles can have a size of about 20 .mu.m to about 50 .mu.m. In
some embodiments, the silk fibroin particles can have a size of
about 50 .mu.m to about 100 .mu.m. In some embodiments, the silk
fibroin particles can have a size of about 100 .mu.m to about 250
.mu.m. In some embodiments, the silk fibroin particles can have a
size of about 500 .mu.m to about 750 .mu.m. In some embodiments,
the silk fibroin particles can have a size of about 750 .mu.m to
about 1000 .mu.m. In some embodiments, the silk fibroin particles
can have a size of about 1000 .mu.m to about 1200 .mu.m. In some
embodiments, the silk fibroin particles can have a size less than 1
.mu.m. In some embodiments, the silk fibroin particles can be
inherently so small than they can be delivered through a needle
and/or a catheter without any prior compression. In such
embodiments, the silk fibroin particles can have a size smaller
than the inner diameter of a needle and/or a catheter so that the
silk fibroin particles need no prior compression before injection
into a tissue through a needle and/or a catheter.
[0044] In some embodiments, the silk fibroin particles can exhibit
a distribution of particle sizes around the indicated "size." In
such embodiments, the term "particle size" as used herein refers to
the mode of a size distribution of silk fibroin particles, i.e.,
the value that occurs most frequently in the size distribution.
Methods for measuring the particle size are known to a skilled
artisan, e.g., by dynamic light scattering (such as
photocorrelation spectroscopy, laser diffraction, low-angle laser
light scattering (LALLS), and medium-angle laser light scattering
(MALLS)), light obscuration methods (such as Coulter analysis
method), or other techniques (such as rheology, and light or
electron microscopy).
[0045] The silk fibroin particles can be produced from
aqueous-based or organic solvent-based silk fibroin solutions. In
some embodiments, the silk fibroin particles produced from organic
solvent-based silk fibroin solution can retain their original
volume for a longer period of time than the aqueous-based silk
fibroin particles. The aqueous- or organic solvent-based silk
fibroin solution used for making silk fibroin particles described
herein can be prepared using any techniques known in the art. The
concentration of silk fibroin in solutions used for soft tissue
repair or augmentation can be suited to the particular volume
retention requirement, e.g., if higher concentrations of silk
fibroin solutions can be used when longer volume retention of the
silk fibroin particles is desired when injected into the tissue to
be repaired or augmented. In some embodiments, the silk fibroin
solution for making the silk fibroin particles described herein can
vary from about 4% (w/v) to about 30% (w/v), inclusive, or about 4%
(w/v) to about 20% (w/v), inclusive. In some embodiments, the silk
fibroin solution can vary from about 6% (w/v) to about 20% (w/v).
In some embodiments, the silk fibroin solution can vary from about
6% (w/v) to about 17% (w/v). Suitable processes for preparing silk
fibroin solution are disclosed, for example, in U.S. Pat. No.
7,635,755; and International Application Nos: WO/2005/012606; and
WO/2008/127401. A micro-filtration step can be used herein. For
example, the prepared silk fibroin solution can be processed
further, e.g., by centrifugation and/or syringe based
micro-filtration before further processing into silk fibroin
particles described herein.
[0046] In some embodiments, the silk fibroin can be also mixed with
other biocompatible and/or biodegradable polymers to form mixed
polymer particles comprising silk fibroin. One or more
biocompatible and/or biodegradable polymers (e.g., two or more
biocompatible polymers) can be added to the silk fibroin solution.
The biocompatible polymer that can be used herein include, but are
not limited to, polyethylene oxide (PEO), polyethylene glycol
(PEG), collagen, fibronectin, keratin, polyaspartic acid,
polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin,
polycaprolactone, polylactic acid, polyglycolic acid,
polyhydroxyalkanoates, dextrans, polyanhydrides, polymer, PLA-PGA,
polyanhydride, polyorthoester, polycaprolactone, polyfumarate,
collagen, chitosan, alginate, hyaluronic acid and other
biocompatible and/or biodegradable polymers. See, e.g.,
International Application Nos.: WO 04/062697; WO 05/012606.
[0047] In some embodiments, at least one active agent described
herein can be added to the silk fibroin solution before further
processing into silk fibroin particles described herein. In some
embodiments, the active agent can be dispersed homogeneously or
heterogeneously within the silk fibroin, dispersed in a gradient,
e.g., using the carbodiimide-mediated modification method described
in the U.S. Patent Application No. US 2007/0212730.
[0048] In some embodiments, the silk fibroin particles can be first
formed and then contacted with (e.g., dipped into) at least one
active agent such that the open surface of the particles can be
coated with at least one active agent.
[0049] In some embodiments, the silk fibroin particles described
herein can be reduced from a solid-state silk fibroin by a
mechanical means. Exemplary mechanical means to obtain silk fibroin
particles include micronizing, milling, pulverizing, crushing,
grinding, freeze-drying or any combination thereof. Methods of
forming a solid-state silk fibroin from a silk fibroin solution are
well known to a skilled artisan, e.g., using a solvent-based or an
aqueous-based silk fibroin solution. See, e.g., Wang Y. et al.
(2008) 29 Biomaterials 3415, U.S. Pat. No. 7,635,755; and
International Application Nos: WO/2005/012606; and
WO/2008/127401.
[0050] In some embodiments, the silk fibroin particles described
herein can comprise porous structures, e.g., to mimic the
structural morphology of a native tissue, to modulate the
degradation rate/volume retention rate of the silk fibroin
particles, and/or to module release profile of an active agent
embedded therein, if any. As used herein, the terms "porous" and
"porosity" are generally used to describe a structure having a
connected network of pores or void spaces (which can, for example,
be openings, interstitial spaces or other channels) throughout its
volume. The term "porosity" is a measure of 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).
[0051] In some embodiments, the porous silk fibroin particles can
be configured to have any porosity, depending on the desired
properties. For example, in some embodiments, the porous silk
fibroin particles can have a porosity of at least about 1%, at
least about 3%, at least about 5%, at least about 10%, at least
about 15%, at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90% or higher. In some embodiments,
the porosity can range from about 50% to about 99%, about 70% to
about 99%, or from about 80% to about 98%. The pore size and total
porosity values can be quantified using conventional methods and
models known to those of skill in the art. For example, the pore
size and porosity can be measured by standardized techniques, such
as mercury porosimetry and nitrogen adsorption. One of ordinary
skill in the art can determine the optimal porosity of the silk
fibroin particles used for various purposes. For example, the
porosity and/or pore size of the silk fibroin particles can be
optimized based on the desired degradation rate or volume retention
rate of the silk fibroin particles, release profiles of an active
agent from the silk fibroin particles, and/or the structural
morphology of the tissue to be repaired or augmented.
[0052] The pores can be adapted to have any shape, e.g., circular,
elliptical, or polygonal. The porous silk fibroin particles can be
adapted to have a pore size of about 10 nm to about 2000 .mu.m,
from about 50 nm to about 1500 .mu.m, from about 0.5 .mu.m to about
1500 .mu.m, from about 1 .mu.m to about 1500 .mu.m, about 2 .mu.m
to about 1500 .mu.m, from about 1 .mu.m to about 1000 .mu.m, from
about 3 .mu.m to 1000 .mu.m, from about 1 .mu.m to about 500 .mu.m,
or from about 3 .mu.m to about 500 .mu.m. In some embodiments, the
pore size of the porous silk fibroin particles can range from about
3 .mu.m to about 500 .mu.m. In some embodiments, the pore size of
the porous silk fibroin particles can range from about 8 .mu.m to
about 1000 .mu.m. In some embodiments, the silk fibroin particles
need not be porous. In such embodiments, the pore size of the silk
fibroin particles can be less than 10 nm or non-detectable. The
term "pore size" as used herein refers to a dimension of a pore. In
some embodiments, the pore size can refer to the longest dimension
of a pore, e.g., a diameter of a pore having a circular cross
section, or the length of the longest cross-sectional chord that
can be constructed across a pore having a non-circular
cross-section. In other embodiments, the pore size can refer the
shortest dimension of a pore.
[0053] Methods for generating porous structures within silk fibroin
matrix, e.g., freeze-drying, salt-leaching, and gas foaming
methods, are well known in the art and have been described in,
e.g., U.S. Pat. No. 7,842,780; and US Patent Application Nos: US
2010/0279112; and US 2010/0279112, the contents of which are
incorporated herein by reference in their entirety.
[0054] In some embodiments, porous silk fibroin particles can be
produced by a porogen-leaching method (e.g., salt-leaching method).
See, e.g., U.S. Pat. No. 7,842,780; and US 2010/0279112. In some
embodiments, the porous silk fibroin particles can be reduced from
a solid-state porous silk fibroin by a mechanical means as
discussed earlier. By way of example only, the silk fibroin
solution can be placed into a non-stick container (e.g., a
Teflon-coated container) containing water-soluble particles, or
porogens that are insoluble in organic solvents. Alternatively, the
porogens can be mixed with the silk fibroin solution prior to
placement in the container. The diameter of the particles
(porogens) can vary in accordance with the pre-determined pore
size. Examples of water-soluble porogens can be used herein
include, NaCl, alkali metals, alkali earth metal halides,
phosphates, and sulfates, sugar crystals, water-soluble
microspheres, polysaccharides and protein microspheres. The dried
silk fibroin matrix can then be immersed in water or other solvent
in which the particles, or porogens are soluble but silk fibroin is
insoluble, to remove the particles (porogens), resulting in a
porous solid-state silk fibroin described herein. The porous
solid-state silk fibroin can then be reduced into the porous silk
fibroin particles described herein, e.g., by a mechanical means
such as micronizing, milling, pulverizing, crushing, grinding,
freeze-drying or any combination thereof.
[0055] Using a porogen-leaching method (e.g., salt-leaching
method), the silk fibroin particles can be created by, for example,
but not limited to, micronizing a solid-state silk fibroin with
pores corresponding to a porogen size ranging from, e.g., about 300
microns to about 500 microns and/or about 850 microns to about 1000
microns. In some embodiments, the pore size range need not be
affected by a micronization process. Depending on how the
solid-state silk fibroin is cut (or micronized), the resultant silk
fibroin particles can have a pore size corresponding to any portion
of the porogen size range. In some embodiments, the pores of the
silk fibroin particles need not be intact pores (e.g., having a
pore size to be a portion of the porogen size range, e.g., at least
about 1%, at least about 5%, at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 95%, at least about 98% or higher, of the
porogen size range). In some embodiments, the pores of the silk
fibroin particles can be intact (e.g., having a pore size
substantially same as the porogen size). In some embodiments, the
intact pores of the silk fibroin particles can remain essentially
the same as the size of porogens used, e.g., between about 300
microns and 500 microns, or between about 850 microns and about
1000 microns. In some embodiments, the smaller silk fibroin
particles need not maintain intact pores, and thus the pores can be
much smaller than the size of the porogens, for example, at least
about 1%, at least about 5%, at least about 10%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 95%, at least about 98% or higher, of the
porogen size range. In some embodiments, the silk fibroin particles
need not be porous. In various embodiments, the silk fibroin
particles can comprise intact pores, partial pores, or a
combination thereof.
[0056] In alternative embodiments, porous silk fibroin particles
can be produced by freeze-drying method. See, e.g., U.S. Pat. No.
7,842,780 and US 2010/0279112. In such embodiments, the silk
fibroin solution placed in a non-stick container can be frozen at
sub-zero temperatures, e.g., from about -80.degree. C. to about
-20.degree. C., for at least about 12 hours, at least about 24
hours, or longer, followed by lyophilization. In one embodiment,
the silk fibroin solution can be frozen from one direction. In some
embodiments, the silk fibroin solution can contain no salt. In some
embodiments, alcohol such as 15%-25% of methanol or propanol can be
added to the silk fibroin solution. The porous solid-state silk
fibroin can then be reduced into the porous silk fibroin particles
described herein, e.g., by a mechanical means such as micronizing,
milling, pulverizing, crushing, grinding, freeze-drying or any
combination thereof. In some embodiments, the porous solid-state
silk fibroin is not produced by freeze-drying method as described
herein.
[0057] In some embodiments, silk fibroin particles or a solid-state
silk fibroin described herein can be subjected to a post-treatment
that will affect at least one silk fibroin property. For example,
post-treatment of silk fibroin particles or a solid-state silk
fibroin can affect silk fibroin properties including .beta.-sheet
content, solubility, active agent loading capacity, degradation
time, drug permeability, or any combinations thereof. Silk
post-processing options include controlled slow drying (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)), 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).
[0058] In some embodiments, post-treatment of the solid-state silk
fibroin or silk fibroin particles, e.g., water-annealing or solvent
immersion, can allow controlling the release of an active agent
from the silk fibroin particles. In some embodiments,
post-treatment of the solid-state silk fibroin or silk fibroin
particles, e.g., water-annealing or solvent immersion, can enable
modulating the degradation or solubility properties of the silk
fibroin particles described herein. In some embodiments,
post-treatment of the solid-state silk fibroin or silk fibroin
particles, e.g., water-annealing or solvent immersion, can enable
modulating the volume retention properties of the silk fibroin
particles described herein.
[0059] In some embodiments, the silk fibroin particles described
herein can be coated with at least one layer of a biocompatible
and/or biodegradable polymer described herein, e.g., to modulate
the degradation and/or volume retention properties of the silk
fibroin particles upon injection into a tissue to be treated and/or
to modulate the rate of active agents released from the silk
fibroin particles. In such embodiments, the biocompatible and/or
biodegradable polymer can comprise at least one active agent.
[0060] In some embodiments, the silk fibroin particles described
herein can be coated with cell adhesion molecules, e.g., but not
limited to, fibronectin, vitronectin, laminin, collagen, any
art-recognized extracellular matrix molecules, and any combinations
thereof.
[0061] In some embodiments, the silk fibroin particles described
herein can be sterilized. Sterilization methods for biomedical
devices are well known in the art, including, but not limited to,
gamma or ultraviolet radiation, autoclaving (e.g., heat/steam);
alcohol sterilization (e.g., ethanol and methanol); and gas
sterilization (e.g., ethylene oxide sterilization).
