U.S. patent application number 16/037559 was filed with the patent office on 2019-08-22 for silk powder compaction for production of constructs with high mechanical strength and stiffness.
The applicant listed for this patent is Trustees of Tufts College. Invention is credited to Rosario Friedman, David L. Kaplan, Gary G. Leisk, Tim Jia-Ching Lo, Fiorenzo Omenetto, Benjamin Partlow.
Application Number | 20190255181 16/037559 |
Document ID | / |
Family ID | 49916588 |
Filed Date | 2019-08-22 |
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United States Patent
Application |
20190255181 |
Kind Code |
A1 |
Kaplan; David L. ; et
al. |
August 22, 2019 |
Silk Powder Compaction for Production of Constructs with High
Mechanical Strength and Stiffness
Abstract
The present disclosure relates generally to compositions and
methods for production of three-dimensional constructs with high
mechanical strength and/or stiffness.
Inventors: |
Kaplan; David L.; (Concord,
MA) ; Omenetto; Fiorenzo; (Lexington, MA) ;
Leisk; Gary G.; (Somerville, MA) ; Lo; Tim
Jia-Ching; (Taoyuan, TW) ; Partlow; Benjamin;
(Marlborough, MA) ; Friedman; Rosario; (Conway,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trustees of Tufts College |
Medford |
MA |
US |
|
|
Family ID: |
49916588 |
Appl. No.: |
16/037559 |
Filed: |
July 17, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14414245 |
Jan 12, 2015 |
10034945 |
|
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PCT/US2013/050520 |
Jul 15, 2013 |
|
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16037559 |
|
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|
61671375 |
Jul 13, 2012 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A43B 1/06 20130101; A61L
31/047 20130101; A61L 27/3604 20130101; A43B 23/0225 20130101; Y10T
29/49 20150115; A61L 27/50 20130101; A43B 1/02 20130101; A43B 13/02
20130101; A61K 47/42 20130101; A43B 23/0205 20130101; B30B 9/28
20130101 |
International
Class: |
A61K 47/42 20060101
A61K047/42; B30B 9/28 20060101 B30B009/28; A61L 31/04 20060101
A61L031/04; A61L 27/50 20060101 A61L027/50; A61L 27/36 20060101
A61L027/36; A43B 23/02 20060101 A43B023/02; A43B 13/02 20060101
A43B013/02; A43B 1/06 20060101 A43B001/06; A43B 1/02 20060101
A43B001/02 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
no. P41 EB002520 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method comprising: (i) providing a composition comprising silk
particles; and (ii) compacting the composition by application of
pressure into a solid state.
2-39. (canceled)
40. A method for increasing compressive strength, clastic modulus,
tlcxural stiffness, or shear stiffness of a silk-based material,
the method comprising: (i) providing a composition comprising silk
particles; and (ii) compacting the composition by application of
pressure into a solid state.
Description
RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.
119(c) of the U.S. Provisional Application No. 61/671,375, filed
Jul. 13, 2012, the content of which is incorporated herein by
reference in its entirety.
TECHNICAL FIELD
[0003] The present disclosure relates generally to compositions and
methods for production of three-dimensional constructs with high
mechanical strength and/or stiffness.
BACKGROUND
[0004] Silk material is produced by thousands of species of spiders
and by worms from various insects such as mites, butterflies, and
moths. Silks produced by silkworms (typically Bombyx mori) and
orb-weaving spiders are widely studied due to their impressive
mechanical properties, environmental stability, biocompatibility,
and tunable degradation. In addition, such silk can be modified to
deliver antibiotics, drugs, and growth factors to enhance healing
in biomedical applications. Biomedical applications have seen
successful introduction of silks, dating to the first usage of silk
sutures centuries ago. See, for example, Vepari, C. and Kaplan, D.
L., "Silk as a Biomaterial," Prog. Polym. Sci. 32 (2007), pp.
991-1007. However, there is no existing technologies that enable
production of three-dimensional silk-based constructs with high
mechanical strength and/or stiffness.
SUMMARY
[0005] Compositions and methods describe herein relate to
fabrication of robust silk material formats using a novel powder
compaction technique. In some embodiments, the process is shown to
generate a variety of construct geometries with greatly enhanced
mechanical performance over existing regenerated silk materials.
The silk-based materials described herein range from monolithic
materials (e.g., silk powder bound and fused together under
elevated temperature and pressure) to composite materials (e.g.,
silk-silk composites made from silk "matrix" and silk reinforcing
phases combined into one consolidated material or part). The
fabrication techniques described herein can be extended to other
protein or non-protein based materials.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIGS. 1A and 1B are photographs of silk construct made using
high-resolution acrylic die insert: the construct (left) and
acrylic die insert (FIG. 1A) and stereomicroscope image of fine
detail on silk construct (FIG. 1B).
[0007] FIGS. 1C and 1D are photographs of silk construct made using
a coin as a die insert: the original coin (FIG. 1) and close-up
silk construct exhibiting fine detail (FIG. 1D).
[0008] FIG. 2 is a schematic representation of a 100% silk
shoe.
[0009] FIGS. 3-5 are schematic representations of a method for
preparing parts of the 100% silk shoe.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0010] While the fiber form of silk is used in suture or
textile-based applications, a solubilized form of silk fibroin is
effective and versatile in creating unique three-dimensional
morphologies and materials for applications that range beyond the
traditional textile-based applications. In a typical protocol, 5
grams of B. mori silkworm coccons are immersed in 1 L of boiling
0.02 M Na.sub.2CO.sub.3 solution for 30 minutes. This degumming
process removes a protein known as sericin, which coats the silk
fibroin and acts as a glue-like substance. Degummed fibers are
collected and rinsed with distilled water three times, then
air-dried. The fibers are then solubilized in 9.3 M LiBr (20% w/v)
at 60.degree. C. for 4 hours. A volume of 15 mL of this solution is
then dialyzed against 1 L of distilled water (water changes after
1, 3, 6, 24, 36, and 48 hours) with a regenerated cellulose
membrane (3500 MWCO, Slide-A-Lyzer, Pierce, Rockford, Ill.). The
solubilized protein solution is then centrifuged twice (9700 PRM,
20 min., 4.degree. C.) to remove insoluble particulates. Protein
concentration is then determined by drying a known volume of the
silk solution under a hood for 12 hours and assessing the mass of
the remaining solids. See, for example, Wray, L. S., Hu, X.,
Gallego, J., Georgakoudi, I., Omenetto, F. G., Schmidt, D. and
Kaplan, D. L., "Effect of Processing on Silk-Based Biomaterials:
Reproducibility and Biocompatibility," Journal of Biomedical
Materials Research Part B: Applied Biomaterials 99B: 1 (2001), pp.
89-10, content of which is incorporated herein by reference in its
entirety.
[0011] Solubilized silk (also referred to as silk solution herein)
can be processed to create a range of material formats, such as
films, foams, fibers, gels, and sponges. Typically, these materials
and or material forms exhibit sort or flexible material response
(e.g., low hardness, low tensile/compressive strength, and low
flexural stiffness). While these responses have not restricted the
usage of silk in implantation or repair applications involving soft
tissue, there is a need to explore the creation of silk materials
and scaffolds that have much better mechanical performance. For
example, silk-based tissue engineering constructs ure being
proposed for bone repair or replacement. For this application,
excellent strength and toughness properties are requisite to
provide structural support within the body.
[0012] Accordingly, in one aspect, the disclosure provides a method
for preparing an article of manufacture. Generally, the method
comprises compacting or consolidating a silk composition. The silk
in the composition can be in an at least partially insoluble state.
After compaction, the composition can be in a solid state. After
compaction, the composition can optionally be processed into a
desired final shape.