[0062] Further, the silk fibroin particles described herein can
take advantage of the many techniques developed to functionalize
silk fibroin (e.g., active agents such as dyes and sensors). See,
e.g., U.S. Pat. No. 6,287,340, Bioengineered anterior cruciate
ligament; WO 2004/000915, Silk Biomaterials & Methods of Use
Thereof; WO 2004/001103, Silk Biomaterials & Methods of Use
Thereof; WO 2004/062697, Silk Fibroin Materials & Use Thereof;
WO 2005/000483, Method for Forming inorganic Coatings; WO
2005/012606, Concentrated Aqueous Silk Fibroin Solution & Use
Thereof; WO 2011/005381, Vortex-Induced Silk fibroin Gelation for
Encapsulation & Delivery; WO 2005/123114, Silk-Based Drug
Delivery System; WO 2006/076711, Fibrous Protein Fusions & Uses
Thereof in the Formation of Advanced Organic/inorganic Composite
Materials; U.S. Application Pub. No. 2007/0212730, Covalently
immobilized protein gradients in three-dimensional porous
scaffolds; WO 2006/042287, Method for Producing Biomaterial
Scaffolds; WO 2007/016524, Method for Stepwise Deposition of Silk
Fibroin Coatings; WO 2008/085904, Biodegradable Electronic Devices;
WO 2008/118133, Silk Microspheres for Encapsulation &
Controlled Release; WO 2008/108838, Microfluidic Devices &
Methods for Fabricating Same; WO 2008/127404, Nanopatterned
Biopolymer Device & Method of Manufacturing Same; WO
2008/118211, Biopolymer Photonic Crystals & Method of
Manufacturing Same; WO 2008/127402, Biopolymer Sensor & Method
of Manufacturing Same; WO 2008/127403, Biopolymer Optofluidic
Device & Method of Manufacturing the Same; WO 2008/127401,
Biopolymer Optical Wave Guide & Method of Manufacturing Same;
WO 2008/140562, Biopolymer Sensor & Method of Manufacturing
Same; WO 2008/127405, Microfluidic Device with Cylindrical
Microchannel & Method for Fabricating Same; WO 2008/106485,
Tissue-Engineered Silk Organs; WO 2008/140562, Electroactive
Biopolymer Optical & Electro-Optical Devices & Method of
Manufacturing Same; WO 2008/150861, Method for Silk Fibroin
Gelation Using Sonication; WO 2007/103442, Biocompatible Scaffolds
& Adipose-Derived Stem Cells; WO 2009/155397, Edible
Holographic Silk Products; WO 2009/100280, 3-Dimensional Silk
Hydroxyapatite Compositions; WO 2009/061823, Fabrication of Silk
Fibroin Photonic Structures by Nanocontact Imprinting; WO
2009/126689, System & Method for Making Biomaterial
Structures.
[0063] In an alternative embodiment, the silk fibroin particles can
include plasmonic nanoparticles to form photothermal elements. This
approach takes advantage of the superior doping characteristics of
silk fibroin. Thermal therapy has been shown to aid in the
transdermal 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 silk fibroin
particles can add specificity to thermal therapy by focusing light
to locally generate heat only via the particles. In some
embodiments, the silk fibroin particles can include photothermal
agents such as gold nanoparticles.
[0064] In some embodiments, the silk fibroin particles can include
an amphiphilic peptide. In other embodiments, the silk fibroin
particles can exclude an amphiphilic peptide. "Amphiphilic
peptides" possess both hydrophilic and hydrophobic properties.
Amphiphilic molecules can generally interact with biological
membranes by insertion of the hydrophobic part into the lipid
membrane, while exposing the hydrophilic part to the aqueous
environment. In some embodiment, the amphiphilic peptide can
comprise a RGD motif. An example of an amphiphilic peptide is a
23RGD peptide having an amino acid sequence:
HOOC-Gly-ArgGly-Asp-Ile-Pro-Ala-Ser-Ser-Lys-Gly-Gly-Gly-Gly-SerArg-Leu-Le-
u-Leu-Leu-Leu-Leu-Arg-NH2. Other examples of amphiphilic peptides
include the ones disclosed in the U.S. Patent App. No.: US
2011/0008406.
Injectable Compositions Comprising Silk Fibroin Particles
[0065] In another aspect, provided herein is an injectable
composition for use in repairing or augmenting a tissue in a
subject comprising a plurality of silk fibroin particles described
herein, wherein at least a portion of the silk fibroin particles
retain their original volume (e.g., at least about 50% or higher)
within the tissue to be repaired or augmented for a period of time
(e.g., at least about 6 weeks or longer).
[0066] As used herein, the term "injectable composition" generally
refers to a composition that can be delivered or administered into
a tissue with a minimally invasive procedure. The term "minimally
invasive procedure" refers to a procedure that is carried out by
entering a subject's body through the skin or through a body cavity
or an anatomical opening, but with the smallest damage possible
(e.g., a small incision, injection). In some embodiments, the
injectable composition can be administered or delivered into a
tissue by injection. In some embodiments, the injectable
composition can be delivered into a tissue through a small incision
on the skin followed by insertion of a needle, a cannula, and/or
tubing, e.g., a catheter. Without wishing to be limited, the
injectable composition can be administered or placed into a tissue
by surgery, e.g., implantation.
[0067] In some embodiments, the injectable compositions can
comprise at least one active agent described herein. In some
embodiments, the injectable compositions can comprise at least one
cell. The term "cells" used herein refers to any cell, prokaryotic
or eukaryotic, including plant, yeast, worm, insect and mammalian.
In some embodiments, the cells can be mammalian cells. Mammalian
cells include, without limitation; primate, human and a cell from
any animal of interest, including without limitation; mouse,
hamster, rabbit, dog, cat, domestic animals, such as equine,
bovine, murine, ovine, canine, feline, etc. The cells can be a wide
variety of tissue types without limitation such as; hematopoietic,
neural, mesenchymal, cutaneous, mucosal, stromal, muscle spleen,
reticuloendothelial, epithelial, endothelial, hepatic, kidney,
gastrointestinal, pulmonary, T-cells etc. Stem cells, pluripotent
stem cells (iPSCs), embryonic stem (ES) cells, ES-derived cells and
stem cell progenitors are also included, including without
limitation, hematopoietic, neural, stromal, muscle, cardiovascular,
hepatic, pulmonary, and gastrointestinal stem cells and
adipose-derived stem cells. In one embodiment, the cells are
adipose-derived stem cells. In some embodiments, the cells can be
ex vivo or cultured cells, e.g. in vitro. For example, for ex vivo
cells, cells can be obtained from a subject, where the subject is
healthy and/or affected with a disease.
[0068] Cells can be obtained, as a non-limiting example, by biopsy
or other surgical means known to those skilled in the art. In some
embodiments, adipose cells can be harvested from a subject by
conventional liposuction or aspiration techniques. In such
embodiments, the cells can be derived from a lipoaspirate. In other
embodiments, the cells can be derived from a bone-marrow aspirate.
Depending on the types of tissues to be repaired or augmented,
cells can be derived from any biological fluid or concentrate,
e.g., a lipoaspirate or a bone-marrow aspirate.
[0069] Accordingly, in some embodiments, the injectable composition
or the silk fibroin particles can be directly delivered with a
biological fluid or concentrate, e.g., a lipoaspirate or a
bone-marrow aspirate. In some embodiments, the injectable
composition or the composition comprising a plurality of silk
fibroin particles can further comprise a biological fluid or
concentrate, such as lipoaspirate, bone marrow aspirate, or any
combinations thereof. In one embodiment, the injectable composition
or the composition comprising a plurality of silk fibroin particles
can further comprise a lipoaspirate.
[0070] In these embodiments, the composition or the injectable
composition can comprise silk fibroin particles and a biological
fluid or concentrate (e.g., a lipoaspirate or a bone-marrow
aspirate) in a volume ratio of about 1:38 to about 12:19, or about
1:19 to about 10:19, or about 2:19 to about 8:19. In one
embodiment, the composition or the injectable composition can
comprise silk fibroin particles and a biological fluid or
concentrate (e.g., a lipoaspirate or a bone-marrow aspirate) in a
volume ratio of about 3:19. In another embodiment, the composition
or the injectable composition can comprise silk fibroin particles
and a biological fluid or concentrate (e.g., a lipoaspirate or a
bone-marrow aspirate) in a volume ratio of about 6:19.
[0071] Cells can be obtained from donors (allogenic) or from
recipients (autologous). Cells can also be of established cell
culture lines, or even cells that have undergone genetic
engineering. Additionally, cells can be collected from a multitude
of hosts including but not limited to human autograft tissues,
transgenic mammals, or bacterial cultures (possibly for use as a
probiotic treatment). In certain embodiments, the injectable
compositions and/or silk fibroin particles can comprise human stem
cells such as, e.g., mesenchymal stem cells, pluripotent stem cells
(iPSCs), synovial derived stem cells, embryonic stem cells, adult
stem cells, umbilical cord blood cells, umbilical Wharton's jelly
cells, osteocytes, fibroblasts, neuronal cells, lipocytes,
adipocytes, bone marrow cells, assorted immunocytes, precursor
cells derived from adipose tissue, bone marrow derived progenitor
cells, peripheral blood progenitor cells, stem cells isolated from
adult tissue and genetically transformed cells or combinations of
the above cells; or differentiated cells such as, e.g., muscle
cells, adipose cells.
[0072] Stem cells can be obtained with minimally invasive
procedures from bone marrow, adipose tissue, or other sources in
the body, are highly expandable in culture, and can be readily
induced to differentiate into adipose tissue forming cells after
exposure to a well-established adipogenic inducing supplement.
Cells can be added to the injectable compositions and/or silk
fibroin particles described herein and cultured in vitro for a
period of time prior to administration to a region of the body, or
added to injectable compositions and/or silk fibroin particles
described herein and administered into a region of the body. The
cells can be seeded on the silk fibroin particles for a short
period of time (less than 1 day) just prior to administration, or
cultured for a longer (more than 1 day) period to allow for cell
proliferation and extracellular matrix synthesis within the seeded
matrix prior to administration.
[0073] When utilized as a source of stem cells, adipose tissue can
be obtained by any method known to a person of ordinary skill in
the art. For example, adipose tissue can be removed from an
individual by suction-assisted lipoplasty, ultrasound-assisted
lipoplasty, and excisional lipectomy. In addition, the procedures
can include a combination of such procedures. Suction assisted
lipoplasty can be desirable to remove the adipose tissue from an
individual as it provides a minimally invasive method of collecting
tissue with minimal potential for stem cell damage that can be
associated with other techniques, such as ultrasound assisted
lipoplasty. The adipose tissue should be collected in a manner that
preserves the viability of the cellular component and that
minimizes the likelihood of contamination of the tissue with
potentially infectious organisms, such as bacteria and/or
viruses.
[0074] In some embodiments, preparation of the cell population can
require depletion of the mature fat-laden adipocyte component of
adipose tissue. This is typically achieved by a series of washing
and disaggregation steps in which the tissue is first rinsed to
reduce the presence of free lipids (released from ruptured
adipocytes) and peripheral blood elements (released from blood
vessels severed during tissue harvest), and then disaggregated to
free intact adipocytes and other cell populations from the
connective tissue matrix. Disaggregation can be achieved using any
conventional techniques or methods, including mechanical force
(mincing or shear forces), enzymatic digestion with single or
combinatorial proteolytic enzymes, such as collagenase, trypsin,
lipase, liberase HI and pepsin, or a combination of mechanical and
enzymatic methods. For example, the cellular component of the
intact tissue fragments can be disaggregated by methods using
collagenase-mediated dissociation of adipose tissue, similar to the
methods for collecting microvascular endothelial cells in adipose
tissue, as known to those of skill in the art. Additional methods
using collagenase that can be used are also known to those of skill
in the art. Furthermore, methods can employ a combination of
enzymes, such as a combination of collagenase and trypsin or a
combination of an enzyme, such as trypsin, and mechanical
dissociation.
[0075] The cell population (processed lipoaspirate) can then be
obtained from the disaggregated tissue fragments by reducing the
presence of mature adipocytes. Separation of the cells can be
achieved by buoyant density sedimentation, centrifugation,
elutriation, differential adherence to and elution from solid phase
moieties, antibody-mediated selection, differences in electrical
charge; immunomagnetic beads, fluorescence activated cell sorting
(FACS), or other means.
[0076] Following disaggregation the active cell population can be
washed/rinsed to remove additives and/or by-products of the
disaggregation process (e.g., collagenase and newly released free
lipid). The active cell population could then be concentrated by
centrifugation. In one embodiment, the cells are concentrated and
the collagenase removed by passing the cell population through a
continuous flow spinning membrane system or the like, such as, for
example, the system disclosed in U.S. Pat. Nos. 5,034,135; and
5,234,608, which are incorporated by reference herein.
[0077] In addition to the foregoing, there are many post-wash
methods that can be applied for further purifying the cell
population. These include both positive selection (selecting the
target cells), negative selection (selective removal of unwanted
cells), or combinations thereof. In another embodiment the cell
pellet could be resuspended, layered over (or under) a fluid
material formed into a continuous or discontinuous density gradient
and placed in a centrifuge for separation of cell populations on
the basis of cell density. In a similar embodiment, continuous flow
approaches such as apheresis and elutriation (with or without
countercurrent) could be used. Adherence to plastic followed by a
short period of cell expansion has also been applied in bone
marrow-derived adult stem cell populations. This approach uses
culture conditions to preferentially expand one population while
other populations are either maintained (and thereby reduced by
dilution with the growing selected cells) or lost due to absence of
required growth conditions. The cells that have been concentrated,
cultured and/or expanded can be incorporated into the silk fibroin
particles and/or injectable compositions described herein.
[0078] In one embodiment, stem cells are harvested, the harvested
cells are contacted with an adipogenic medium for a time sufficient
to induce differentiation into adipocytes, and the adipocytes are
loaded onto a biocompatible matrix which is implanted. In
additional embodiments, at least some of the stem cells can be
differentiated into adipocytes so that a mixture of both cell types
is initially present that changes over time to substantially only
adipocytes, with stem cells being present in small to undetectable
quantities. Adipose tissue is fabricated in vivo by the stem cells
or prepared ex vivo by the stem cells.
[0079] A number of different cell types or combinations thereof can
be employed in the injectable compositions, depending upon the
types of tissues to be repaired or augmented. These cell types
include, but are not limited to: smooth muscle cells, skeletal
muscle cells, cardiac muscle cells, epithelial cells, endothelial
cells, urothelial cells, fibroblasts, myoblasts, chondrocytes,
chondroblasts, osteoblasts, osteoclasts, keratinocytes,
hepatocytes, bile duct cells, pancreatic islet cells, thyroid,
parathyroid, adrenal, hypothalamic, pituitary, ovarian, testicular,
salivary gland cells, adipocytes, and precursor cells. By way of
example only, smooth muscle cells and endothelial cells can be
employed when the injectable compositions are used to repair or
augment muscular and/or vascular tissues, such as vascular,
esophageal, intestinal, rectal, or ureteral tissues; chondrocytes
can be included in injectable compositions for cartilaginous
tissues; fibroblasts can be included in injectable compositions
intended to replace and/or enhance any of the wide variety of
tissue types (e.g., skin) that contains extracellular matrix, e.g.,
collagen; adipocytes can be included in injectable compositions
intended to repair or augment any of the wide variety of adipose
tissues. In general, any cells that are found in the natural tissue
can be included in the injectable compositions used for
corresponding tissue. In addition, progenitor cells, such as
myoblasts or stem cells, can be included to produce their
corresponding differentiated cell types.