[0013] In some embodiments, the silk fibroin composition is in form
of a powder, i.e., the composition comprises silk particles. The
silk particles can be nanoparticles or microparticles. As used
herein, the term "particle" includes spheres; rods; shells; and
prisms; and these particles can be part of a network or an
aggregate. Without limitations, the particle can have any size from
nm to millimeters. In some embodiments, the particles can have a
size ranging from about 0.01 .mu.m to about 1000 .mu.m, about 0.05
.mu.m to about 500 .mu.m, about 0.1 .mu.m to about 250 .mu.m, about
0.25 .mu.m to about 200 .mu.m, or about 0.5 .mu.m to about 100
.mu.m. Further, the silk particle can be of any shape or form,
e.g., spherical, rod, elliptical, cylindrical, capsule, or disc. In
some embodiments, the silk particle is a microparticle or a
nanoparticle. As used herein, the term "microparticle" refers to a
particle having a particle size of about 1 nm to about 1000 .mu.m.
As used herein, the term "nanoparticle" refers to particle having a
particle size of about 0.1 nm to about 1000 nm.
[0014] Without wishing to be bound by a theory, particle size can
greatly determine microscopic and macroscopic properties of the
final product. Particle size is dependent on a number of process
parameters, including, but not limited to, the size of the ceramic
balls used, the amount of silk placed in each ball mill cup, the
rotational speed (RPM) of the machine, and the duration of ball
milling. Particle size in the powder can be predicted based on some
of these process parameters, e.g., with mathematical modeling and
or experimentation to determine the correlation. For example, this
can be done by milling a given volume of silk fibroin for varying
ball mill speeds and duration. Scanning Electron Microscopy (SEM)
can be performed on representative samples from each experiment to
determine particle size. Additional tests can be run on each sample
to determine the effect of process parameters on the color,
molecular weight, viscosity in a solution, and solubility in water
of the resulting constructs.
[0015] In some embodiments, the silk particles comprise silk
fibroin substantially free of sericin. In some embodiments, the
silk particles comprise non-degummed silk or partially degummed
silk (i.e., silk having some amount of sericin). Silk fibers are
composed of fibroin and sericin proteins. For biomedical
applications, the sericin can be removed before implantation to
prevent immunogenic responses. This can be done through a process
known as degumming. For example, 5 grams of B. mori silkworm
cocoons can be immersed in 1 L of boiling 0.02 M Na.sub.2CO.sub.3
solution for 30 minutes. Degummed fibers can be collected and
rinsed with distilled water (e.g., three times) and air-dried. A
calibrated inspection tool can be developed to measure sericin. In
some embodiments, a tunable amount of sericin can be left in the
silk material after degumming. This can eliminate the need to mix
together silk particles prepared from degummed and non-degummed
silk fibroin.
[0016] Silk powder can be useful in many applications, for example,
as a filler in silk gels or other silk forms or possibly as a
crystallite-like material to enhance (acting as a catalyst)
conversion of a silk solution into a hydrogel. For the hard silk
material described in the present process, in some embodiments, a
liquid binder can be added to the silk particle composition. Taking
advantage of the ability of sericin protein to act as a glue-like
binder in silkworm cocoons, in some embodiments, the method
disclosed herein comprises mixing in a combination of silk
particles made from degummed and silk particles made from
non-degummed silk fibroin. To control the amount of sericin, a
specific proportion of each can be weighed and mixed together. The
particles are mixed together vigorously to ensure that the final
mixture is homogeneous.
[0017] The mixture can comprise from 100% non-degummed silk to 100%
degummed silk. Without wishing to be bound by theory, the
consolidation ability, level of bonding, and strength properties of
the final construct can be likely highly dependent on the sericin
content. Another variation that can occur in this step includes the
addition of other silk (and non-silk) materials to reinforce the
construct. Various composite architectures can be used, for
example, from chopped or continuous fiber reinforcement, to the
embedding of textile-like reinforcing layers. In addition to
mechanical reinforcing phases, there are many art-recognized
additives that can be used, each of which can affect the final
product differently.
[0018] In some embodiments, the composition comprises a mixture of
silk particles comprising degummed silk and silk particles
comprising non-degummed silk. Ratio of degummed silk to
non-degummed silk in the composition can range from about 50:1
(w/w) to about 1:50 (w/w). In some embodiments, ratio of degummed
silk to non-degummed silk in the composition can range from about
25:1 (w/w) to about 1:25 (w/w), from about 20:1 (w/w) to about 1:20
(w/w), from about 15:1 (w/w) to about 1:15 (w/w), from about 10:1
(w/w) to about 1:10 (w/w), from about 5:1 (w/w) to about 1:25
(w/w), from about 1:1 (w/w) to about 1:20, from about 1:1 (w/w) to
about 1:15 (w/w), or from about 1:2.5 (w/w) to about 1:10
(w/w).
[0019] Various methods of producing silk particles (e.g.,
nanoparticles and microparticles) are known in the art. For
example, a milling machine (e.g., a Retsch planetary ball mill) can
be used to produce silk powder. Generally, the ball mill consists
of either two of four sample cups arranged around a central axis,
which is geared such that each cup rotates both centrally and
locally. Each ceramic cup is filled with small ceramic spheres. A
range of sizes is available; balls with a diameter of 10
millimeters were are used for the milling operations described in
the present disclosure. As the cups spin, the spheres crush
material in the cups to a small characteristic size. Both degummed
and non-degummed silk can be converted from pulverized material to
powder form in the ball mill.
[0020] Before milling, a pulverization step can be used to break up
silk fibroin in the form of whole cocoons or bave silk before
introduction to a ball mill. If the cocoons are not shredded, it is
possible that the ball mill can take a significant amount of time
to crush the cocoons into powder. One issue, however, can be
related to the degradation (decreased molecular weight of silk
fibroin) from pulverization. Testing with SDS-PAGE (gel
electrophoresis) has shown that pulverizing silk before degumming
can degrade molecular weight significantly, when compared to silk
that was not pulverized. While this can have a negative impact on
the final properties achievable in the silk constructs, elimination
of this step may not provide a significant benefit. Without wishing
to be bound by theory, the ball milling operation can degrade the
silk material as well. In some embodiments, the milling can be used
to produce powders. In other embodiments, alternative powder
formation techniques can be used (e.g., lyophilization or flash
freezing and crushing). In other embodiments, alternative grates on
the pulverizer, with larger holes, can be used. This can generate
larger silk particle sizes.
[0021] Generally, for pulverization, dried silk is placed into a
pulverizer, e.g., Fritsch Pulverisette 19, which "pulverizes" the
silk by forcing it through a grate by the rotating action of a
5-bladed milling cutter. To ensure proper flow of the silk material
through the pulverizer (e.g., Pulverisette), a vacuum (e.g., an
industrial vacuum) can be attached to the outflow tube on the
bottom of the grate. Pulverized silk can then be collected from the
inside of the industrial vacuum. Generally, the resulting silk
material is chopped and fluffy, made up of fairly short silk
particles. Given the availability of additional grates with unique
perforation size, silk particles of varying length can be
produced.
[0022] In some embodiments, the silk particles can be produced by a
polyvinyl alcohol (PVA) phase separation method as described in,
e.g., International App. No. WO 2011/041395, the content of which
is incorporated herein by reference in its entirety. Other methods
for producing silk fibroin particles are described, for example, in
U.S. App. Pub. No. U.S. 2010/0028451 and PCT App. Pub. No.: WO
2008/118133 (using lipid as a template for making silk microspheres
or nanospheres), and in Wenk et al. J Control Release, Silk fibroin
spheres as a platform for controlled drug delivery, 2008; 132:
26-34 (using spraying method to produce silk microspheres or
nanospheres), content of all of which is incorporated herein by
reference in its entirety.
[0023] In some embodiments, silk particles can be produced using a
freeze-drying method as described in U.S. Provisional Application
Ser. No. 61/719,146, filed Oct. 26, 2012, content of which is
incorporated herein by reference in its entirety. Specifically,
silk foam can be produced by freeze-drying a silk solution. The
foam then can be reduced to particles. For example, a silk solution
can be cooled to a temperature at which the liquid earner
transforms into a plurality of solid crystals or particles and
removing at least some of the plurality of solid crystals or
particles to leave a porous silk material (e.g., silk foam). After
cooling, liquid carrier can be removed, at least partially, by
sublimation, evaporation, and/or lyophilization. In some
embodiments, the liquid carrier can be removed under reduced
pressure. After formation, the silk fibroin foam can be subjected
to grinding, cutting, crushing, or any combinations thereof to form
silk particles. For example, the silk fibroin foam can be blended
in a conventional blender or milled in a ball mill to form silk
particles of desired size.