[0080] In some embodiments, the injectable compositions can further
comprise a pharmaceutically acceptable carrier. As used herein, the
term "pharmaceutically acceptable carrier" refers to a
pharmaceutically-acceptable material, composition or vehicle for
administration of the silk fibroin particles, and optionally an
active agent. Pharmaceutically acceptable carriers include any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, and isotonic and absorption delaying agents,
which are compatible with the silk fibroin particles and the
activity of the active agent, if any, and are physiologically
acceptable to the subject. The pharmaceutical formulations suitable
for injection include sterile aqueous solutions or dispersions. The
carrier can be a solvent or dispersing medium containing, for
example, water, cell culture medium, buffers (e.g., phosphate
buffered saline), polyol (for example, glycerol, propylene glycol,
liquid polyethylene glycol, and the like), suitable mixtures
thereof. In some embodiments, the pharmaceutical carrier can be a
buffered solution (e.g. PBS).
[0081] Additionally, various additives which enhance the stability,
sterility, and isotonicity of the injectable compositions,
including antimicrobial preservatives, antioxidants, chelating
agents, and buffers, can be added. Prevention of the action of
microorganisms can be ensured by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, and the like. In many cases, it may be desirable to
include isotonic agents, for example, sugars, sodium chloride, and
the like. The injectable compositions can also contain auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents, gelling or viscosity enhancing additives, preservatives,
colors, and the like, depending upon the preparation desired.
Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th
edition, 1985, incorporated herein by reference, may be consulted
to prepare suitable preparations, without undue
experimentation.
[0082] Viscosity of the injectable compositions can be maintained
at the selected level using a pharmaceutically acceptable
thickening agent. In one embodiment, methylcellulose is used
because it is readily and economically available and is easy to
work with. Other suitable thickening agents include, for example,
xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose,
carbomer, and the like. The preferred concentration of the
thickener will depend upon the agent selected, and the desired
viscosity for injection. The important point is to use an amount
which will achieve the selected viscosity, e.g., addition of such
thickening agents into some embodiments of the injectable
compositions.
[0083] Typically, any additives (in addition to the silk fibroin
particles described herein and/or additional active agents) can be
present in an amount of 0.001 to 50 wt % dry weight or in a
buffered solution. In some embodiments, the active agent can be
present in the order of micrograms to milligrams to grams, such as
about 0.0001 to about 5 wt %, about 0.0001 to about 1 wt %, about
0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, about
0.01 to about 10 wt %, and about 0.05 to about 5 wt %. For any
pharmaceutical composition to be administered to a subject in need
thereof, it is preferred to determine toxicity, such as by
determining the lethal dose (LD) and LD50 in a suitable animal
model e.g., rodent such as mouse; and, the dosage of the
composition(s), concentration of components therein and timing of
administering the composition(s), which elicit a suitable response.
Such determinations do not require undue experimentation from the
knowledge of the skilled artisan.
Active Agents
[0084] In some embodiments, the injectable composition and/or the
silk fibroin particles described herein can further comprise at
least one active agent. The active agent can be mixed, dispersed,
or suspended in the injectable composition, and/or it can be
distributed or embedded in the silk fibroin particles. In some
embodiments, the active agent can be distributed, embedded or
encapsulated in the silk fibroin particles. In some embodiments,
the active agent can be coated on surfaces of the silk fibroin
particles. In some embodiments, the active agent can be mixed with
the silk fibroin particles to form an injectable composition. The
term "active agent" can also encompass combinations or mixtures of
two or more active agents, as described below. Examples of active
agents include, but are not limited to, a biologically active agent
(e.g., a therapeutic agent), a cosmetically active agent (e.g., an
anti-aging agent), a cell attachment agent (e.g., integrin-binding
molecules), and any combinations thereof.
[0085] The term "biologically active agent" as used herein refers
to any molecule which exerts at least one biological effect in
vivo. For example, the biologically active agent can be a
therapeutic agent to treat or prevent a disease state or condition
in a subject. Examples of biologically active agents include,
without limitation, peptides, peptidomimetics, aptamers, antibodies
or a portion thereof, antibody-like molecules, nucleic acids (DNA,
RNA, siRNA, shRNA), polysaccharides, enzymes, receptor antagonists
or agonists, hormones, growth factors, autogenous bone marrow,
antibiotics, antimicrobial agents, small molecules and therapeutic
agents. The biologically active agents can also include, without
limitations, anti-inflammatory agents, anesthetics, active agents
that stimulate issue formation, and/or healing and regrowth of
natural tissues, and any combinations thereof.
[0086] Anti-inflammatory agents can include, but are not limited
to, naproxen, sulindac, tolmetin, ketorolac, celecoxib, ibuprofen,
diclofenac, acetylsalicylic acid, nabumetone, etodolac,
indomethacin, piroxicam, cox-2 inhibitors, ketoprofen, antiplatelet
medications, salsalate, valdecoxib, oxaprozin, diflunisal,
flurbiprofen, corticosteroids, MMP inhibitors and leukotriene
modifiers or combinations thereof.
[0087] Agents that increase formation of new tissues and/or
stimulates healing or regrowth of native tissue at the site of
injection can include, but are not limited to, fibroblast growth
factor (FGF), transforming growth factor-beta (TGF-.beta.,
platelet-derived growth factor (PDGF), epidermal growth factors
(EGFs), connective tissue activated peptides (CTAPs), osteogenic
factors including bone morphogenic proteins, heparin, angiotensin
II (A-II) and fragments thereof, insulin-like growth factors, tumor
necrosis factors, interleukins, colony stimulating factors,
erythropoietin, nerve growth factors, interferons, biologically
active analogs, fragments, and derivatives of such growth factors,
and any combinations thereof.
[0088] Anesthetics can include, but are not limited to, those used
in caudal, epidural, inhalation, injectable, retrobulbar, and
spinal applications, such as bupivacaine, lidocaine, benzocaine,
cetacaine, ropivacaine, and tetracaine, or combinations
thereof.
[0089] In some embodiments, the active agents can be cosmetically
active agents. By the term "cosmetically active agent" is meant a
compound that has a cosmetic or therapeutic effect on the skin,
hair, or nails, e.g., anti-aging agents, anti-free radical agents,
lightening agents, whitening agents, depigmenting agents, darkening
agents such as self-tanning agents, colorants, anti-acne agents,
shine control agents, anti-microbial agents, anti-inflammatory
agents, anti-mycotic agents, anti-parasite agents, external
analgesics, sun-blocking agents, photoprotectors, antioxidants,
keratolytic agents, detergents/surfactants, moisturizers,
nutrients, vitamins, energy enhancers, anti-perspiration agents,
astringents, deodorants, hair removers, firming agents,
anti-callous agents, muscle relaxants, agents for hair, nail,
and/or skin conditioning, and any combination thereof.
[0090] In one embodiment, the cosmetically active agent can be
selected from, but not limited to, the group consisting of hydroxy
acids, benzoyl peroxide, sulfur resorcinol, ascorbic acid,
D-panthenol, hydroquinone, octyl methoxycinnamate, titanium
dioxide, octyl salicylate, homosalate, avobenzone, polyphenolics,
carotenoids, free radical scavengers, ceramides, polyunsaturated
fatty acids, essential fatty acids, enzymes, enzyme inhibitors,
minerals, hormones such as estrogens, steroids such as
hydrocortisone, 2-dimethylaminoethanol, copper salts such as copper
chloride, coenzyme Q10, lipoic acid, amino acids such a proline and
tyrosine, vitamins, lactobionic acid, acetyl-coenzyme A, niacin,
riboflavin, thiamin. ribose, electron transporters such as NADH and
FADH2, and other botanical extracts such as aloe vera, feverfew,
and soy, and derivatives and mixtures thereof. Examples of vitamins
include, but are not limited to, vitamin A, vitamin Bs (such as
vitamin B3, vitamin B5, and vitamin B12), vitamin C, vitamin K, and
vitamin E, and derivatives thereof.
[0091] In one embodiment, the cosmetically active agents can be
antioxidants. Examples of antioxidants include, but are not limited
to, water-soluble antioxidants such as sulfhydryl compounds and
their derivatives (e.g., sodium metabisulfite and
N-acetyl-cysteine), lipoic acid and dihydrolipoic acid,
resveratrol, lactoferrin, ascorbic acid, and ascorbic acid
derivatives (e.g., ascorbyl palmitate and ascorbyl polypeptide).
Oil-soluble antioxidants suitable for use in the compositions
described herein include, but are not limited to, butylated
hydroxytoluene, tocopherols (e.g., tocopheryl acetate),
tocotrienols, and ubiquinone. Natural extracts containing
antioxidants suitable for use in the injectable compositions
described herein, include, but not limited to, extracts containing
flavonoids and isoflavonoids and their derivatives (e.g., genistein
and diadzein), and extracts containing resveratrol. Examples of
such natural extracts include grape seed, green tea, pine bark, and
propolis. Other examples of antioxidants can be found on pages
1612-13 of the ICI Handbook.
[0092] In some embodiments, the active agents can be cell
attachment agents. Examples of cell attachment agents include, but
are not limited to, hyaluronic acid, collagen, crosslinked
hyaluronic acid/collagen, an integrin-binding molecule, chitosan,
elastin, fibronectin, vitronectin, laminin, proteoglycans, any
derivatives thereof, and any combinations thereof.
[0093] In some embodiments, the injectable compositions and/or silk
fibroin particles can further comprise at least one additional
material for soft tissue augmentation, e.g., dermal filler
materials, including, but not limited to, poly(methyl methacrylate)
microspheres, hydroxylapatite, poly(L-lactic acid), collagen,
elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal
filler products such as BOTOX.RTM. (from Allergan), DYSPORT.RTM.,
COSMODERM.RTM., EVOLENCE.RTM., RADIESSE.RTM., RESTYLANE.RTM.,
JUVEDERM.RTM. (from Allergan), SCULPTRA.RTM., PERLANE.RTM., and
CAPTIQUE.RTM., and any combinations thereof.
[0094] In some embodiments, the injectable composition and/or silk
fibroin particles can comprise metallic nanoparticles (e.g., but
not limited to, gold nanoparticles), optical molecules (e.g., but
not limited to, fluorescent molecules, and/or quantum dots), and
any other art-recognized contrast agent, e.g., for biomedical
imaging.
[0095] In various embodiments, the injectable compositions can be
stored or transported dried or hydrated.
[0096] When the active agents are embedded in the silk fibroin
particles, the bioactivity of the active agents (e.g., at least
about 30% of the bioactivity of the active agents) can be
stabilized for a period of time (e.g., days, weeks, or months)
under specific conditions. Such conditions can include, but are not
limited to, a state-changing cycle (e.g., freeze-thaw cycles),
temperatures (e.g., above 0.degree. C.), air pressures, humidity,
and light exposure. See U.S. Application Ser. No. 61/477,737. Some
embodiments of the injectable composition can be stored or
transported between about 0.degree. C. and about 60.degree. C.,
about 10.degree. C. and about 60.degree. C., or about 15.degree. C.
and about 60.degree. C. In these embodiments, the injectable
compositions can be stored or transported at room temperatures
while the bioactivity of the active agents (e.g., at least about
30% of the bioactivity of the active agents) can be stabilized for
a period of time, e.g., at least about 3 weeks or longer.
Applications of Injectable Compositions and Silk Fibroin Particles
Described Herein
[0097] The injectable compositions described herein can be used in
a variety of medical uses, including, without limitation, fillers
for tissue space, templates for tissue reconstruction or
regeneration, scaffolds for cells in tissue engineering
applications, or as a vehicle/carrier for drug delivery. A
plurality of silk fibroin particles injected into a tissue to be
repaired or augmented can act as a scaffold to mimic the
extracellular matrices (ECM) of the body, and/or promote tissue
regeneration. The scaffold can serve as both a physical support
and/or an adhesive template for cells to proliferate therein. In
some embodiments, the silk fibroin particles can contain no cells.
Yet the silk fibroin particles can be coated with cell attachment
agents, e.g., collagen, and/or chemoattractants, e.g., growth
factors, that can attract host cells to the silk fibroin particles
and support the cell proliferation. In some embodiments, the silk
fibroin particles can be seeded with cells prior to administration
to a target tissue to be repaired or augmented.
[0098] In some embodiments, provided herein are injectable
compositions that can be used to fill, volumize, and/or regenerate
a tissue in need thereof. The injectable compositions can generally
be used for tissue filling or volumizing, soft tissue augmentation,
replacement, cosmetic enhancement and/or tissue repair in a
subject. Additionally, the injectable compositions can be used for
filling of any tissue void or indentation that are either naturally
formed (e.g., aging) or created by surgical procedure for removal
of tissue (e.g., a dermal cyst or a solid tumor), corticosteroid
treatment, immunologic reaction resulting in lipoatrophy, tissue
damage resulting from impact injuries or therapeutic treatment
(e.g., radiotherapy or chemotherapy). The injectable compositions
can also be used to raise scar depressions.
[0099] In certain embodiments, the injectable compositions can be
used for soft tissue augmentation. As used herein, by the term
"augmenting" or "augmentation" is meant increasing, filling in,
restoring, enhancing or replacing a tissue. In some embodiments,
the tissue can lose its elasticity, firmness, shape and/or volume.
In some embodiments, the tissue can be partially or completely lost
(e.g., removal of a tissue) or damaged. In those embodiments, the
term "augmenting" or "augmentation" can also refer to decreasing,
reducing or alleviating at least one symptom or defect in a tissue
(for example, but not limited to, loss of elasticity, firmness,
shape and/or volume in a tissue; presence of a void or an
indentation in a tissue; loss of function in a tissue) by injecting
into the tissue with at least one injectable composition described
herein. In such embodiments, at least one symptom or defect in a
tissue can be decreased, reduced or alleviated by at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80% or higher, as compared to no
treatment. In some embodiments, at least one symptom or defect in a
tissue can be decreased, reduced or alleviated by at least about
90%, at least about 95%, at least about 97%, or higher, as compared
to no treatment. In some embodiments, at least one symptom or
defect in a tissue can be decreased, reduced or alleviated by 100%
(defect-free or the defect is undetectable by one of skill in the
art), as compared to no treatment. In other embodiments, the tissue
can be augmented to prevent or delay the onset of defect
manifestation in a tissue, e.g., loss of elasticity, firmness,
shape and/or volume in a tissue, or signs of wrinkles. As used
herein, the phrase "soft tissue augmentation" is generally used in
reference to altering a soft tissue structure, including but not
limited to, increasing, filling in, restoring, enhancing or
replacing a tissue, e.g., to improve the cosmetic or aesthetic
appearance of the soft tissue. For example, breast augmentation
(also known as breast enlargement, mammoplasty enlargement,
augmentation mammoplasty) alters the size and shape of a woman's
breasts to improve the cosmetic or aesthetic appearance of the
woman. Examples of soft tissue augmentation includes, but is not
limited to, dermal tissue augmentation; filling of lines, folds,
wrinkles, minor facial depressions, and cleft lips, especially in
the face and neck; correction of minor deformities due to aging or
disease, including in the hands and feet, fingers and toes;
augmentation of the vocal cords or glottis to rehabilitate speech;
dermal filling of sleep lines and expression lines; replacement of
dermal and subcutaneous tissue lost due to aging; lip augmentation;
filling of crow's feet and the orbital groove around the eye;
breast augmentation; chin augmentation; augmentation of the cheek
and/or nose; bulking agent for periurethral support, filling of
indentations in the soft tissue, dermal or subcutaneous, due to,
e.g., overzealous liposuction or other trauma; filling of acne or
traumatic scars; filling of nasolabial lines, nasoglabellar lines
and intraoral lines. In some embodiments, the injectable
compositions and/or silk fibroin particles described herein can be
used to treat facial lipodystrophies. In some embodiments, the
injectable compositions can be used for breast augmentation and/or
reconstruction.