[0024] The term "compacting" can be understood to mean reduce in
volume and/or increase in density. One way of compacting the silk
fibroin composition can be by applying pressure to the composition.
Accordingly, in some embodiments, the method comprises providing a
silk composition, wherein the silk fibroin can be in an at least
partially insoluble state; and applying pressure to the
composition.
[0025] The pressure can be applied using a press, e.g., designed
specifically for this purpose. In one non-limiting example, the
press is composed of 4 parts--a base plate, a cavity plate, a top
plate, and a piston. The base plate is attached to the cavity plate
by four 1/4''-20 bolts to form a well. The silk composition is
deposited in the well, and the piston is inserted into position.
The piston is machined to just fit inside the well to minimize the
amount the composition that can leak out upon compaction. Next the
top plate is bolted onto the cavity plate, and the bolls are
tightened using a torque wrench such that there is a specific
amount of pressure on the material inside the press. It is
important for the pressure to be sufficient and for the consistency
of the compound to be correct, otherwise the compound can leak, or
the resulting material can be inconsistent and non-homogenous.
[0026] Applying adequate pressure is desirable during the
compaction process. With insufficient pressure, the final construct
can be porous and easily crack. With over-pressure, as with the
addition of too much binder, e.g., water, the compound can leak out
of the press, generating a final construct with poor geometric
stability and poor mechanical performance. Accordingly, in some
embodiments, an integrated, one-piece bottom plate/cavity plate can
be developed. This can prevent leakage at the base of the well.
However, removal of the final construct can become more difficult.
Alternatively, the well and piston can be fabricated with draft
angles, which can allow for easier construct removal.
[0027] The pressure to be applied to the composition can be a
pressure of about 0.05 bar, about 0.1 bar, about 0.15 bar, about
0.2 bar, about 0.25 bar, about 0.3 bar, about 0.35 bar, about 0.4
bar, about 0.45 bar, about 0.5 bar, about 0.55 bar, about 0.6 bar,
about 0.65 bar, about 0.7 bar, about 0.75 bar or higher. For
example, the pressure can be about 1 bar, 1.25 bar, 1.5 bar, 1.75
bar, 2 bar, 2.25 bar, 2.5 bar, 2.75 bar, 3 bar, 3.25 bar, 3.5 bar,
3.75 bar, 4 bar, 4.25 bar, 4.5 bar, 4.75 bar, 5 bar, 5.25 bar, 5.5
bar, 5.75 bar, 6 bar, 7.25 bar, 7.5 bar, 7.75 bar, 8 bar, 8.25 bar,
8.5 bar, 8.75 bar, 9 bar, 9.25 bar, 9.5 bar, 9.75 bar, 10 bar, or
higher. In some embodiments, the pressure is about 1 bar or
higher.
[0028] It is to be noted, that the method disclosed herein differs
from the methods wherein the composition is incubated under
pressure but a pressure is not directly applied to the composition.
In the method disclosed herein, the silk fibroin composition is
compacted by applying a pressure directly to the composition.
[0029] As used herein the term "insoluble state" when used in
reference to a silk fibroin refers to the formation of or state of
being in a substantially amorphous, primarily beta-sheet
conformation. The term "formed into an insoluble state" is not
intended to reflect polymerization of silk monomers into a silk
polymer. Rather, it is intended to reflect the conversion of
soluble silk fibroin to a water insoluble state. As used herein,
silk fibroin is in an "insoluble state" if it can be pelleted by
centrifugation or if it cannot be dissolved by immersion in or
rinsing with water at 37.degree. C. or less.
[0030] Without limitation, compaction can be carried out at any
desired temperature. In some embodiments, compaction is at room
temperature. In some other embodiments, compaction is at an
elevated temperature. As used herein, the term "elevated
temperature" means a temperature higher that room temperature.
Generally, the elevated temperature is a temperature higher than
about 25.degree. C. For example, the elevated temperature can be
temperature of about 30.degree. C. or higher, about 35.degree. C.
or higher, about 40.degree. C. or higher, about 45.degree. C. or
higher, about 50.degree. C. or higher, about 55.degree. C. or
higher, about 60.degree. C. or higher, about 65.degree. C. or
higher, about 70.degree. C. or higher, about 75.degree. C. or
higher, about 80.degree. C. or higher, about 85.degree. C. or
higher, about 90.degree. C. or higher, about 95.degree. C. or
higher, about 100.degree. C. or higher, about 105.degree. C. or
higher, about 110.degree. C. or higher, about 115.degree. C. or
higher, about 120.degree. C. or higher, about 125.degree. C. or
higher, about 130.degree. C. or higher, about 135.degree. C. or
higher, about 140.degree. C. or higher, about 145.degree. C. or
higher, or about 150.degree. C. or higher. In some embodiments,
compaction can be at room temperature, about 60.degree. C., or
about 120.degree. C.
[0031] In some embodiments, with the composition under pressure in
a compaction press, the entire press can be placed in a preheated
oven for a specific amount of time.
[0032] Without wishing to be bound by a theory, mechanistically,
the consolidation process that occurs with the silk powder is
likely related to the glass transition temperature (Tg) of the
polymer involved. While it is widely reported that the Tg for silk
fibroin is in the range of 190.degree. C. to 210.degree. C., the Tg
can shift depending on molecular weight. Given the degradation that
occurs due to the pulverizing and ball milling operations, the silk
powder generated likely has a much lower Tg. The Tg of silk before
and after pulverizing and ball milling can be determined using
analytical techniques, such as Differential Scanning Calorimetry
(DSC). The temperature used during the consolidation process can
affect the mechanical property of the final construct. If the
temperature is too high or the material is left in the oven too
long, sample burning can occur. If the temperature is too low or
the material is not maintained at elevated temperature long enough,
the sample could be soft and not fully dry, leading to construct
deformation, inhomogeneity, and poor mechanical robustness.
[0033] Without limitation, compaction can be for any desired period
of time. For example, the compaction can be for a period of
minutes, hours, or days. For example, the compaction can be for a
period of about one hour, two hours, three hours, four hours, five
hours, six hours, twelve hours, one day, two days, three days or
longer.
[0034] The compaction time and/or temperature can affect the sample
greatly. For example, if the temperature is too low or the heating
time too short, the sample typically does not consolidate well (not
cooked through). If the temperature is too high or the heating time
too long, the sample appears to overheat and even burn
(over-cooking). In either case, the resulting construct can have
poor geometric stability and limited mechanical robustness.
[0035] If the compaction is at an elevated temperature, it can be
desirable to cool the compacted composition before removal from
removing it from the press. Cooling (e.g., complete cooling) can be
desirable before removal of the compacted composition from the
press or the compacted composition can warp as it cools outside of
the press. The compacted composition can be cooled for any desired
period of time before removal from the press. In some embodiments,
the press can be removed from the oven and placed in a fume hood to
cool by convection with room temperature air. Once completely cool,
the bolts can be released and the sample removed.
[0036] In some embodiments, the silk composition can further
comprise a binder. As used herein, the term "binder" includes any
additive which imparts cohesive qualities and is used for the
purpose of binding or holding together powdered components in a
solid compacted form. Suitable binders depend on the individual
application and are known to, and can be determined by, the person
skilled in the art. Without wishing to be bound by theory,
hydration of the sericin and possibly fibroin can cause the
material to become slightly sticky; e.g., recapitulating the
glue-like response of sericin naturally produced by silkworms.
[0037] In some embodiments, the binders contemplated are liquids,
e.g., water, salt solutions, and the like. Amount of liquid binder
in the silk composition can range from about 0.1% (w/w) to about
75% (w/w) of the total of the composition. In some embodiments,
amount of the liquid binder in the silk composition can range from
about 5% (w/w) to about 65% (w/w) from about 10% (w/w) to about 60%
(w/w), from about 15% (w/w) to about 50% (w/w), from about 20%
(w/w) to about 45% (w/w), or from about 25% (w/w) to about 40%
(w/w). In some embodiments, it is can be desirable to use a ratio
of 3 to 6 grams of silk particles for every 2 ml of liquid binder.