[0100] In some embodiments, the injectable compositions can be used
for soft tissue repair. The term "repair" or "repairing" as used
herein, with respect to a tissue, refers to any correction,
reinforcement, reconditioning, remedy, regenerating, filling of a
tissue that restores volume, shape and/or function of the tissue.
In some embodiments "repair" includes full repair and partial
repair. For example, the volume, shape and/or function of a tissue
to be repaired can be restored by at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80% or higher, as compared to no treatment. In
some embodiments, the volume, shape and/or function of a tissue to
be repaired can be restored by at least about 90%, at least about
95%, at least about 97%, or higher, as compared to no treatment. In
some embodiments, the volume, shape and/or function of a tissue to
be repaired can be restored by 100% (defect-free or the defect is
undetectable by one of skill in the art), as compared to no
treatment. In various embodiments, the injectable compositions can
be used to repair any soft tissues discussed earlier, e.g., breast,
skin, and any soft tissues amenable for soft tissue augmentation.
In some embodiments, the term "repair" or "repairing" are used
herein interchangeably with the term "regeneration" or "regenerate"
when used in reference to tissue treatment.
[0101] In some embodiments, the injectable compositions can be used
for soft tissue reconstruction. As used herein, the phrase "soft
tissue reconstruction" refers to rebuilding a soft tissue structure
that was severely damaged or lost, e.g., by a dramatic accident or
surgical removal. For example, breast reconstruction is the
rebuilding of a breast, usually in women. Conventional methods of
construct a natural-looking breast generally involve using
autologous tissue or prosthetic material. In some embodiments, such
breast reconstruction can include reformation of a natural-looking
areola and nipple, wherein such procedure can involve the use of
implants or relocated flaps of the patient's own tissue. In some
embodiments, administration of injectable compositions and/or silk
fibroin particles into a soft tissue region to be reconstructed can
maintain the shape and/or size of the reconstructed soft tissue
structure for a period of time, e.g., at least 6 weeks, at least
about 2 months, at least about 3 months or longer.
[0102] Without wishing to be bound, some embodiments of the
injectable compositions can be used for hard tissue
(musculoskeletal) augmentation or repair, such as augmentation or
repair of bone, cartilage and ligament.
[0103] The injectable compositions and silk fibroin particles
described herein can also be used for filling a tissue located at
or near a prosthetic implant, for example, but not limited to, a
conventional breast implant or knee replacement implant. In some
embodiments, the injectable compositions and silk fibroin particles
can be used to interface between a prosthetic implant and a tissue,
e.g., to fill a void between the prosthetic implant and the tissue,
and/or to prevent the tissue in direct contact with the prosthetic
implant. By way of example only, after placing a prosthetic implant
(e.g., a breast implant) in a subject, an injectable composition
described herein can be introduced at or adjacent to the implant to
fill any void between the implant and the tissue (e.g., breast
tissue) and/or "sculpt" the tissue for a more natural look.
[0104] In any of the uses described herein, silk fibroin particles
could be combined with cells for purposes of a biologically
enhanced repair. Cells could be collected from a multitude of hosts
including but not limited to human autograft tissues, or transgenic
mammals. More specifically, human cells used can comprise cells
selected from stem cells (e.g., adipocyte-derived stem cells),
osteocytes, fibroblasts, lipocytes, assorted immunocytes, cells
from lipoaspirate or any combinations thereof. In some embodiments,
the cells can be added after rinsing of the silk fibroin particles
themselves. They can be blended into the silk fibroin particles,
carrier solution, or mixture of silk fibroin particles and carrier
solution prior to injection.
[0105] In some embodiments, administering the cells (e.g., stem
cells or lipoaspirate) with silk fibroin particles or an injectable
composition described herein can enhance or accelerate host
integration and/or tissue formation over time. The cells can be
mixed with the silk fibroin particles or an injectable composition
described herein, or they can be administered prior to,
concurrently with, or after the silk fibroin particles or an
injectable composition is introduced into a target site. Without
wishing to be bound by theory, the cells can secrete pro-angiogenic
factors and/or growth factors at the target site. As the tissue
regenerates or remodels to fill up a void or repair a defect, the
silk fibroin particles can degrade accordingly. In some
embodiments, the silk fibroin particles can integrate with the
regenerated host tissue.
[0106] In addition, active agents such as therapeutic agents,
pharmaceuticals, or specific growth factors added to the silk
fibroin particles for purposes of improved outcome can be
introduced at any or a combination of several points throughout the
silk fibroin particle production process. In some embodiments,
these factors can be added to silk fibroin solution or the
accelerant phase prior to drying and solidification, they can be
soaked into the silk fibroin matrix during the accelerant rinsing
process, or they can be coated onto the bulk silk fibroin following
rinsing. The active-agent containing solid-state silk fibroin can
then be reduced into particles using the methods described herein.
In some embodiments, a solid-state silk fibroin can be reduced into
particles before introducing an active agent into the silk fibroin
particles. For example, an active agent can be soaked into the silk
fibroin particles, coated onto the silk fibroin particles, or
introduced into a carrier fluid before or after blending with the
silk fibroin particles.
[0107] In some aspects, the injectable composition and silk fibroin
particles described herein can be used as tissue space fillers. In
one embodiment, the tissue space filler is a dermal filler. The
dermal filler can be used to improve skin appearance or condition,
including, but not limited to, rehydrating the skin, providing
increased elasticity to the skin, reducing skin roughness, making
the skin tauter, reducing or eliminating stretch lines or marks,
giving the skin better tone, shine, brightness, and/or radiance,
reducing or eliminating wrinkles in the skin, providing wrinkle
resistance to the skin and replacing loss of soft tissue.
[0108] A dermal filler comprising silk fibroin particles can be
modulated for particle hardness and opacity through alteration of
silk fibroin concentration and formulation method. In one
embodiment, a dermal filler can be produced by forming a
solid-state silk fibroin from a silk fibroin solution of about 6%
(w/v) to about 20% (w/v), followed by reducing the solid-state silk
fibroin into silk fibroin particles such that they can be injected
into a tissue through a needle or a cannula. The needle or cannula
can have an outer diameter of no larger than 3 mm, no larger than 2
mm, no larger than 1 mm, no larger than 0.8 mm, no larger than 0.6
mm, no larger than 0.4 mm, no larger than 0.2 mm or no larger than
0.1 mm. In some embodiments, the needle or cannula gauge can range
from 12 to 34, 15 to 34, 20 to 32, or 25 to 30. The size of the
needle or cannula can be determined to allow for an appropriate
extrusion force of less than 40 N (nominal deliverable injection
force for a human hand).
[0109] Accordingly, another aspect described herein provides a
method of improving a condition and/or appearance of skin in a
subject in need thereof. Non-limiting examples of a skin condition
or and/or appearance include dehydration, lack of skin elasticity,
roughness, lack of skin tautness, skin stretch line and/or marks,
skin paleness, and skin wrinkles. The method comprises injecting an
injectable composition described herein into a dermal region of the
subject, wherein the injection improves the skin condition and/or
appearance. For example, improving a skin appearance include, but
are not limited to, rehydrating the skin, providing increased
elasticity to the skin, reducing skin roughness, making the skin
tauter, reducing or eliminating stretch lines or marks, giving the
skin better tone, shine, brightness and/or radiance to reduce
paleness, reducing or eliminating wrinkles in the skin, and
providing wrinkle resistance to the skin.
[0110] As used herein, the term "dermal region" refers to the
region of skin comprising the epidermal-dermal junction and the
dermis including the superficial dermis (papillary region) and the
deep dermis (reticular region). The skin is composed of three
primary layers: the epidermis, which provides waterproofing and
serves as a barrier to infection; the dermis, which serves as a
location for the appendages of skin; and the hypodermis
(subcutaneous adipose layer). The epidermis contains no blood
vessels, and is nourished by diffusion from the dermis. The main
type of cells which make up the epidermis include, but are not
limited to, keratinocytes, melanocytes, Langerhans cells and
Merkels cells.
[0111] The dermis is the layer of skin beneath the epidermis that
consists of connective tissue and cushions the body from stress and
strain. The dermis is tightly connected to the epidermis by a
basement membrane. It also harbors many mechanoreceptor/nerve
endings that provide the sense of touch and heat. It contains the
hair follicles, sweat glands, sebaceous glands, apocrine glands,
lymphatic vessels and blood vessels. The blood vessels in the
dermis provide nourishment and waste removal from its own cells as
well as from the Stratum basale of the epidermis. The dermis is
structurally divided into two areas: a superficial area adjacent to
the epidermis, called the papillary region, and a deep thicker area
known as the reticular region.
[0112] The papillary region is composed of loose areolar connective
tissue. It is named for its fingerlike projections called papillae
that extend toward the epidermis. The papillae provide the dermis
with a "bumpy" surface that interdigitates with the epidermis,
strengthening the connection between the two layers of skin. The
reticular region lies deep in the papillary region and is usually
much thicker. It is composed of dense irregular connective tissue,
and receives its name from the dense concentration of collagenous,
elastic, and reticular fibers that weave throughout it. These
protein fibers give the dermis its properties of strength,
extensibility, and elasticity. Also located within the reticular
region are the roots of the hair, sebaceous glands, sweat glands,
receptors, nails, and blood vessels. Stretch marks from pregnancy
are also located in the dermis.
[0113] The hypodermis is not part of the skin, and lies below the
dermis. Its purpose is to attach the skin to underlying bone and
muscle as well as supplying it with blood vessels and nerves. It
consists of loose connective tissue and elastin. The main cell
types are fibroblasts, macrophages and adipocytes (the hypodermis
contains 50% of body fat). Fat serves as padding and insulation for
the body.
[0114] In one embodiment, provided herein is a method of treating
skin dehydration, which comprises injecting to a dermal region
suffering from skin dehydration one or more embodiments of an
injectable composition described herein. For example, the
injectable composition can comprise a plurality of silk fibroin
particles, and optionally a biological fluid carrier (e.g., a
lipoaspirate) and/or an active agent, wherein the injection of the
composition rehydrates the skin, thereby treating skin
dehydration.
[0115] In another embodiment, a method of treating a lack of skin
elasticity comprises injecting to a dermal region suffering from a
lack of skin elasticity one or more embodiments of an injectable
composition described herein. For example, the injectable
composition can comprise a plurality of silk fibroin particles, and
optionally a biological fluid carrier (e.g., a lipoaspirate) and/or
an active agent, wherein the injection of the composition increases
the elasticity of the skin, thereby treating a lack of skin
elasticity.
[0116] In yet another embodiment, a method of treating skin
roughness comprises injecting to a dermal region suffering from
skin roughness one or more embodiments of an injectable composition
described herein. For example, the injectable composition can
comprise a plurality of silk fibroin particles, and optionally a
biological fluid carrier (e.g., a lipoaspirate) and/or an active
agent, wherein the injection of the composition decreases skin
roughness, thereby treating skin roughness.
[0117] In still another embodiment, a method of treating a lack of
skin tautness comprises injecting to a dermal region suffering from
a lack of skin tautness one or more embodiments of an injectable
composition described herein. For example, the injectable
composition can comprise a plurality of silk fibroin particles, and
optionally a biological fluid carrier (e.g., a lipoaspirate) and/or
an active agent, wherein the injection of the composition makes the
skin tauter, thereby treating a lack of skin tautness.
[0118] In a further embodiment, a method of treating a skin stretch
line or mark comprises injecting to a dermal region suffering from
a skin stretch line or mark one or more embodiments of an
injectable composition described herein. For example, the
injectable composition can comprise a plurality of silk fibroin
particles, and optionally a biological fluid carrier (e.g., a
lipoaspirate) and/or an active agent, wherein the injection of the
composition reduces or eliminates the skin stretch line or mark,
thereby treating a skin stretch line or mark.
[0119] In another embodiment, a method of treating skin paleness
comprises injecting to a dermal region suffering from skin paleness
one or more embodiments of an injectable composition described
herein. For example, the injectable composition can comprise a
plurality of silk fibroin particles, and optionally a biological
fluid carrier (e.g., a lipoaspirate) and/or an active agent,
wherein the injection of the composition increases skin tone or
radiance, thereby treating skin paleness.
[0120] In another embodiment, a method of treating skin wrinkles
comprises injecting to a dermal region suffering from skin wrinkles
one or more embodiments of an injectable composition described
herein. For example, the injectable composition can comprise a
plurality of silk fibroin particles, and optionally a biological
fluid carrier (e.g., a lipoaspirate) and/or an active agent,
wherein the injection of the composition reduces or eliminates skin
wrinkles, thereby treating skin wrinkles.
[0121] In yet another embodiment, a method of treating, preventing
or delaying the formation of skin wrinkles comprises injecting to a
dermal region susceptible to, or showing signs of wrinkles one or
more embodiments of an injectable composition described herein. For
example, the injectable composition can comprise a plurality of
silk fibroin particles, and optionally a biological fluid carrier
(e.g., a lipoaspirate) and/or an active agent, wherein the
injection of the composition makes the skin resistant to skin
wrinkles, thereby treating, preventing or delaying the formation of
skin wrinkles.
[0122] The effective amount and administration schedule of silk
fibroin particles injected into a dermal region can be determined
by a person of ordinary skill in the art taking into account
various factors, including, without limitation, the type of skin
condition, the location of the skin condition, the cause of the
skin condition, the severity of the skin condition, the degree of
relief desired, the duration of relief desired, the particular silk
fibroin particle formulation used, the rate of degradation or
volume retention of the particular silk fibroin particle
formulation used, the pharmacodynamics of the particular silk
fibroin particle formulation used, the nature of the other
compounds included in the particular silk fibroin particle
formulation used, the particular characteristics, history and risk
factors of the individual, such as, e.g., age, weight, general
health, and any combinations thereof. In some embodiments, the silk
fibroin particles can be injected into a dermal region every 3
months, every 6 months, every 9 months, every year, every two years
or longer.
[0123] In another aspect, the injectable compositions can be used
as a dermal filler for dermal bulking to reconstruct or augment a
soft tissue body part, such as, e.g., a lip, a breast, a breast
part such as the nipple, a muscle, or any other soft body part
where adipose and/or connective tissue is used to provide shape,
insulation, or other biological function. In fillers used for these
applications, the silk fibroin concentration and/or the amount of a
carrier (e.g., saline) added to silk fibroin particle mixture can
be adjusted for the relevant constraints of a given biological
environment. For example, silk fibroin particles for breast
augmentation can be adapted for particle hardness and volume
retention through alteration of silk fibroin concentration and
processing method. For example, about 4% (w/v) to about 8% (w/v)
silk fibroin concentration, optionally containing an active agent,
e.g., adipose cells such adipose-derived stem cells or cells from
lipoaspirate, can be used to produce the silk fibroin particles.