Generally, the amount of the liquid binder in the composition is
sufficient to provide a silk composition of a desired
viscosity.
[0038] In some embodiments, the binder is a solubilized silk
solution. Given the ability to easily adjust concentration (silk
fibroin-to-water ratio), this provides additional flexibility for
preparing the silk composition comprising the binder. Silk solution
can act as a good binder for other forms of silk. There can be a
number of potential benefits, beyond improved mechanical
performance. The concentration, viscosity, molecular weight, and
conformational makeup of the silk fibroin/water solution likely can
have effects on the consistency and properties of the material
during the process and the final constructs.
[0039] The consistency of the liquid binder comprising composition
needs to be correct. With an insufficient quantity of liquid
binder, the compacted composition can likely become inhomogeneous
and possibly develop cracks and exhibit poor mechanical properties.
With too much binder, the composition viscosity can likely become
too low and prevent proper consolidation in the press (leakage from
under the piston and likely development of voids or geometrical
unstable constructs.
[0040] In some embodiments, the silk composition has a paste (or
paste-like) consistency. In some embodiments, paste (or paste-like)
consistency means that the composition is malleable or moldable.
Paste consistency can be stated in terms of the viscosity of the
solution. In some embodiments, viscosity of the composition can
range from about 0.1 to about 250 Pas, from about 0.2 to about 150
Pas, from about 0.3 to about 100 Pas, from about 0.4 to about 50
Pas, or from about 0.5 to about 25 Pas. Compositions with overly
high viscosity can be difficult to spread, smooth, and shape, while
those with excessively low viscosity can be difficult to handle for
molding purposes. Without wishing to be bound by a theory,
compositions of higher viscosity can be used without a mold. For
example, a composition of higher viscosity can be formed into a
simple geometric shape by mechanical means, e.g. hands.
Compositions of lower viscosity can be used for injection molding
into molds of predetermined shape or into molds of simple geometric
shapes. Compositions of higher viscosity can also be used for
injection molding into predetermined shapes or simple geometric
shapes.
[0041] Viscosity can be measured with various types of viscometers
and rheometers. A rheometer is generally used for those fluids
which cannot be defined by a single value of viscosity and
therefore require more parameters to be set and measured than is
the case for a viscometer. In some embodiments, viscosity can be
determined at room temperature.
[0042] In some embodiments, a small amount of distilled water is
measured and added to the silk composition comprising silk
particles, e.g., with a 1 ml syringe. For example, a few drops of
water can be added at a time and mixed with the silk particles.
Once all water is added, a thorough mixing yields a viscous and
sticky compound that has the consistency of smooth peanut
butter.
[0043] In some embodiments, the compacted composition is a hard
material, with a ceramic-like feel. Mechanical response varies
widely depending on the parameters selected throughout the
process.
[0044] After compaction, the compacted composition can be processed
into the final desired shape to obtain an article of manufacture.
As used herein, the term "processing" with reference to processing
into the desired shape should be understood to include any method
or process used to provide the final shape of the manufactured
article. Without limitation, such processing can include, but is
not limited to, mechanical and chemical means. For example,
processing can be selected from the group consisting of machining,
turning (lathe), rolling, thread rolling, drilling, milling,
sanding, punching, die cutting, blanking, broaching, extruding,
chemical etching, and any combinations thereof. As used herein, the
term "machining" should be understood to include all types of
machining operations including, but run limited to, CNC machining,
cutting, milling, turning, drilling, shaping, planing, broaching,
sawing, burnishing, grinding, and the like. One or more of the
processing methods can be used in combination to obtain more
complex, intricate geometries. The term "machinable" means a
material which can be readily subjected to machining.
[0045] Accordingly, in some embodiments, the method comprises: (i)
providing a composition comprising silk particles; (ii) compacting
the composition by application of pressure; and (iii) processing
the compacted composition to a desired shape.
[0046] In some embodiments, the composition is in a mold. As used
herein, the term "mold" is intended to encompass any mold,
container or substrate capable of shaping, holding or supporting
the silk composition. Thus, the mold in its simplest form could
simply comprise a supporting surface. The mold can be of any
desired shape, and can be fabricated from any suitable material
including polymers (such as polysulphone, polypropylene,
polyethylene), metals (such as stainless steel, titanium, cobalt
chrome), ceramics (such as alumina, zirconia), glass ceramics, and
glasses (such as borosilicate glass). In some embodiments, the mold
can provide a scaffold of simple geometry, which can be processed
into the final desired shape, i.e., the mold can be used to provide
a blank which can be processed to the final shape.
[0047] As used herein, the term "silk fibroin" or "fibroin"
includes silkworm fibroin and insect or spider silk proiein. See
e.g., Lucas et al., 13 Adv. Protein Chem. 107 (1958). Any type of
silk fibroin can be used according to aspects of the present
invention. 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 can be attained by
extracting sericin from the cocoons of B. mori. Organic silkworm
cocoons are also commercially available. There are many different
silks, however, including spider silk (e.g., obtained from Nephila
clavipes), transgenic silks, genetically engineered silks
(recombinant silk), such as silks from bacteria, yeast, mammalian
cells, transgenic animals, or transgenic plants, and variants
thereof, that can be used. See for example, WO 97/08315 and U.S.
Pat. No. 5,245,012, content of both of which is incorporated heroin
by reference in its entirety. In some embodiments, silk fibroin can
be derived from other sources such as spiders, other silkworms,
bees, and bioengineered variants thereof. In some embodiments, silk
fibroin can be extracted from a gland of silkworm or transgenic
silkworms. See for example, WO2007/098951, content of which is
incorporated herein by reference in its entirety. In some
embodiments, silk fibroin is free, or essentially free of sericin,
i.e., silk fibroin is a substantially sericin-depleted silk
fibroin.
[0048] Degummed silk can be prepared by any conventional method
known to one skilled in the art. For example, B. mori cocoons are
boiled for about up to 60 minutes, generally about 30 minutes, in
an aqueous solution. In one embodiment, the aqueous solution is
about 0.02M Na.sub.2CO.sub.3. The cocoons are rinsed, for example,
with water to extract the sericin proteins. The degummed silk can
be dried and used for preparing silk powder. Alternatively, the
extracted silk can dissolved in an aqueous salt solution. Salts
useful for this purpose include lithium bromide, lithium
thiocyanate, calcium nitrate or other chemicals capable of
solubilizing silk. In some embodiments, the extracted silk can
dissolved in about 8M-12 M LiBr solution. The salt is consequently
removed using, for example, dialysis.
[0049] If necessary, the solution can then be concentrated using,
for example, dialysis against a hygroscopic polymer, for example,
PEG, a polyethylene oxide, amylose or sericin. In some embodiments,
the PEG is of a molecular weight of 8,000-10,000 g/mol and has a
concentration of about 10% to about 50% (w/v). A slide-a-lyzer
dialysis cassette (Pierce, MW CO 3500) can be used. However, any
dialysis system can be used. The dialysis can be performed for a
time period sufficient to result in a final concentration of
aqueous silk solution between about 10% to about 30%. In most cases
dialysis for 2-12 hours can be sufficient. See, for example,
International Patent Application Publication No. WO 2005/012606,
the content of which is incorporated herein by reference in its
entirety.
[0050] The silk fibroin solution can be produced using organic
solvents. Such methods have been described, for example, in Li, M.,
et al., J. Appl. Poly Sci. 2001, 79, 2192-2199; Min, S., et al.,
Sen'l Gakkaishi 1997, 54, 85-92; Nazarov, R. et al.,
Biomacromolecules 2004 May-June; 5(3):718-26, content of all which
is incorporated herein by reference in their entirety. An exemplary
organic solvent that can be used to produce a silk solution
includes, but is not limited to, hexafluoroisopropanol (HFIP). See,
for example, International Application No. WO2004/000915, content
of which is incorporated herein by reference in its entirety. In
some embodiments, the silk solution is free or essentially free of
organic solvents, i.e., solvents other than water.