Carrier content in the case of saline can be on the order of 0% to
25% (v/v). Other factors such as, e.g., defect type, defect size
and needs for a specific depth of injection of the filler, should
be also considered.
[0124] Without wishing to be bound, while injection is
minimally-invasive, other administration method can be also be
used, e.g., implantation, when needed, e.g., to repair or argument
a large defect area. For example, for dermal injection and lip
augmentation, a syringe needle sized 26 g-30 g can be used. In
applications involving large quantities of filler, e.g., breast
reconstruction or breast augmentation, a larger particle size and a
larger bore needle such as 23 g-27 g or smaller needle gauge can be
used to administer the filler. In some embodiments, surgery, e.g.,
implantation, can also be employed to administer large quantities
of filler and/or to reach a certain depth of tissues.
[0125] Accordingly, yet another aspect described herein provide a
method of soft tissue reconstruction, repair, or augmentation, the
method comprising administering one or more embodiments of an
injectable composition described herein to a soft tissue region of
an individual in need thereof. For example, the injectable
composition can comprise a plurality of silk fibroin particles, and
optionally an active agent and/or a carrier (e.g., a biological
fluid carrier such as a lipoaspirate). Administration methods of an
injectable composition described herein can be determined by an
ordinary artisan. In some embodiments, the administration method
can be injection. In some embodiments, the administration method
can be surgery, e.g., implantation.
[0126] While injectable compositions and/or silk fibroin particles
described herein can be directly applied on a target region (e.g.,
injection or surgery), in some embodiments, an injectable
composition and/or silk fibroin particles disclosed herein can also
be used to fill an expandable implantable medical device, such as,
e.g., an expandable breast implant shell, which is placed in a
defect area. In such embodiments, provided herein is a method of
soft tissue reconstruction, repair or augmentation, the method
comprising placing an implantable medical device into a soft tissue
region of an individual at the desired location; and expanding the
device by filling the device with silk fibroin particles and/or
injectable compositions described herein, wherein expansion of the
medical device by filling it with silk fibroin particles and/or
injectable compositions described herein can reconstruct or augment
the soft tissue.
[0127] The silk fibroin particles or injectable compositions
disclosed herein can be also used in conjunction with
interventional radiology embolization procedures for blocking
abnormal blood (artery) vessels (e.g., for the purpose of stopping
bleeding) or organs (to stop the extra function e.g. embolization
of the spleen for hypersplenism) including uterine artery
embolization for percutaneous treatment of uterine fibroids.
Modulation of silk fibroin particle hardness and volume retention
rate can be done through alteration of silk fibroin concentration
and processing methods as described earlier.
[0128] The silk fibroin particles or injectable compositions
disclosed herein can be used to repair void space in a spine, e.g.,
created by spine disk nucleus removal surgery, to help maintain the
normal distance between the adjacent vertebral bodies. In some
embodiments, a vertebral disc filler comprising a plurality of silk
fibroin particles can be used to repair void space present in the
spine, e.g., between vertebral bodies, and/or in a ruptured spine
disk. In such embodiments, a silk fibroin concentration of about 4%
(w/v) to about 10% (w/v) can be used to fabricate the silk fibroin
particles described herein. Accelerant and/or active agents can
also be mixed with silk fibroin particles and/or injectable
compositions before, during, or after injection into the site of
interest.
[0129] The silk fibroin particles or injectable compositions
disclosed herein can be used to fill up the vitreous cavity to
support the eyeball structure and maintain the retina's position.
The viscosity of the injectable composition described herein can be
adjusted for the viscosity of vitreous fluid in the eye by one of
skill in the art.
[0130] In some embodiments, the silk fibroin particles and/or
injectable compositions can be used as a template for tissue
reconstruction or augmentation, e.g., soft tissue reconstruction or
augmentation (e.g., breast augmentation), or even for small bone or
cartilage defects such as fractures. The administration of silk
fibroin particles or injectable compositions described herein can
be used to facilitate cartilage/bone cell ingrowth and
proliferation and support collagen matrix deposition thus to
improve cartilage/bone repair. In another aspect, prior to
administration, donor cartilage cells can be seeded or mixed with
silk fibroin particles and/or injectable compositions described
herein to expand cell population and thus to promote the
development of cartilage tissue. In some embodiments, specific
growth factors such as TGF-.beta. or bone morphogenic proteins
(BMPs) which support cartilage or bone tissue formation,
respectively, can be added into silk fibroin particles.
[0131] In another embodiment, the silk fibroin particles and/or
injectable compositions described herein can be used for facial
plastic surgery, such as, e.g., nose reconstruction. The
reconstruction strategy discussed above for repairing a
cartilage/bone defect can also be applicable for facial plastic
surgery.
[0132] In some embodiments, the silk fibroin particles and/or
injectable compositions described herein can be used as scaffolds
to support cell growth for tissue engineering. For example, the
silk fibroin particles and/or injectable compositions described
herein can be administered into an incision or wound site to
promote wound healing or wound disclosure. The methods generally
comprise administering an injectable composition or silk fibroin
particles described herein, at the wound or incision site and
allowing the wound or incision to heal while the silk fibroin
particles is eroded or absorbed in the body and is replaced with
the individual's own viable tissue. The methods can further
comprise seeding the silk fibroin particles or mixing the
injectable composition with viable cellular material, either from
the individual or from a donor, prior to or during
administration.
[0133] In another aspect, the injectable composition comprising
silk fibroin particles can be used, directly or indirectly, in
methods of repairing, augmenting, or reconstructing a tissue in a
subject, e.g., augmenting or reconstructing the breast of a human
being. In some embodiments, the injectable compositions or silk
fibroin particles can be directly placed into a tissue (e.g., a
breast tissue) to be repaired or augmented, e.g., by injection. The
injectable compositions or silk fibroin particles can be injected
into a tissue (e.g., a breast tissue) every 6 months, every year,
every 2 years, every 3 years, or longer. In other embodiments, the
injectable compositions or silk fibroin particles can be used to
enhance support of a conventional tissue implant, e.g., by
enhancing support of the lower pole position of a breast implant.
In alternative embodiments, the method can generally comprise
administering an injectable composition and/or silk fibroin
particles near or in proximity to a tissue implant, for example, a
conventional breast implant, and seeding the injectable composition
and/or silk fibroin particles with viable cellular material prior
to or during administration. In yet another embodiment, an
injectable composition and/or silk fibroin particles can be used to
partially or completely cover a tissue implant (e.g., a breast
implant) to provide a beneficial interface with host tissue and to
reduce the potential for malpositioning or capsular
contracture.
[0134] In some embodiments, the silk fibroin particles and/or
injectable compositions described herein can be used as fillers to
promote or support adipogenesis, e.g., to treat facial
lipodystrophies. In such embodiments, the injectable compositions
and/or silk fibroin particles can be seeded or mixed with
adipose-associated cells, such adipose-derived stem cells or
lipoaspirate, prior to or concurrently with the injection to a
target area suffering from facial lipodystrophies in a subject. In
some embodiments, the silk fibroin particles can be injected every
3 months, every 6 months, every 9 months, every year, every two
years or longer, to maintain the treatment.
[0135] In still another embodiment, the silk fibroin particles
and/or injectable compositions described herein can be used as the
scaffold for cells useful for peripheral nerve repair. Silk fibroin
particles can be delivered (e.g., via injection) to the location of
the nerve defect with or without additional device to aid the
connection to the nerve ends. For such purpose, specific growth
factors such as nerve growth factor (NGF), which supports nerve
regeneration can be added into injectable compositions and/or mixed
with silk fibroin particles prior to or during administration. In
such embodiments, softer silk fibroin particles, e.g. using a silk
fibroin concentration of about 0.5 (w/v) to about 3% (w/v), can be
used. Depending on the brain microenvironment, harder silk fibroin
particles can also be used. The silk fibroin particles and/or
injectable compositions can be infused with or added with
appropriate therapeutic factors according to the methods described
above.
[0136] Any cells described herein can be seeded upon a surface of
silk fibroin particles described herein. For example, silk fibroin
particles can be submersed in an appropriate growth medium for the
cells of interest, and then directly exposed to the cells. The
cells are allowed to proliferate on the surface and interstices of
the silk fibroin particles. The silk fibroin particles are then
removed from the growth medium, washed if necessary, and
administered. Alternatively, the cells can be placed in a suitable
buffer or liquid growth medium and drawn through silk fibroin
particles by using vacuum filtration. Cells can also be admixed
with silk fibroin solution prior to forming silk fibroin particles,
capturing at least some of the cells within the silk fibroin
particles. In another embodiment, the cells of interest can be
dispersed into an appropriate solution (e.g., a growth medium or
buffer) and then sprayed onto silk fibroin particles. For example,
electro-spraying involves subjecting a cell-containing solution
with an appropriate viscosity and concentration to an electric
field sufficient to produce a spray of small charged droplets of
solution that contain cells.
[0137] In some embodiments, the silk fibroin particles or
injectable compositions comprising at least one active agent can be
used as a platform for drug delivery. For example, the silk fibroin
particles can be formed with a pharmaceutical agent either
entrained in or bound to the particles and then administered into
the body (e.g., injection, implantation or even oral
administration). In some embodiments, an active agent can be mixed
with silk fibroin particles and/or injectable compositions and then
administered into the body (e.g., injection, implantation or even
oral administration). For extended or sustained release, silk
fibroin particles can manipulated, e.g., to modulate its beta-sheet
content, for its volume retention and/or degradation rate. To
further control the drug release profile, the
pharmaceutically-active drug-containing silk fibroin particles can
be mixed with an additional silk fibroin gel phase acting as a
carrier either with or without a viscosity inducing component, a
surfactant, and/or an included lubricant fluid like saline. The
therapeutic-bound silk fibroin particles can also be further
crosslinked to enhance the stability to extend the release period.
In an alternative approach, silk fibroin particles can be mixed
with other polymers, for examples, hyaluronic acid, to prolong the
release of certain growth factors or cytokines and to stabilize the
functionality. Furthermore, the silk fibroin particles and/or
injectable compositions can also be used for coating coaxial drug
delivery systems, e.g., by spraying.
[0138] As used herein, the term "sustained release" refers to the
release of a pharmaceutically-active drug over a period of about
seven days or more. In aspects of this embodiment, a drug delivery
platform comprising the silk fibroin particles and/or injectable
compositions releases a pharmaceutically-active drug over a period
of, e.g., at least about 7 days after administration, at least
about 15 days after administration, at least about 30 days after
administration, at least about 45 days after administration, at
least about 60 days after administration, at least about 75 days
after administration, or at least about 90 days after
administration.
[0139] As used herein, the term "extended release" refers to the
release of a pharmaceutically-active drug over a period of time of
less than about seven days. In such embodiments, a drug delivery
platform comprising the silk fibroin particles and/or injectable
compositions described herein can release a pharmaceutically-active
drug over a period of, e.g., about 1 day after administration,
about 2 days after administration, about 3 days after
administration, about 4 days after administration, about 5 days
after administration, or about 6 days after administration.
[0140] Depending on the formulation and processing methods of the
silk fibroin particles and the associated applications, the
injectable compositions or silk fibroin particles can be
administered (e.g., by injection) periodically, for example, every
3 months, every 4 months, every 5 months, every 6 months, every 7
months, every 8 months, every 9 months, every 10 months, every 11
months, every year, every 2 years or longer.
[0141] In some embodiments of any of the applications described
herein, the injectable compositions or silk fibroin particles can
be at least partially dry when administered in a tissue to be
repaired or augmented. In some embodiments, the injectable
compositions or silk fibroin particles can be dried (e.g., in the
absence of a carrier) when administered in a tissue to be repaired
or augmented.
[0142] In some embodiments of any of the applications described
herein, the injectable compositions or silk fibroin particles can
be at least partially hydrated when administered in a tissue to be
repaired or augmented. In some embodiments, the injectable
compositions or silk fibroin particles can be hydrated (e.g., in
the presence of an injection carrier, e.g., a buffered solution
and/or lipoaspirate) when administered in a tissue to be repaired
or augmented.
[0143] In some embodiments of any of the applications described
herein, the injectable compositions or silk fibroin particles can
be injected subcutaneously, submuscularly, or intramuscularly.
[0144] In some embodiments, the methods and/or compositions
described herein can be used in the dermal region. In some
embodiments, the methods and/or compositions described herein can
be used in the epidermal layer, dermal layer, hypodermis layer, or
any combinations thereof.
Delivery Devices and Kits Comprising Silk Fibroin Particles
[0145] Delivery devices comprising an injectable composition or
silk fibroin particles described herein are also provided herein.
Delivery devices can be any conventional delivery device used for
injection purposes, e.g., a syringe. Accordingly, a further aspect
provided herein is a syringe comprising an injectable composition
or silk fibroin particles.
[0146] In some embodiments, the delivery device (e.g., a syringe)
can further comprise a needle. In some embodiments, the delivery
device (e.g., a syringe) can further comprise a catheter or a
cannula.
[0147] In various embodiments, the delivery device (e.g., a
syringe) can include an injection carrier, e.g., a buffered
solution.
[0148] In various embodiments, the delivery device (e.g., a
syringe) can include an anesthetic.
[0149] Further provided herein is a kit comprising one embodiment
of an injectable composition or silk fibroin particles packaged in
a syringe with a needle or a cannula. In some embodiments, a local
anesthetic can be blended with the injectable composition or silk
fibroin particles insides the syringe. In alternative embodiments,
a local anesthetic can be packaged in a separate container or in a
separate syringe. For example, it is desirable to apply a local
anesthetic to a target tissue to be treated prior to further
treatment. An exemplary anesthetic includes, but is not limited to,
lidocane. Dependent upon application, the kit can include syringes
sizes from 0.5 mL to 60 mL, where applications requiring larger
volumes (e.g., bone fillers, disc fillers) are supplied in a larger
size syringe. Additionally, needle gauge can adjusted according to
injection site with an acceptable range of 10 g to 30 g needles.
For example, 26 g to 30 g needles can be used for intradermal
injections.
[0150] In some embodiments, the kit can further comprise a
plurality of syringes (each with a needle) containing an injectable
composition or silk fibroin particles described herein. Each
syringe can be individually packaged.
[0151] In some embodiments, the kit can further comprise a
container containing a buffered solution or an injection
carrier.
[0152] In some embodiments, the kit can further comprise at least
one additional empty syringe. In some embodiments, the kit can
further comprise at least one additional needle. In some
embodiments, the kit can further comprise at least one catheter or
cannula.
[0153] Embodiments of the various aspects described herein can be
illustrated by the following numbered paragraphs. [0154] 1. A
method for repairing or augmenting a tissue in a subject
comprising: injecting in the tissue to be repaired or augmented a
composition comprising a plurality of silk fibroin particles,
wherein at least a portion of the silk fibroin particles retain at
least about 50% of their original volume within the tissue for at
least about 6 weeks. [0155] 2. The method of paragraph 1, wherein
said at least a portion of the silk fibroin particles retain at
least about 50% of their original volume within the tissue for at
least about 3 months. [0156] 3. The method of paragraph 2, wherein
said at least a portion of the silk fibroin particles retain at
least about 50% of their original volume within the tissue for at
least about 6 months. [0157] 4. The method of any of paragraphs
1-3, wherein said at least a portion of the silk fibroin particles
retain at least about 60% of their original volume within the
tissue for at least about 6 weeks. [0158] 5. The method of
paragraph 4, wherein said at least a portion of the silk fibroin
particles retain at least about 70% of their original volume within
the tissue for at least about 6 weeks. [0159] 6. The method of
paragraph 5, wherein said at least a portion of the silk fibroin
particles retain at least about 80% of their original volume within
the tissue for at least about 6 weeks. [0160] 7. The method of any
of paragraphs 1-6, wherein said at least a portion of the silk
fibroin particles retain at least about 70% of their original
volume within the tissue for at least 3 months. [0161] 8. The
method of any of paragraphs 1-7, wherein said at least a portion of
the silk fibroin particles are adapted to degrade no more than 50%
of their original volume in at least about 6 weeks. [0162] 9. The
method of paragraph 8, wherein said at least a portion of the silk
fibroin particles are adapted to degrade no more than 50% of their
original volume in at least about 3 months. [0163] 10. The method
of any of paragraphs 1-9, wherein said at least a portion of the
silk fibroin particles are adapted to degrade no more than 30% of
their original volume in at least about 6 weeks. [0164] 11. The
method of paragraph 10, wherein said at least a portion of the silk
fibroin particles are adapted to degrade no more than 10% of their
original volume in at least about 6 weeks. [0165] 12. The method of
any of paragraphs 1-11, wherein said at least a portion of the silk
fibroin particles are adapted to degrade no more than 30% of their
original volume in at least about 3 months. [0166] 13. The method
of any of paragraphs 1-12, wherein the silk fibroin particles are
porous. [0167] 14. The method of paragraph 13, wherein the porous
silk fibroin particles have a porosity of at least about 1%, at
least about 5%, at least or 10%, at least about 15%, or at least
about 30%. [0168] 15. The method of paragraph 14, wherein the
porous silk fibroin particles have a porosity of at least about
50%. [0169] 16. The method of paragraph 15, wherein the porous silk
fibroin particles have a porosity of at least about 70%. [0170] 17.
The method of any of paragraphs 13-16, wherein the pores have a
size of about 10 nm to about 1000 .mu.m. [0171] 18. The method of
paragraph 17, wherein the pores have a size of about 1 .mu.m to
about 1000 .mu.m. [0172] 19. The method of any of paragraphs 1-18,
wherein the silk fibroin particles have a size of about 500 nm to
about 5000 .mu.m. [0173] 20. The method of paragraph 19, wherein
the silk fibroin particles have a size of about 1 .mu.m to about
2000 .mu.m. [0174] 21. The method of paragraph 20, wherein the silk
fibroin particles have a size of about 10 .mu.m to about 1500
.mu.m. [0175] 22. The method of any of paragraphs 1-21, wherein the
porous silk fibroin particles are reduced from a solid-state porous
silk fibroin by a mechanical means. [0176] 23. The method of
paragraph 22, wherein the mechanical means is selected from the
group consisting of micronizing, milling, pulverizing, crushing,
grinding, cutting, and any combinations thereof. [0177] 24. The
method of any of paragraphs 22-23, wherein the solid-state porous
silk fibroin is formed by a porogen-leaching method. [0178] 25. The
method of any of paragraphs 1-24, wherein the composition or the
silk fibroin particles further comprise(s) at least one active
agent. [0179] 26. The method of paragraph 25, wherein the at least
one active agent is a biologically active agent, a cosmetically
active agent, a cell attachment agent, a contrast agent, or any
combinations thereof. [0180] 27. The method of paragraph 26,
wherein the biologically active agent is selected from the group
consisting of a drug, a therapeutic agent, an anesthetic, a cell
growth factor, a peptide, a peptidomimetic, an antibody or a
portion thereof, an antibody-like molecule, nucleic acid, a
polysaccharide, and any combinations thereof. [0181] 28. The method
of paragraph 26, wherein the cell attachment agent is selected from
the group consisting of hyaluronic acid, collagen, crosslinked
hyaluronic acid/collagen, an integrin-binding molecule, chitosan,
elastin, fibronectin, vitronectin, laminin, proteoglycans, any
derivatives thereof, and any combinations thereof. [0182] 29. The
method of paragraph 26, wherein the cosmetically active agent is
selected from the group consisting of an anti-aging agent, an
anti-free radical agent, an anti-oxidant, a hydrating agent, a
whitening agent, a colorant, a depigmenting agent, a sun-blocking
agent, a muscle relaxant, and any combinations thereof. [0183] 30.
The method of any of paragraphs 1-29, wherein the composition
further comprises a cell. [0184] 31. The method of paragraph 30,
wherein the cell is a stem cell. [0185] 32. The method of any of
paragraphs 1-31, wherein the composition further comprises a
biological fluid or concentrate. [0186] 33. The method of paragraph
32, wherein the biological fluid or concentrate is lipoaspirate,
bone marrow aspirate, or any combinations thereof. [0187] 34. The
method of paragraph 32, wherein the biological fluid or concentrate
is lipoaspirate. [0188] 35. The method of any of paragraphs 32-34,
wherein the volume ratio of the silk fibroin particles to the
biological fluid or concentrate ranges from about 1:19 to about
12:19. [0189] 36. The method of paragraphs 32-34, wherein the
volume ratio of the silk fibroin particles to the biological fluid
or concentrate ranges from about 3:19 to about 6:19. [0190] 37. The
method of any of paragraphs 1-36, wherein the composition or the
silk fibroin particles further comprise(s) a hydrogel. [0191] 38.
The method of any of paragraphs 1-37, wherein the composition or
the silk fibroin particles further comprise(s) a dermal filler
material. [0192] 39. The method of paragraph 38, wherein the dermal
filler material is selected from the group consisting of
poly(methyl methacrylate) microspheres, hydroxylapatite,
poly(L-lactic acid), hyaluronic acid, collagen, and any
combinations thereof. [0193] 40. The method of any of paragraphs
1-39, wherein the composition further comprises a
pharmaceutically-acceptable carrier. [0194] 41. The method of any
of paragraphs 1-40, wherein the injection is performed
subcutaneously, submuscularly, or intramuscularly. [0195] 42. The
method of any of paragraphs 1-41, wherein the injection is
performed with a needle with a gauge of about 25-26. [0196] 43. The
method of any of paragraphs 1-42, wherein the composition is at
least partially dry when injected in the tissue. [0197] 44. The
method of any of paragraphs 1-43, wherein the composition is at
least partially hydrated when injected in the tissue. [0198] 45.
The method of any of paragraphs 1-44, wherein the tissue is a soft
tissue. [0199] 46. The method of paragraph 45, wherein the soft
tissue is selected from the group consisting of a tendon, a
ligament, skin, a breast tissue, a fibrous tissue, a connective
tissue, a muscle, and any combinations thereof. [0200] 47. The
method of paragraph 46, wherein the soft tissue is skin. [0201] 48.
The method of paragraph 46, wherein the soft tissue is a breast
tissue. [0202] 49. The method of any of paragraphs 1-48, wherein
the subject is a mammalian subject. [0203] 50. The method of
paragraph 49, wherein the mammalian subject is a human. [0204] 51.
The method of any of paragraphs 1-50, wherein the composition is
stored or transported dried. [0205] 52. The method of paragraph 51,
wherein the composition is stored or transported at a temperature
between about 0.degree. C. and about 60.degree. C. [0206] 53. The
method of paragraph 52, wherein the composition is stored or
transported at a temperature between about 10.degree. C. and about
60.degree. C. [0207] 54. The method of paragraph 53, wherein the
composition is stored or transported at a temperature between about
15.degree. C. and about 60.degree. C. [0208] 55. The method of any
of paragraphs 1-54, wherein the silk fibroin particles exclude an
amphiphilic peptide. [0209] 56. The method of paragraph 55, wherein
the amphiphilic peptide comprises a RGD motif. [0210] 57. An
injectable composition for use in repairing or augmenting a tissue
in a subject, comprising a plurality of silk fibroin particles,
wherein at least a portion of the silk fibroin particles retain at
least about 50% of their original volume within the tissue to be
repaired or augmented for at least about 6 weeks. [0211] 58. The
composition of paragraph 57, wherein the silk fibroin particles
exclude an amphiphilic peptide. [0212] 59. The composition of
paragraph 58, wherein the amphiphilic peptide comprises a RGD
motif. [0213] 60. The composition of any of paragraphs 57-59,
wherein said at least a portion of the silk fibroin particles
retain at least about 50% of their original volume within the
tissue for at least about 3 months. [0214] 61. The composition of
any of paragraphs 57-60, wherein said at least a portion of the
silk fibroin particles retain at least about 50% of their original
volume within the tissue for at least about 6 months. [0215] 62.
The composition of any of paragraphs 57-61, wherein said at least a
portion of the silk fibroin particles retain at least about 60% of
their original volume within the tissue for at least about 6 weeks.
[0216] 63. The composition of paragraph 62, wherein said at least a
portion of the silk fibroin particles retain at least about 70% of
their original volume within the tissue for at least about 6 weeks.
[0217] 64. The composition of paragraph 63, wherein said at least a
portion of the silk fibroin particles retain at least about 80% of
their original volume within the tissue for at least about 6 weeks.
[0218] 65. The composition of any of paragraphs 57-64, wherein said
at least a portion of the silk fibroin particles retain at least
about 70% of their original volume within the tissue for at least 3
months. [0219] 66. The composition of any of paragraphs 57-65,
wherein said at least a portion of the silk fibroin particles are
adapted to degrade no more than 50% of their original volume in at
least about 6 weeks. [0220] 67. The composition of any of
paragraphs 57-66, wherein said at least a portion of the silk
fibroin particles are adapted to degrade no more than 50% of their
original volume in at least about 3 months. [0221] 68. The
composition of any of paragraphs 57-67, wherein said at least a
portion of the silk fibroin particles are adapted to degrade no
more than 30% of their original volume in at least about 6 weeks.
[0222] 69. The composition of any of paragraphs 57-68, wherein said
at least a portion of the silk fibroin particles are adapted to
degrade no more than 10% of their original volume in at least about
6 weeks. [0223] 70. The composition of any of paragraphs 57-69,
wherein said at least a portion of the silk fibroin particles are
adapted to degrade no more than 30% of their original volume in at
least about 3 months. [0224] 71. The composition of any of
paragraphs 57-70, wherein the silk fibroin particles are porous.
[0225] 72. The composition of paragraph 71, wherein the porous silk
fibroin particles have a porosity of at least about 1%, at least
about 5%, at least about 10%, at least about 15%, or at least about
30%. [0226] 73. The composition of paragraph 72, wherein the porous
silk fibroin particles have a porosity of at least about 50%.
[0227] 74. The composition of paragraph 73, wherein the porous silk
fibroin particles have a porosity of at least about 70%. [0228] 75.
The composition of any of paragraphs 71-74, wherein the pores have
a size of about 10 nm to about 1000 .mu.m. [0229] 76. The
composition of paragraph 75, wherein the pores have a size of about
1 .mu.m to about 1000 .mu.m. [0230] 77. The composition of any of
paragraphs 57-76, wherein the silk fibroin particles have a size of
about 500 nm to about 5000 .mu.m. [0231] 78. The composition of
paragraph 77, wherein the silk fibroin particles have a size of
about 1 .mu.m to about 2000 .mu.m. [0232] 79. The composition of
paragraph 78, wherein the silk fibroin particles have a size of
about 10 .mu.m to about 1500 .mu.m. [0233] 80. The composition of
any of paragraphs 71-79, wherein the porous silk fibroin particles
are reduced from a solid-state porous silk fibroin by a mechanical
means. [0234] 81. The composition of paragraph 80, wherein the
mechanical means is selected from the group consisting of
micronizing, milling, pulverizing, crushing, grinding, cutting, and
any combinations thereof. [0235] 82. The composition of paragraph
80 or 81, wherein the solid-state porous silk fibroin porous is
formed by a porogen-leaching method. [0236] 83. The composition of
any of paragraphs 57-82, wherein the injectable composition or the
silk fibroin particles further comprise(s) at least one active
agent. [0237] 84. The composition of paragraph 83, wherein the at
least one active agent is a biologically active agent, a
cosmetically active agent, a cell attachment agent, a contrast
agent, or any combinations thereof. [0238] 85. The composition of
paragraph 84, wherein the biologically active agent is selected
from the group consisting of a therapeutic agent, an anesthetic, a
cell growth factor, a peptide, a peptidomimetic, an antibody or a
portion thereof, an antibody-like molecule, nucleic acid, a
polysaccharide, and any combinations thereof. [0239] 86. The
composition of paragraph 84, wherein the cell attachment agent is
selected from the group consisting of hyaluronic acid, collagen,
crosslinked hyaluronic acid/collagen, an integrin-binding molecule,
chitosan, elastin, fibronectin, vitronectin, laminin,
proteoglycans, any derivatives thereof, and any combinations
thereof. [0240] 87. The composition of paragraph 84, wherein the
cosmetically active agent is selected from the group consisting of
an anti-aging agent, an anti-free radical agent, an anti-oxidant, a
hydrating agent, a whitening agent, a colorant, a depigmenting
agent, a sun-blocking agent, a muscle relaxant, and any
combinations thereof.
[0241] 88. The composition of any of paragraphs 57-87, further
comprising a cell. [0242] 89. The composition of paragraph 88,
wherein the cell is a stem cell. [0243] 90. The composition of any
of paragraphs 57-89, further comprising a biological fluid or
concentrate. [0244] 91. The composition of paragraph 90, wherein
the biological fluid or concentrate is lipoaspirate, bone marrow
aspirate, or any combinations thereof. [0245] 92. The composition
of paragraph 90, wherein the biological fluid or concentrate is
lipoaspirate. [0246] 93. The composition of any of paragraphs
90-92, wherein the volume ratio of the silk fibroin particles to
the biological fluid or concentrate ranges from about 3:19 to about
6:19. [0247] 94. The composition of any of paragraphs 57-93,
wherein the injectable composition or the silk fibroin particles
further comprise(s) a hydrogel. [0248] 95. The composition of any
of paragraphs 57-94, wherein the injectable composition or the silk
fibroin particles further comprise(s) a dermal filler material.