[0051] Generally, any amount of silk fibroin can be present in the
solution. For example, amount of silk in the solution or the
composition prepared therefrom can be from about 1% (w/v) to about
50% (w/v) of silk, e.g., silk fibroin. In some embodiments, the
amount of silk in the solution or the composition prepared
therefrom can be from about 1% (w/v) to about 35% (w/v), from about
1% (w/v) to about 30% (w/v), from about 1% (w/v) to about 25%
(w/v), from about 1% (w/v) to about 20% (w/v), from about 1% (w/v)
to about 15% (w/v), from about 1% (w/v) to about 10% (w/v), from
about 5% (w/v) to about 25% (w/v), from about 5% (w/v) to about 20%
(w/v), from about 5% (w/v) to about 15% (w/v). In some embodiments,
the silk in the silk solution is about 25% (w/v). In some
embodiments, the silk in the silk solution is about 6% (w/v) to
about 8% (w/v). Exact amount of silk in the silk solution can be
determined by drying a known amount of the silk solution and
measuring the mass of the residue to calculate the solution
concentration.
[0052] The silk fibroin can be used to fabricate a silk
fibroin-based scaffold which can then be used to produce silk
particles for use in the disclosed method. For example, the silk
fibroin solution can be formed into silk fibroin-based scaffold
such as a fiber, film, gel, hydrogel, foam, mesh, mat, or non-woven
mat. The silk fibroin-based scaffold (e.g., fiber, film, gel,
hydrogel, foam, mesh, mat, or non-woven mat) can be processed by
subjecting the silk fibroin-bused scaffold to milling, grinding,
cutting, crushing, or any combinations thereof to form silk
particles. For example, the silk fibroin-based scaffold can be
blended in a conventional blender or milled in a ball mill to form
silk particles of desired size.
[0053] The silk fibroin-based scaffold can be in any form, shape or
size. Accordingly, in some embodiments, the silk fibroin-based
material is in the form of a fiber. As used herein, the term
"fiber" means a relatively flexible, unit of matter having a high
ratio of length to width across its cross-sectional perpendicular
to its length. Methods for preparing silk fibroin fibers are well
known in the art. A fiber can be prepared by electrospinning a silk
solution, drawing a silk solution, and the like. Electrospun silk
materials, such as fibers, and methods for preparing the same are
described, for example in WO2011/008842, content of which is
incorporated herein by reference in its entirety. Micron-sized silk
fibers (e.g., 10-600 .mu.m in size) and methods for preparing the
same are described, for example in Mandal et al., PNAS, 2012, doi:
10.1073/pnas.1119474109; U.S. Provisional Application No.
61/621,209, filed Apr. 6, 2012, and PCT application no.
PCT/US13/35389, filed Apr. 5, 2013, content of all of which is
incorporated herein by reference
[0054] In some embodiments, the silk fibroin-based scaffold can be
in the form of a film, e.g., a silk film. As used herein, the term
"film" refers to a flat or tubular flexible structure. It is to be
noted that the term "film" is used in a generic sense to include a
web, film, sheet, laminate, or the like. In some embodiments, the
film is a patterned film, e.g., nanopatterned film. Exemplary
methods for preparing silk fibroin films are described in, for
example, WO 2004/000915 and WO 2005/012606, content of both of
which is incorporated herein by reference in its entirety.
[0055] In some embodiments, the silk fibroin-based scaffold can be
in the form of a gel or hydrogel. The term "hydrogel" is used
herein to mean a silk-based material which exhibits the ability to
swell in water and to retain a significant portion of water within
its structure without dissolution. Methods for preparing silk
fibroin gels and hydrogels are well known in the art. Methods for
preparing silk fibroin gels and hydrogels include, but are not
limited to, sonication, vortexing, pH titration, exposure to
electric field, solvent immersion, water annealing, water vapor
annealing, and the like. Exemplary methods for preparing silk
fibroin gels and hydrogels are described in, for example, WO
2005/012606, content of which is incorporated herein by reference
in its entirety. In some embodiments, the silk fibroin-based
scaffold can be in the form of a sponge or foam. Methods for
preparing silk fibroin gels and hydrogels are well known in the
art. In some embodiments, the foam or sponge is a patterned foam or
sponge, e.g., nanopatterned foam or sponge. Exemplary methods for
preparing silk foams and sponges are described in, for example, WO
2004/000915, WO 2004/000255, and WO 2005/012606, content of all of
which is incorporated herein by reference in its entirety.
[0056] In some embodiments, the silk fibroin-based scaffold can be
in the form of a cylindrical matrix, e.g., a silk tube. The silk
tubes can be made using any method known in the art. For example,
tubes can be made using molding, dipping, electrospinning, gel
spinning, and the like. Gel spinning is described in Lovett et al.
(Biomaterials, 29(35):4650-4657 (2008)) and the construction of
gel-spun silk tubes is described in PCT application no.
PCT/US2009/039870, filed Apr. 8, 2009, content of both of which is
incorporated herein by reference in their entirety. Construction of
silk tubes using the dip-coating method is described in PCT
application no. PCT/US2008/072742, filed Aug. 11, 2008, content of
which is incorporated herein by reference in its entirety.
Construction of silk fibroin tubes using the film-spinning method
is described in PCT application No. PCT/US2013/030206, filed Mar.
11, 2013 and U.S. Provisional application No. 61/613,185, filed
Mar. 20, 2012.
[0057] In some embodiments, the silk fibroin-based scaffold can be
porous. For example, the silk fibroin-matrix can have a porosity of
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%, or higher. As
used herein, 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).
Determination of porosity is well known to a skilled artisan, e.g.,
using standardized techniques, such as mercury porosimetry and gas
adsorption, e.g., nitrogen adsorption.
[0058] The porous silk-based scaffold can have any pore size. As
used herein, the term "pore size" refers to a diameter or an
effective diameter of the cross-sections of the pores. The term
"pore size" can also refer to an average diameter or an average
effective diameter of the cross-sections of the pores, based on the
measurements of a plurality of pores. The effective diameter of a
cross-section that is not circular equals the diameter of a
circular cross-section that has the same cross-sectional area as
that of the non-circular cross-section.
[0059] Methods for forming pores in silk fibroin-based scaffolds
are known in the art and include, but are not limited,
porogen-leaching methods, freeze-drying methods, and/or gas-forming
method. Exemplary methods for forming pores in a silk-based
material are described, for example, in U.S. Pat. App. Pub. Nos.:
US 2010/0279112 and US 2010/0279112; U.S. Pat. No. 7,842,780; and
WO2004062697, content of all of which is incorporated herein by
reference in its entirety.
[0060] Though not meant to be bound by a theory, silk fibroin-based
scaffold's porosity, structure, and mechanical properties can be
controlled via different post-spinning processes such as vapor
annealing, heat treatment, alcohol treatment, air-drying,
lyophilization and the like. Additionally, any desirable release
rates, profiles or kinetics of a molecule encapsulated in the
matrix can be controlled by varying processing parameters, such as
matrix thickness, silk molecular weight, concentration of silk in
the matrix, beta-sheet conformation structures, silk II beta-sheet
crystallinity, or porosity and pore sizes.
[0061] In some embodiments, the method further comprises inducing a
conformational change in silk fibroin to make the silk fibroin at
least partially insoluble. Without wishing to be bound by a theory,
the induced conformational change alters the crystallinity of the
silk fibroin, e.g., Silk II beta-sheet crystallinity. The
conformational change can be induced by any methods known in the
art, including, but not limited to, alcohol immersion (e.g.,
ethanol, methanol), water annealing, shear stress, ultrasound
(e.g., by sonication), pH reduction (e.g., pH titration and/or
exposure to an electric field) and any combinations thereof. For
example, the conformational change can be induced by one or more
methods, including but not limited to, controlled slow drying (Lu
et al., 10 Macromolecules 1032 (2009)); water annealing (Jin et
al., Water-Stable Silk Films with Reduced .beta.-Sheet Content, 15
Adv. Funct. Mats. 1241 (2005); Hu et al. Regulation of Silk
Material Structure by Temperature-Controlled Water Vapor Annealing,
12 Biomacromolecules 1686 (2011)); 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;
solvent immersion, including methanol (Hofmann et al., Silk fibroin
as an organic polymer for controlled drug delivery, 111 J Control
Release. 219 (2006)), ethanol (Miyairi et al., Properties of
b-glucosidase immobilized in sericin membrane. 56 J. Fermen. Tech.