[0249] 96. The composition of paragraph 95, wherein the dermal
filler material is selected from the group consisting of
poly(methyl methacrylate) microspheres, hydroxylapatite,
poly(L-lactic acid), hyaluronic acid, collagen, and any
combinations thereof. [0250] 97. The composition of any of
paragraphs 57-96, wherein the injectable composition further
comprises a pharmaceutically-acceptable carrier. [0251] 98. The
composition of any of paragraphs 57-97, wherein the injectable
composition is stored or transported dried. [0252] 99. The
composition of paragraph 98, wherein the injectable composition is
stored or transported at a temperature between about 0.degree. C.
and about 60.degree. C. [0253] 100. The composition of paragraph
99, wherein the injectable composition is stored or transported at
a temperature between about 10.degree. C. and about 60.degree. C.
[0254] 101. The composition of paragraph 100, wherein the
injectable composition is stored or transported at a temperature
between about 15.degree. C. and about 60.degree. C. [0255] 102. A
delivery device comprising an injectable composition of any of
paragraphs 57-101. [0256] 103. The delivery device of paragraph
102, further comprising a syringe. [0257] 104. The delivery device
of paragraph 103, wherein the syringe further comprises a needle.
[0258] 105. The delivery device of any of paragraphs 102-104,
further comprising a catheter. [0259] 106. The delivery device of
any of paragraphs 102-105, further comprising an injection carrier.
[0260] 107. A syringe comprising an injectable composition of any
of paragraphs 57-101. [0261] 108. The syringe of paragraph 107,
further comprising a needle. [0262] 109. The syringe of paragraph
107 or 108, further comprising a catheter. [0263] 110. The syringe
of any of paragraphs 107-109, further comprising an injection
carrier.
Some Selected Definitions of Terms
[0264] 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 certain embodiments of
the aspects described herein, the subject is a mammal, e.g., a
primate, e.g., a human. A subject can be male or female.
Preferably, the subject is a mammal. The mammal can be a human,
non-human primate, mouse, rat, dog, cat, horse, or cow, but are not
limited to these examples. Mammals other than humans can be
advantageously used as subjects that represent animal models of
tissue repair, regeneration and/or reconstruction. In addition, the
methods and compositions described herein can be used to treat
domesticated animals and/or pets.
[0265] The term "statistically significant" or "significantly"
refers to statistical significance and generally means a two
standard deviation (2SD) below or above a reference level. The term
refers to statistical evidence that there is a difference. It is
defined as the probability of making a decision to reject the null
hypothesis when the null hypothesis is actually true. The decision
is often made using the p-value.
[0266] As used herein, the terms "proteins" and "peptides" are used
interchangeably herein to designate a series of amino acid residues
connected to the other by peptide bonds between the alpha-amino and
carboxy groups of adjacent residues. The terms "protein", and
"peptide", which are used interchangeably herein, refer to a
polymer of protein amino acids, including modified amino acids
(e.g., phosphorylated, glycated, etc.) and amino acid analogs,
regardless of its size or function. Although "protein" is often
used in reference to relatively large polypeptides, and "peptide"
is often used in reference to small polypeptides, usage of these
terms in the art overlaps and varies. The term "peptide" as used
herein refers to peptides, polypeptides, proteins and fragments of
proteins, unless otherwise noted. The terms "protein" and "peptide"
are used interchangeably herein when referring to a gene product
and fragments thereof. Thus, exemplary peptides or proteins include
gene products, naturally occurring proteins, homologs, orthologs,
paralogs, fragments and other equivalents, variants, fragments, and
analogs of the foregoing.
[0267] The term "nucleic acids" used herein refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA), polymers thereof in either
single- or double-stranded form. Unless specifically limited, the
term encompasses nucleic acids containing known analogs of natural
nucleotides, which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions)
and complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer, et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka, et al., J. Biol. Chem. 260:2605-2608
(1985), and Rossolini, et al., Mol. Cell. Probes 8:91-98 (1994)).
The term "nucleic acid" should also be understood to include, as
equivalents, derivatives, variants and analogs of either RNA or DNA
made from nucleotide analogs, and, single (sense or antisense) and
double-stranded polynucleotides.
[0268] The term "short interfering RNA" (siRNA), also referred to
herein as "small interfering RNA" is defined as an agent which
functions to inhibit expression of a target gene, e.g., by RNAi. An
siRNA can be chemically synthesized, it can be produced by in vitro
transcription, or it can be produced within a host cell. siRNA
molecules can also be generated by cleavage of double stranded RNA,
where one strand is identical to the message to be inactivated. The
term "siRNA" refers to small inhibitory RNA duplexes that induce
the RNA interference (RNAi) pathway. These molecules can vary in
length (generally 18-30 base pairs) and contain varying degrees of
complementarity to their target mRNA in the antisense strand. Some,
but not all, siRNA have unpaired overhanging bases on the 5' or 3'
end of the sense 60 strand and/or the antisense strand. The term
"siRNA" includes duplexes of two separate strands, as well as
single strands that can form hairpin structures comprising a duplex
region.
[0269] The term "shRNA" as used herein refers to short hairpin RNA
which functions as RNAi and/or siRNA species but differs in that
shRNA species are double stranded hairpin-like structure for
increased stability. The term "RNAi" as used herein refers to
interfering RNA, or RNA interference molecules are nucleic acid
molecules or analogues thereof for example RNA-based molecules that
inhibit gene expression. RNAi refers to a means of selective
post-transcriptional gene silencing. RNAi can result in the
destruction of specific mRNA, or prevents the processing or
translation of RNA, such as mRNA.
[0270] The term "enzymes" as used here refers to a protein molecule
that catalyzes chemical reactions of other substances without it
being destroyed or substantially altered upon completion of the
reactions. The term can include naturally occurring enzymes and
bioengineered enzymes or mixtures thereof. Examples of enzyme
families include kinases, dehydrogenases, oxidoreductases, GTPases,
carboxyl transferases, acyl transferases, decarboxylases,
transaminases, racemases, methyl transferases, formyl transferases,
and .alpha.-ketodecarboxylases.
[0271] As used herein, the term "aptamers" means a single-stranded,
partially single-stranded, partially double-stranded or
double-stranded nucleotide sequence capable of specifically
recognizing a selected non-oligonucleotide molecule or group of
molecules. In some embodiments, the aptamer recognizes the
non-oligonucleotide molecule or group of molecules by a mechanism
other than Watson-Crick base pairing or triplex formation. Aptamers
can include, without limitation, defined sequence segments and
sequences comprising nucleotides, ribonucleotides,
deoxyribonucleotides, nucleotide analogs, modified nucleotides and
nucleotides comprising backbone modifications, branchpoints and
nonnucleotide residues, groups or bridges. Methods for selecting
aptamers for binding to a molecule are widely known in the art and
easily accessible to one of ordinary skill in the art.
[0272] As used herein, the term "antibody" or "antibodies" refers
to an intact immunoglobulin or to a monoclonal or polyclonal
antigen-binding fragment with the Fc (crystallizable fragment)
region or FcRn binding fragment of the Fc region. The term
"antibodies" also includes "antibody-like molecules", such as
fragments of the antibodies, e.g., antigen-binding fragments.
Antigen-binding fragments can be produced by recombinant DNA
techniques or by enzymatic or chemical cleavage of intact
antibodies. "Antigen-binding fragments" include, inter alia, Fab,
Fab', F(ab')2, Fv, dAb, and complementarity determining region
(CDR) fragments, single-chain antibodies (scFv), single domain
antibodies, chimeric antibodies, diabodies, and polypeptides that
contain at least a portion of an immunoglobulin that is sufficient
to confer specific antigen binding to the polypeptide. Linear
antibodies are also included for the purposes described herein. The
terms Fab, Fc, pFc', F(ab') 2 and Fv are employed with standard
immunological meanings (Klein, Immunology (John Wiley, New York,
N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of
Modern Immunology (Wiley & Sons, Inc., New York); and Roitt, I.
(1991) Essential Immunology, 7th Ed., (Blackwell Scientific
Publications, Oxford)). Antibodies or antigen-binding fragments
specific for various antigens are available commercially from
vendors such as R&D Systems, BD Biosciences, e-Biosciences and
Miltenyi, or can be raised against these cell-surface markers by
methods known to those skilled in the art.
[0273] As used herein, the term "Complementarity Determining
Regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino
acid residues of an antibody variable domain the presence of which
are necessary for antigen binding. Each variable domain typically
has three CDR regions identified as CDR1, CDR2 and CDR3. Each
complementarity determining region may comprise amino acid residues
from a "complementarity determining region" as defined by Kabat
(i.e. about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the
light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (i.e. about residues 26-32
(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain
and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). In some instances, a complementarity determining region
can include amino acids from both a CDR region defined according to
Kabat and a hypervariable loop.
[0274] The expression "linear antibodies" refers to the antibodies
described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995).
Briefly, these antibodies comprise a pair of tandem Fd segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions. Linear
antibodies can be bispecific or monospecific.
[0275] The expression "single-chain Fv" or "scFv" antibody
fragments, as used herein, is intended to mean antibody fragments
that comprise the VH and VL domains of antibody, wherein these
domains are present in a single polypeptide chain. Preferably, the
Fv polypeptide further comprises a polypeptide linker between the
VH and VL domains which enables the scFv to form the desired
structure for antigen binding. (Pl{umlaut over (.upsilon.)}ckthun,
The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).
[0276] The term "diabodies," as used herein, refers to small
antibody fragments with two antigen-binding sites, which fragments
comprise a heavy-chain variable domain (VH) Connected to a
light-chain variable domain (VL) in the same polypeptide chain
(VH-VL). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. (EP 404,097; WO 93/11161; Hollinger et
ah, Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993)).
[0277] As used herein, the term "small molecules" refers to natural
or synthetic molecules including, but not limited to, peptides,
peptidomimetics, amino acids, amino acid analogs, polynucleotides,
polynucleotide analogs, aptamers, nucleotides, nucleotide analogs,
organic or inorganic compounds (i.e., including heteroorganic and
organometallic compounds) having a molecular weight less than about
10,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 5,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 1,000
grams per mole, organic or inorganic compounds having a molecular
weight less than about 500 grams per mole, and salts, esters, and
other pharmaceutically acceptable forms of such compounds.
[0278] The term "antibiotics" is used herein to describe a compound
or composition which decreases the viability of a microorganism, or
which inhibits the growth or reproduction of a microorganism. As
used in this disclosure, an antibiotic is further intended to
include an antimicrobial, bacteriostatic, or bactericidal agent.
Exemplary antibiotics include, but are not limited to, penicillins,
cephalosporins, penems, carbapenems, monobactams, aminoglycosides,
sulfonamides, macrolides, tetracyclins, lincosides, quinolones,
chloramphenicol, vancomycin, metronidazole, rifampin, isoniazid,
spectinomycin, trimethoprim, and sulfamethoxazole.
[0279] The term "therapeutic agents" is art-recognized and refers
to any chemical moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or
systemically in a subject. Examples of therapeutic agents, also
referred to as "drugs", are described in well-known literature
references such as the Merck Index, the Physicians Desk Reference,
and The Pharmacological Basis of Therapeutics, and they include,
without limitation, medicaments; vitamins; mineral supplements;
substances used for the treatment, prevention, diagnosis, cure or
mitigation of a disease or illness; substances which affect the
structure or function of the body; or pro-drugs, which become
biologically active or more active after they have been placed in a
physiological environment. Various forms of a therapeutic agent may
be used which are capable of being released from the subject
composition into adjacent tissues or fluids upon administration to
a subject. Examples include steroids and esters of steroids (e.g.,
estrogen, progesterone, testosterone, androsterone, cholesterol,
norethindrone, digoxigenin, cholic acid, deoxycholic acid, and
chenodeoxycholic acid), boron-containing compounds (e.g.,
carborane), chemotherapeutic nucleotides, drugs (e.g., antibiotics,
antivirals, antifungals), enediynes (e.g., calicheamicins,
esperamicins, dynemicin, neocarzinostatin chromophore, and
kedarcidin chromophore), heavy metal complexes (e.g., cisplatin),
hormone antagonists (e.g., tamoxifen), non-specific (non-antibody)
proteins (e.g., sugar oligomers), oligonucleotides (e.g., antisense
oligonucleotides that bind to a target nucleic acid sequence (e.g.,
mRNA sequence)), peptides, proteins, antibodies, photodynamic
agents (e.g., rhodamine 123), radionuclides (e.g., I-131, Re-186,
Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67 and
Cu-64), toxins (e.g., ricin), and transcription-based
pharmaceuticals.
[0280] As used herein, the term "hormones" generally refers to
naturally or non-naturally occurring hormones, analogues and mimics
thereof. In certain embodiments, the term "hormones" refers to any
hormones used in therapeutic treatment, e.g., growth hormone
treatment. As used herein, "growth hormone" or "GH" refers to
growth hormone in native-sequence or in variant form, and from any
source, whether natural, synthetic, or recombinant. Examples
include human growth hormone (hGH), which is natural or recombinant
GH with the human native sequence (somatotropin or somatropin), and
recombinant growth hormone (rGH), which refers to any GH or variant
produced by means of recombinant DNA technology, including
somatrem, somatotropin, and somatropin. In one embodiment, hormones
include insulin.
[0281] As used herein, a "contrast agent" can be any chemical
moiety that is used to increase the degree of difference between
the lightest and darkest part of a scan or an imaging, e.g., during
medical scan or imaging, relative to a scan performed without the
use of a contrast agent. For example, contrast agents can include
imaging agents containing radioisotopes such as indium or
technetium; dyes containing iodine, gadolinium or cyanine; enzymes
such as horse radish peroxidase, GFP, alkaline phosphatase, or
.beta.-galactosidase; fluorescent substances such as europium
derivatives; luminescent substances such as N-methylacrydium
derivatives or the like. In some embodiments, contrast agents can
include gold nanoparticles and/or quantum dots.
[0282] As used herein, the term "substantially" means a proportion
of at least about 60%, or preferably at least about 70% or at least
about 80%, or at least about 90%, at least about 95%, at least
about 97% or at least about 99% or more, or any integer between 70%
and 100%. In some embodiments, the term "substantially" means a
proportion of at least about 90%, at least about 95%, at least
about 98%, at least about 99% or more, or any integer between 90%
and 100%. In some embodiments, the term "substantially" can include
100%.
[0283] As used herein, the term "comprising" means that other
elements can also be present in addition to the defined elements
presented. The use of "comprising" indicates inclusion rather than
limitation.
[0284] The term "consisting of" refers to the components thereof as
described herein, which are exclusive of any element not recited in
that description of the embodiment.
[0285] 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 of the
invention
[0286] 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 may mean .+-.1%.
[0287] 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."
[0288] Unless otherwise explained, 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 disclosure belongs.
Definitions of common terms in diseases and disorders, separation
and detection techniques can be found in The Merck Manual of
Diagnosis and Therapy, 18th Edition, published by Merck Research
Laboratories, 2006 (ISBN 0-911910-18-2); Robert S. Porter et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0289] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0290] All patents and other publications identified throughout the
specification 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 present invention. 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 are based on the information available
to the applicants and do not constitute any admission as to the
correctness of the dates or contents of these documents.
[0291] Some embodiments described herein are further illustrated by
the following example which should not be construed as
limiting.