303 (1978)), glutaraldehyde (Acharya et al., Performance evaluation
of a silk protein-based matrix for the enzymatic conversion of
tyrosine to L-DOPA. 3 Biotechnol J. 226 (2008)), and
1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) (Bayraktar et
al., Silk fibroin as a novel coating material for controlled
release of theophylline. 60 Eur J Pharm Biopharm. 373 (2005)); pH
adjustment, e.g., pH titration and/or exposure to an electric field
(see, e.g., U.S. Patent App. No. US2011/0171239); heat treatment;
shear stress (see, e.g., International App. No.: WO 2011/005381),
ultrasound, e.g., sonication (see, e.g., U.S. Patent Application
Publication No. U.S. 2010/0178304 and International App. No.
WO2008/150861); and any combinations thereof. Content of all of the
references listed above is incorporated herein by reference in
their entirety.
[0062] In some embodiments, the conformation of the silk fibroin
can be altered by water annealing. Without wishing to be bound by a
theory, it is believed that physical temperature-controlled water
vapor annealing (TCWVA) provides a simple and effective method to
obtain refined control of the molecular structure of silk
biomaterials. The silk materials can be prepared with control of
crystallinity, from a low content using conditions at 4.degree. C.
(.alpha. helix dominated silk I structure), to highest content of
.about.60% crystallinity at 100.degree. C. (.beta.-sheet dominated
silk II structure). This physical approach covers the range of
structures previously reported to govern crystallization during the
fabrication of silk materials, yet offers a simpler, green
chemistry, approach with tight control of reproducibility.
Temperature controlled water vapor annealing is described, for
example, in Hu et al., Rergulation of Silk Material Structure By
Temperature Controlled Water Vapor Annealing, Biomacromolecules,
2011, 12(5): 1686-1696, content of which is incorporated herein by
reference in its entirety.
[0063] In some embodiments, alteration in the conformation of the
silk fibroin can be induced by immersing in alcohol, e.g.,
methanol, ethanol, etc. The alcohol concentration can be at least
10%, at least 20%, at least 30%, at least 40%, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90% or 100%. In
some embodiment, alcohol concentration is 100%. If the alteration
in the conformation is by immersing in a solvent, the silk
composition can be washed, e.g., with solvent-water gradient to
remove any of the residual solvent that is used for the immersion.
The washing can be repeated one, e.g., one, two, three, four, five,
or more times.
[0064] Alternatively, the alteration in the conformation of the
silk fibroin can be induced with sheer stress. The sheer stress can
be applied, for example, by passing the silk composition through a
needle. Other methods of inducing conformational changes include
applying an electric field, applying pressure, or changing the salt
concentration.
[0065] The treatment time for inducing the conformational change
can be any period of time to provide a desired silk II (beta-sheet
crystallinity) content. In some embodiments, the treatment time can
range from about 1 hour to about 12 hours, from about 1 hour to
about 6 hours, from about 1 hour to about 5 hours, from about 1
hour to about 4 hours, or from about 1 hour to about 3 hours. In
some embodiments, the sintering time can range from about 2 hours
to about 4 hours or from 2.5 horus to about 3.5 hours.
[0066] When inducing the conformational change is by solvent
immersion, treatment time can range from minutes to hours. For
example, immersion in the solvent can be for a period of at least
about 15 minutes, at least about 30 minutes, at least about 1 hour,
at least about 2 hours, at least 3 hours, at least about 6 hours,
at least about 18 hours, at least about 12 hours, at least about 1
day, at least about 2 days, at least about 3 days, at least about 4
days, at least about 5 days, at least about 6 days, at least about
7 days, at least about 8 days, at least about 9 days, at least
about 10 days, at least about 11 days, at least about 12 days, at
least about 13 days, or at least about 14 days. In some
embodiments, immersion in the solvent can be for a period of about
12 hours to about seven days, about 1 day to about 6 days, about 2
to about 5 days, or about 3 to about 4 days.
[0067] After the treatment to induce the conformutionul change,
silk fibroin can comprise a silk II beta-sheet crystallinity
content of 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%, or at least about 95% but not 100% (i.e., all the silk
is present in a silk II beta-sheet conformation). In some
embodiments, silk is present completely in a silk II beta-sheet
conformation, i.e., 100% silk II beta-sheet crystallinity.
[0068] In some embodiments, the silk composition for compaction can
comprise one or more (e.g., one, two, three, four, five or more)
additives. Without wishing to be bound by a theory additive can
provide one or more desirable properties to an article of
manufacture, e.g., strength, flexibility, case of processing and
handling, biocompatibility, bioresorability, lack of air bubbles,
surface morphology, and the like, prepared from the compacted
composition. The additive can be covalently or non-covalently
linked with silk and can be integrated homogenously or
heterogeneously within the silk composition.
[0069] An additive can be selected from small organic or inorganic
molecules; saccharines; oligosaccharides; polysaccharides;
biological macromolecules, e.g., peptides, proteins, and peptide
analogs and derivatives; peptidomimetics; antibodies and antigen
binding fragments thereof; nucleic acids; nucleic acid analogs and
derivatives; glycogens or other sugars; immunogens; antigens; an
extract made from biological materials such as bacteria, plants,
fungi, or animal cells; animal tissues; naturally occurring or
synthetic compositions; and any combinations thereof. Furthermore,
the additive can be in any physical form. For example, the additive
can be in the form of a particle, a fiber, a film, a gel, a mesh, a
mat, a non-woven mat, a powder, a liquid, or any combinations
thereof. In some embodiments, the additive is a particle.
[0070] Total amount of additives in the composition can be from
about 0.1 wt % to about 99 wt %, from about 0.1 wt % to about 70 wt
%, from about 5 wt % to about 60 wt %, from about 10 wt % to about
50 wt %, from about 15 wt % to about 45 wt %, or from about 20 wt %
to about 40 wt %, of the total silk composition. In some
embodiments, ratio of silk to additive in the composition can range
from about 50:1 (w/w) to about 1:50 (w/w), from about 25:1 (w/w) to
about 1:25 (w/w), from about 20:1 (w/w) to about 1:20 (w/w), from
about 10:1 (w/w) to about 1:10 (w/w), or from about 5:1 (w/w) to
about 1:5 (w/w).
[0071] In some embodiments, the additive is a calcium phosphate
material (CaP). As used herein, the term "calcium phosphate
material" refers to any material composed of calcium and phosphate
ions. The term "calcium phosphate material" is intended to include
naturally occurring and synthetic materials composed of calcium and
phosphate ions. The ratio of calcium to phosphate ions in the
calcium phosphate materials is preferably selected such that the
resulting material is able to perform its intended function. For
convenience, the calcium to phosphate ion ratio is abbreviated as
the "Ca/P ratio." In some embodiments, the Ca/P ratio can range
from about 1:1 to about 1.67 to 1. In some embodiments, the calcium
phosphate material can be calcium deficient. By "calcium deficient"
is meant a calcium phosphate material with a calcium to phosphate
ratio of less than about 1.6 as compared to the ideal
stoichiometric value of approximately 1.67 for hydroxyapatite
[0072] The calcium phosphate material can be in the form of
particles. Without limitations, the calcium phosphate material
particles can be of any desired size. In some embodiments, the
calcium phosphate material particles can have a size ranging from
about 0.01 .mu.m to about 1000 .mu.m, about 0.05 .mu.m to about 500
.mu.m, about 0.1 .mu.m to about 250 .mu.m, about 0.25 .mu.m to
about 200 .mu.m, or about 0.5 .mu.m to about 100 .mu.m. Further,
the calcium phosphate material particle can be of any shape or
form, e.g., spherical, rod, elliptical, cylindrical, capsule, or
disc.