[0292] The contents of all references cited throughout this
application, examples, as well as the figures and tables are
incorporated herein by reference in their entirety.
EXAMPLES
Example 1. Comparison of Injectable Silk Fibroin Scaffold Particles
with Other Silk Fibroin Scaffolds or Hydrogels
Exemplary Materials and Methods
[0293] Materials:
[0294] All chemicals and solvents used in the Examples for silk
scaffold preparation were purchased from Sigma Aldrich (St. Louis,
Mo.) unless otherwise noted. Sterile water and saline were
purchased from Invitrogen (Carlsbad, Calif.). It should be noted
that equivalent materials can be used and purchased from other
commercial vendors.
[0295] Preparation of Silk Fibroin Solutions:
[0296] Bombyx mori silkworm cocoons were purchased from Tajimia
Shoji Co. (Yokohama, Japan). Coccons were cut into pieces, and
boiled in 0.02 M Na.sub.2CO.sub.3 for about 10-60 minutes, and
preferably for about 30 minutes. The resulting silk fibroin fibers
were rinsed in distilled water and let dried. The dried silk
fibroin fibers were re-solubilized in 9.3 M LiBr at 60.degree. C.,
for about 1-4 hours, until dissolved. The silk fibroin solution was
dialyzed, with a molecular weight cutoff of 3500 Daltons, against
distilled water for at least 6 water changes. The aqueous silk
fibroin solution was lyophilized till dry and then dissolved in
hexa-fluoro-iso-propanol (HFIP) to yield a 17% (w/v) solvent based
silk fibroin solution.
[0297] Preparation of Silk Fibroin Porous Scaffolds:
[0298] Silk fibroin porous scaffolds were formed from either
aqueous or solvent based silk fibroin solutions (e.g., 6%-20% w/v).
Porogens (e.g., salts such as sodium chloride NaCl) with a size
ranging from 100 microns to 1.2 millimeters were used. Porogens
were packed into a Teflon coated container, and silk fibroin
solution was poured atop the salt. The container was covered and
allowed to sit at room temperature for about 1-3 days. The
container was then left uncovered for about 1 day. The container
was placed into distilled water to leach out the salt.
Alternatively, the scaffold can be placed in a methanol bath or
water-annealed for about 1 hour to about 1 day to further induce
beta-sheet formation. In some embodiments, the silk fibroin porous
scaffolds can be further coated with extracellular matrix
molecules, such as laminin, to facilitate cell attachment. In some
embodiments, the silk fibroin porous scaffolds can be coated with
no extracellular matrix molecules.
[0299] Preparation of Injectable Silk Fibroin Porous Scaffolds:
[0300] Aqueous or solvent based silk fibroin porous scaffolds were
chopped manually or by mechanical means, for example, with a
rotating blade, such as the ones in conventional food processors.
Chopped scaffolds were passed through sieves of various openings to
obtain the desired range of sizes. The chopped scaffolds were
allowed to dry and then autoclaved to sterilize. The dry,
autoclaved, chopped scaffolds (termed as "silk fibroin scaffold
particles" below) are stored at room temperature until use.
[0301] Preparation of Injectable Silk Fibroin Hydrogels ("Silk
Fibroin Vortexed Gels"):
[0302] Aqueous silk fibroin solution (e.g., .about.4% w/v) was
first sterilized by being passed through a 0.22 micron filter unit.
The sterile silk fibroin solution was then concentrated, for
example, to .about.8%, .about.10%, and .about.12% w/v solutions,
using centrifugal filter units (Amicon Ultra-15 Centrifugal Filter
Unit, Millipore, Billerica, Mass.) according to manufacturers'
protocols. To form the injectable silk fibroin hydrogels, a
vortexing method was employed (Yucel et al, 2009. Biophys J. 97:
2044). Briefly, the aqueous silk fibroin solutions were vortexed
(Vortex-Genie 2, Fisher Scientific, Pittsburgh, Pa.) with varying
power and time, depending on the sample volume and concentration,
until the clear solution became turbid. The turbid solution was
placed in .about.37.degree. C. for about 30 minutes to further
induce gelation. After 30 minutes, the hydrogel alone or mixed with
lipoaspirate was loaded into a syringe and injected.
[0303] Injection Methods:
[0304] Silk fibroin scaffolds were injected via different methods,
e.g., subcutaneously, intramuscularly, or submuscularly, in a
variety of preparations. Exemplary preparations include, but are
not limited to, dry state, dry state mixed with lipoaspirate,
hydrated state (e.g., in sterile water or normal saline), or
hydrated state mixed with lipoaspirate.
[0305] In Vivo Injections:
[0306] A female nude rat model was used for assessing the silk
fibroin scaffolds and hydrogels described herein. Other mammalian
models (e.g., mouse, rabbit, canine, or porcine models) can also be
used depending on the applications of the injectable silk fibroin
scaffolds and the tissues to be modeled for treatment. Six month
old rats were weighed and anesthetized with isoflurane in oxygen
prior to injection. A total of about 1 ml was used per injection.
Briefly, dry silk fibroin scaffold particles were immersed in
saline or lipoaspirate immediately before loading into a syringe.
The filled syringe was attached to a cannula no larger than 2 mm
inner diameter. Subcutaneous injections were performed above the
pectoral muscles. Intramuscular and submuscular injections were
performed between the pectoralis major and pectoralis minor muscles
or underneath the pectoral muscles, respectively. A fanning
subcutaneous injection method was performed in the dorsus of the
rat. See, for example, FIG. 1. Injected samples were explanted, and
evaluated for weight, volume retention and histological outcomes
after 1, 2, 10, 30 days. Volume retention was performed by 2
methods, e.g., scale measurements and volume displacement. In some
embodiments, whole silk fibroin porous scaffolds (5 mm
diameter.times.2 mm height) were implanted in the same locations
for comparison.
[0307] Histology:
[0308] Explants were cut in half and fixed in 10% formalin
overnight at 4.degree. C. One group was embedded in OCT freezing
media, cryosectioned into 10 micron sections, and stained with Oil
Red O. The remaining half were placed through a series of
dehydration steps, embedded in paraffin and cut in 10 micron
sections. Three continuous sections were placed on each slide, and
stained according to standard histological methods for hematoxylin
and eosin (H&E), or processed for immunohistochemistry. After
antigen retrieval, the sections were incubated with primary
anti-rat CD31, CD68, CD80, CD163, anti-human nucleus antibodies for
about 1 hour. The sections were then incubated with secondary
biotinylated antibodies raised in the species of the primary
antibodies for about 1 hour. The Vector Labs ABC antibody detection
kit was used along with a DAB substrate to enhance colorimetric
expression.
Results
[0309] In some embodiments, silk fibroin vortexed gels, alone or
mixed with lipoaspirate, can be resorbed faster than lipoaspirate
alone in a 6-week study. Accordingly, some embodiments of the silk
fibroin vortexed gels may not be ideal for applications, which
require size and/or shape retention for an extended time such as 6
weeks or longer. However, in some embodiments, the volume retention
properties of the silk fibroin vortexed gel can be adjusted, for
example, by modulating the silk fibroin concentration, and/or
vortexing or shearing rate of the silk fibroin solution.
[0310] Implanted silk fibroin porous scaffolds can retain their
shape and size over an extended period of time. However, the
implanted silk fibroin porous scaffolds generally require one or
more incisions to be placed. In addition, the implanted silk
fibroin porous scaffolds are generally cast to fill a particular
anatomical void prior to surgery, and/or they cannot be molded
during surgery to fill an irregular shaped void.
[0311] Unlike silk fibroin vortexed gels or implanted silk fibroin
porous scaffolds, an injectable silk fibroin porous scaffold (e.g.,
silk fibroin particles described herein) can serve as a therapy
that is both minimally invasive and capable of sustaining its size
and shape for at least about 1 month, at least about 2 months, at
least about 3 months, at least about 4 months, at least about 6
months, or at least about 1 year or longer, while still being an
off-the-shelf product. While the injectable silk fibroin scaffolds
(e.g., silk fibroin particles described herein) can reduce the
administration to a minimally invasive procedure, they can also
allow the surgeon to flexibly mold the injectable silk fibroin
scaffolds (e.g., in form of particles) into any shape or size
defect.
[0312] As shown in Table 1, different combinations of various
parameters related to scaffold parameters and/or injection methods
were used to assess the in vivo application of injectable silk
fibroin scaffolds.
TABLE-US-00001 TABLE 1 Various combinations of exemplary parameters
related to injectable scaffold particle properties and injection
methods Pore size of a silk Particle fibroin Diameter scaffold
Injection Hydration Injection Time (.mu.m) (.mu.m) carrier state
site points Submicron 300-500 No carrier Dry Subcuta- 1 day 1-5
850-1000 Saline Hydrated neous 2 day 5-20 Lipoaspi- (saline)
intramus- 10 day 20-50 rate cular 14 day 50-100 Submus- 1 month
100-250 cular 500-750 750-1000 1000-2000
[0313] In some embodiments, two different methods of processing
silk fibroin were employed to alter the degradation rate of silk
fibroin. It has been previously shown that silk fibroin porous
scaffold implants in a subcutaneous rat model degrade within 3-6
months when prepared with the aqueous method, but remain for at
least up to 2 years when prepared with solvent based method (Wang
et al. 2008. Biomaterials. 29: 3415). Accordingly, either method
can be used to prepare injectable silk fibroin scaffolds, depending
on desired properties of the scaffolds, applications and/or tissues
to be treated. In some embodiments, aqueous methods for preparing
injectable silk fibroin scaffolds can be used. In other
embodiments, solvent based methods for preparing injectable silk
fibroin scaffolds can be used. In this Example, injectable silk
fibroin scaffolds prepared by both aqueous and solvent base methods
were studied to evaluate integration after the silk fibroin
material is completely gone and long-term integration of the
material. Early time points (e.g., 1-2 days) can be used to
evaluate signs of acute inflammation, while later time points are
evaluated for overall tissue integration, vascularity, volume
retention, and scaffold degradation.
[0314] Another variable to be assessed was pore size range. Smaller
pores, 300-500 microns were compared to larger pores, 850-1000
microns. Further, various diameters of the injectable silk fibroin
scaffold particles, as shown in Table 1, were assessed. In some
embodiments, injectable silk fibroin scaffold particles smaller
than several microns can also be produced. In some embodiments, as
the silk fibroin scaffold particles are degrading, they do not
produce an adverse inflammatory response due to their size. As
shown in Table 1, the silk fibroin scaffold particles can be dry or
hydrated. One of the advantages of using dry silk fibroin scaffold
particles is that they can be used off the shelf. Another benefit
of the dry particles is their ability to infuse into the scaffold
particles while eliminating the need to pre-hydrate.
[0315] The different injection locations, as shown in Table 1, are
meant to represent examples of clinical injection sites for a
variety of applications, but they should not be construed as
limiting. For example, subcutaneous injections can be
representative of the soft tissue defect fillers; subcutaneous,
submuscular and intramuscular injections can be representative of
breast reconstructions.
[0316] In some embodiments, the injectable silk fibroin scaffolds
(e.g., unseeded or cell-seeded) can also increase vascularity in a
tissue to be treated by at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90% or higher, as
compared to a non-treated tissue or a tissue treated with non-silk
injectable compositions.
[0317] In some embodiments, the injectable silk fibroin scaffolds
seeded with cells, e.g., ASCs or lipoaspirate, can also increase
its integration with the host tissue by at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90% or higher, as compared to a tissue treated with unseeded
injectable silk fibroin scaffolds.
[0318] In some embodiments, the injectable silk fibroin scaffolds
seeded with cells, e.g., ASCs or lipoaspirate, can increase adipose
tissue regeneration by at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90% or higher, as
compared to a tissue treated with unseeded injectable silk fibroin
scaffolds.
Example 2. In Vivo Studies of Injectable Silk Fibroin Porous
Scaffolds
Exemplary Materials and Methods
[0319] Preparation of Injectable Silk Fibroin Porous Scaffolds:
[0320] In particular embodiments, silk fibroin porous scaffolds
were formed from solvent based silk solutions (17% w/v). Porogens
can be used to create pores with the scaffolds. In certain
embodiments, NaCl porogens with a size ranging from about 300-500
microns were used. In certain embodiments, NaCl porogens with a
size ranging from about 850-1000 microns were used. NaCl porogens
were packed into a Teflon coated container, and silk solution was
poured atop the salt. The container was covered and allowed to sit
at room temperature for 1-3 days. The container was then left
uncovered for 1 day. The container was placed into distilled water
to leach out the salt. In some embodiments, the silk resultant
scaffolds were placed in a methanol bath for 1 day to further
induce beta-sheet formation. Injectable silk fibroin porous
scaffolds can then be produced using the methods described in
Example 1, e.g., by reducing the silk fibroin porous scaffold into
smaller pieces. See, for example, FIG. 2.
[0321] Preparation of Processed Lipoaspirate:
[0322] Lipoaspirate from elective plastic surgery was obtained on
the same day of scaffold injection. Lipoaspirate was transported
aseptically at room temperature just after surgery. Approximately
30 ml of lipoaspirate was added to a 50 ml conical tube and
centrifuged at room temperature at 1000 rpm for 10 minutes. The
blood and free lipids were removed. The remaining tissue was placed
in sterile Petri dishes. The injectable silk fibroin porous
scaffolds were placed into the processed lipoaspirate for 1 hour
prior to injection.
[0323] Injection Methods:
[0324] One ml of chopped silk fibroin porous scaffolds and
lipoaspirate was drawn up into a 1 ml syringe. The final volume
ratios of lipoaspirate to silk scaffolds could range from 19:3 (low
dose) to 19:6 (high dose). In some embodiments, the injectable
scaffold and lipoaspirate mixture was injected subcutaneously
through a 24 gauge needle, in a hydrated state with lipoaspirate,
on the back of an athymic mouse. See, e.g., FIG. 1.
[0325] Histology:
[0326] Explant sample and constructs were processed according
standard histology protocols. Formalin fixed samples were put
through a series of dehydration solvents and finally paraffin using
an automated tissue processor. Samples were embedded in paraffin,
cut in 10 micron sections, and let to adhere on glass slides. The
sections were rehydrated and stained with hematoxylin and eosin,
and imaged.
Results
[0327] At 6 weeks post-injection, the mice were sacrificed and the
injected materials were harvested. The explanted masses were
measured for their weight and mass. No differences in relative
weight change or mass change were found with silk fibroin particles
produced from solid-state porous silk fibroin of varying porosities
(e.g., 300-500 micron pores vs. 850-1000 micron pores) or relative
ratios of silk fibroin scaffold to lipoaspirate (e.g., 3:19 vs
6:19) at such time point.
[0328] As shown in FIG. 3, the injected lipoaspirate (line arrows)
was detected in all groups. Within the injected lipoaspirate,
injectable silk fibroin scaffold pieces were detected (dotted
arrows). The injected silk fibroin materials did not elicit a
pro-inflammatory response; however macrophages were detected at the
periphery of the injected mass.
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