[0073] In some embodiments, the calcium phosphate material particle
is a microparticle or a nanoparticle. In some embodiments, the
calcium phosphate material particle has a particle size of about
0.01 .mu.m to about 1000 .mu.m, about 0.05 .mu.m to about 750
.mu.m, about 0.1 .mu.m to about 500 .mu.m, about 0.25 .mu.m to
about 250 .mu.m, or about 0.5 .mu.m to about 100 .mu.m. In some
embodiments, the silk particle has a particle size of about 0.1 nm
to about 1000 nm, about 0.5 nm to about 500 nm, about 1 nm to about
250 nmm, about 10 nm to about 150 nm, or about 15 nm to about 100
nm.
[0074] The calcium phosphate material can be selected, for example,
from one or more of brushite, octacalcium phosphate, tricalcium
phosphate (also referred to as tricalcic phosphate and calcium
orthophosphate), calcium hydrogen phosphate, calcium dihydrogen
phosphate, apatite, and/or hydroxyapatite. Further, tricalcium
phosphate (TCP) can be in the alpha or the beta crystal form. In
some embodiments, the calcium phosphate material is beta-tricalcium
phosphate or apatite, e.g., hydroxyapatite (HA).
[0075] The amount of the calcium phosphate material in the silk
composition can range from about 1% to about 99% (w/w or w/v). In
some embodiments, the amount of the calcium phosphate material in
the silk composition can be from about 5% to about 95% (w/w or
w/v), from about 10% to about 90% (w/w or w/v), from about 15% to
about 80% (w/w or w/v), from about 20% to about 75% (w/w or w/v),
from about 25% to about 60% (w/w or w/v), or from about 30% to
about 50% (w/w or w/v). In some embodiments, the amount of the
calcium phosphate material in the silk composition can be less than
20%.
[0076] Generally, the silk composition can comprise any ratio of
silk to calcium phosphate material. For example, the ratio of silk
to calcium phosphate material in the composition can range from
about 1000:1 to about 1:1000. The ratio can be based on weight or
moles. In some embodiments, the ratio of silk to calcium phosphate
material in the solution can range from about 500:1 to about 1:500
(w/w), from about 250:1 to about 1:250 (w/w), from about 50:1 to
about 1:200 (w/w), from about 10:1 to about 1:150 (w/w) or from
about 5:1 to about 1:100 (w/w).
[0077] In some embodiments, the additive can be a silk-based
material. The silk-based material can be selected from the group
consisting of silk fibers, micro-sized silk fibers, unprocessed
silk fibers, silk particles, and any combinations thereof.
[0078] In some embodiments, the additive is a silk fiber. While the
use of silk fibers is described in for example, US patent
application publication no. US20110046686, the previously described
materials do not provide machinable silk materials as disclosed in
the present disclosure.
[0079] In some embodiments, the silk fibers are microfibers or
nanofibers. In some embodiments, the additive is micron-sized silk
fiber (10-600 .mu.m). Micron-sized silk fibers can be obtained by
hydrolyzing the degummed silk fibroin or by increasing the boing
time of the degumming process. Alkali hydrolysis of silk fibroin to
obtain micron-sized silk fibers is described for example in Mandal
et al., PNAS, 2012, doi: 10.1073/pnas.119474109; U.S. Provisional
Application No. 61/621,209, filed Apr. 6, 2012; and PCT application
no. PCT/US13/35389, filed Apr. 5, 2013, content of all of which is
incorporated herein by reference. Because regenerated silk fibers
made from HFIP silk solutions are mechanically strong, the
regenerated silk fibers can also be used as additive.
[0080] In some embodiments, the silk fiber is an unprocessed silk
fiber, e.g., raw silk or raw silk fiber. The term "raw silk" or
"raw silk fiber" refers to silk fiber that has not been treated to
remove sericin, and thus encompasses, for example, silk fibers
taken directly from a cocoon. Thus, by unprocessed silk fiber is
meant silk fibroin, obtained directly from the silk gland. When
silk fibroin, obtained directly from the silk gland, is allowed to
dry, the structure is referred to as silk I in the solid state.
Thus, an unprocessed silk fiber comprises silk fibroin mostly in
the silk I conformation. A regenerated or processed silk fiber on
the other hand comprises silk fibroin having a substantial silk II
or beta-sheet crystallinity.
[0081] Because implantation and post-surgical imaging of current
resorbable fixation devices is a problem, the article of
manufacture, e.g., medical devices such as orthopedic screws or
other fasteners can be enhanced with iron particles. Accordingly,
in some embodiments, the additive is an iron particle. Without
wishing to be bound by a theory, it is believed that the iron
particles can help the surgeon during implantation due to a
magnetic screw that can be attracted to a screw driver head.
Further, once the surgery is complete, the surgeon could quickly
check that all components are properly placed and have not migrated
or failed with a simple magnetic sensor. This would allow for a
first pass check of surgical errors and allow the surgeon to reopen
the wound and fix the problem before the patient leaves the
operating room. This would save on time, money, and recovery
time.
[0082] In some embodiments, the additive is a biocompatible
polymer. Exemplary biocompatible polymers include, but are not
limited to, a poly-lactic acid (PLA), poly-glycolic acid (PGA),
poly-lactide-co-glycolide (PLGA), polyesters, poly(ortho ester),
poly(phosphazine), poly(phosphate ester), polycaprolactone,
gelatin, collagen, fibronectin, keratin, polyaspartic acid,
alginate, chitosan, chitin, hyaluronic acid, pectin,
polyhydroxyalkanoates, dextrans, and polyanhydrides, polyethylene
oxide (PEO), poly(ethylene glycol) (PEG), triblock copolymers,
polylysine, alginate, polyaspartic acid, any derivatives thereof
and any combinations thereof. Other exemplary biocompatible
polymers amenable to use according to the present disclosure
include those described for example in U.S. Pat. Nos. 6,302,848;
6,395,734; 6,127,143; 5,263,992; 6,379,690; 5,015,476; 4,806,355;
6,372,244; 6,310,188; 5,093,489; 387,314; 6,325,810; 6,337,198;
6,267,776; 5,576,881; 6,245,537; 5,902,800; and 5,270,419, content
of all of which is incorporated herein by reference.
[0083] In some embodiments, the biocompatible polymer is PEG or
PEO. As used herein, the term "polyethylene glycol" or "PEG" means
an ethylene glycol polymer that contains about 20 to about 2000000
linked monomers, typically about 50-1000 linked monomers, usually
about 100-300. PEG is also known as polyethylene oxide (PEO) or
polyoxyethylene (POE), depending on its molecular weight. Generally
PEG, PEO, and POE are chemically synonymous, but historically PEG
has tended to refer to oligomers and polymers with a molecular mass
below 20,000 g/mol, PEO to polymers with a molecular mass above
20,000 g/mol, and POE to a polymer of any molecular mass. PEG and
PEO are liquids or low-melting solids, depending on their molecular
weights. PEGs are prepared by polymerization of ethylene oxide and
are commercially available over a wide range of molecular weights
from 300 g/mol to 10,000,000 g/mol. While PEG and PEO with
different molecular weights find use in different applications, and
have different physical properties (e.g. viscosity) due to chain
length effects, their chemical properties are nearly identical.
Different forms of PEG are also available, depending on the
initiator used for the polymerization process--the most common
initiator is a monofunctional methyl ether PEG, or
methoxypoly(ethylene glycol), abbreviated mPEG.
Lower-molecular-weight PEGs are also available as purer oligomers,
referred to as monodisperse, uniform, or discrete PEGs are also
available with different geometries.
[0084] As used herein, the term PEG is intended to be inclusive and
not exclusive. The term PEG includes poly(ethylene glycol) in any
of its forms, including alkoxy PEG, difunctional PEG, multiarmed
PEG, forked PEG, branched PEG, pendent PEG (i.e., PEG or related
polymers having one or more functional groups pendent to the
polymer backbone), or PEG With degradable linkages therein.
Further, the PEG backbone can be linear or branched. Branched
polymer backbones are generally known in the art. Typically, a
branched polymer has a central branch core moiety and a plurality
of linear polymer chains linked to the central brunch core. PEG is
commonly used in branched forms that can be prepared by addition of
ethylene oxide to various polyols, such as glycerol,
pentaerythritol and sorbitol. The central branch moiety can also be
derived from several amino acids, such as lysine. The branched
poly(ethylene glycol) can be represented in general form as
R(-PEG-OH)m in which R represents the core moiety, such as glycerol
or pentaerythritol, and m represents the number of arms.
Multi-armed PEG molecules, such as those described in U.S. Pat. No.
5,932,462, which is incorporated by reference herein in its
entirety, can also be used as biocompatible polymers.
[0085] Some exemplary PEGs include, but are not limited to, PEG20,
PEG30, PEG40, PEG60, PEG80, PEG100, PEG115, PEG200, PEG300, PEG400,
PEG500, PEG600, PEG1000, PEG1500, PEG2000, PEG3350, PEG4000,
PEG4600, PEG5000, PEG6000, PEG8000, PEG11000, PEG12000, PEG15000,
PEG20000, PEG250000, PFG500000, PEG100000, PEG2000000 and the like.
In some embodiments, PEG is of MW 10,000 Dalton. In some
embodiments, PEG is of MW 100,000, i.e. PEO of MW 100,000.
[0086] In some embodiments, the additive is an enzyme that
hydrolyzes silk fibroin. Without wishing to be bound by a theory,
such enzymes can be used to control the degradation of the article
of manufacture.
Article of Manufacture
[0087] Silk-based materials can be used to produce tissue scaffolds
for tissue engineering applications. While these tissue scaffolds
take advantage of the biocompatibilily, tunable degradation, and
other properties of silk. In some embodiments, they typically
cannot withstand the loading conditions experienced by structural
tissue (e.g., bone) in the body. Accordingly, mechanically robust
silk materials are developed, and such material formats can range
from monolithic to composite structures (silk-silk composites: silk
reinforcing phase bound by a second silk material phase).
[0088] One application area for robust monolithic and composite
silk material is in creating tissue engineering scaffolds for human
tissue repair/replacement in areas where in vivo physiological
loading conditions may be significant. For example, such material
can be used to replace the traditional metal plate and screw
components used in a reconstructive orthopedic surgery. Other
biomedical applications include usage as an internal fracture
stabilizer (smart splint used as an in in vivo brace) or void
filling where bone defects or disease have compromised mechanical
stability.
[0089] Hard, strong, lightweight, and biodegradable monolithic and
composite silk materials are not limited to biomedical
applications. Machine components, such as nuts, bolts, and gears
could potentially be constructed of silk. Everyday consumer items,
such as biodegradable dishware, plastic ware, or food containers
could be silk-based. Given the ability to mold the silk materials
described, fairly complex shapes can be created, along with the
ability to emboss and imprint images, numbers, and codes. The
properties of the silk material can be enhanced and specifically
tailored through the addition of other material phases. For
example, short or continuous silk have fiber can be incorporated in
a composite construct to enhance material toughness. Optically
clear fiber (including silk-based material) can be embedded to
provide sensing and information transmission capability. By
combining multiple silk material formats, entirely unique products
can be fabricated, e.g., construction of a 100% silk apparel and/or
accessories. In one embodiment, a 100% silk shoe can be fabricated
by combining multiple silk material formats: for example, the hard
monolithic and composite silk materials can be combined with silk
foams, films, and fibers to make the desired shoe form.
[0090] Accordingly, the disclosure also provides an article of
manufacture prepared by the method described herein. The article of
manufacture prepared according the method described herein is
biocompatible and/or at least partially bioresorbable. As used
herein, the term "biocompatible" refers to a material that does not
elicit a substantial immune response in the host.
[0091] By "bioresorbable" is meant the ability of a material to be
resorbed or remodeled in vivo. The resorption process involves
degradation and elimination of the original implant material
through the action of body fluids, enzymes or cells. The resorbed
materials can be used by the host in the formation of new tissue,
or it can be otherwise re-utilized by the host, or it can be
excreted. The article of manufacture described herein can have a
resorption half-life of approximately 6 months to approximately 12
months. In some embodiments, the article of manufacture has a
resorption half-life of approximately 9 months. The article of
manufacture can be completely resorbed in approximately 12 months
to approximately 24 months. In some embodiments the material is
completely resorbed in approximately 12 months.
[0092] In some embodiments, the article of manufacture described
herein has compressive strength, compressive toughness and
compressive elastic modulus values approximate to those of healthy
human bone and enables immediate load-bearing. Without wishing to
be bound by a theory, load-bearing properties can also prevent
unwanted resorption of adjacent bone resulting from high local
stress concentration or stress-shielding.
[0093] Compressive toughness is the capacity of a material to
resist fracture when subjected to axially directed pushing forces.
Bu definition, the compressive toughness of a material is the
ability to absorb mechanical (or kinetic) energy up to the point of
failure. Toughness is measured in units of joules per cubic meter
(Jm.sup.-3) and can be measured as the area under a stress-strain
curve. In some embodiments, the article of manufacture described
herein has a compressive toughness of about 1 kJ m.sup.-3 to about
20 kJm.sup.-3 or about 1 kJm.sup.-3 to approximately 5 kJm.sup.-3
at 6% strain as measured by the J-integral method. In one
embodiment, article of manufacture has a compressive toughness of
about 1.3 kJm.sup.-3, which is the approximate compressive
toughness of healthy bone.
[0094] Compressive strength is the capacity of a material to
withstand axially directed pushing forces. By definition, the
compressive strength of a material is that value of uniaxial
compressive stress reached when the material fails completely. A
stress-strain curve is a graphical representation of the
relationship between stress derived from measuring the load applied
on the sample (measured in MPa) and strain derived from measuring
the displacement as a result of compression of the sample. The
ultimate compressive strength of the material can depend upon the
target site of implantation. For example, if the material is for
placement next to osteoporotic cancellous bone, to avoid high
stress accumulation and stress shielding, the material can comprise
a compressive strength (stress to yield point) of approximately 0.1
MPa to approximately 2 MPa. If the material is intended for
placement next to healthy cancellous bone, the material can
comprise an ultimate compressive strength (stress to yield point)
of approximately 5 MPa. Alternatively, if the material is intended
for placement next to cortical bone, the material can comprise an
ultimate compressive strength (stress to yield point) of at least
40 MPa.
[0095] Generally, the article of manufacture described herein
comprises an ultimate compressive strength (stress to yield point)
of at least 5 MPa, at least 10 MPa, at leaste 15 MPa, at least 20
MPa, at least 25 MPa, at least 30 MPa, at least 35 MPa, at least 40
MPa, at least 45 MPa, at least 50 MPa, at least 55 MPa, at least 60
MPa, at least 65 MPa, at least 70 MPa, at least 75 MPa, at least 80
MPa, at least 85 MPa, at least 90 MPa, at least 95 MPa, at least
100 MPa, at least 105 MPa, at least 110 MPa, at least 115 MPa, at
least 120 MPa, at least 125 MPa, at least 130 MPa, at least 135
MPa, at least 140 MPa, at least 145 MPa, at least 150 MPa, or at
least 155 MPa.
[0096] For example, the article of manufacture described herein
comprises an ultimate compressive strength of about 5 MPa to about
140 MPa, about 20 MPa to about 130 MPa, from about 60 MPa to about
125 MPa, or from about 90 to about 120 MPa. In some embodiments,
the article of manufacture described herein comprises an ultimate
compressive strength (stress to yield point) of at least 100 MPa.
In one embodiment, the article of manufacture described herein
comprises an ultimate compressive strength (stress to yield point)
of approximately 104 MPa. In some embodiment, the article of
manufacture described herein has a compressive strength of from
about 20 MPa to about 130 MPa at 5% strain.
[0097] Compressive elastic modulus is the mathematical description
of the tendency of a material to be deformed elastically (i.e.
non-permanently) when a force is applied to it. The Young's modulus
(E) describes tensile elasticity, or the tendency of a material to
deform along an axis when opposing forces are applied along that
axis; it is defined as the ratio of tensile stress to tensile
strain (measured in MPa) and is otherwise known as a measure of
stiffness of the material. The elastic modulus of an object is
defined as the slope of the stress-strain curve in the elastic
deformation region. The article of manufacture described herein can
comprise a compressive elastic modulus of between approximately 100
MPa and approximately 5,000 MPa GPa at 5% strain. In some
embodiments, the article of manufacture described herein comprises
a compressive elastic modulus of between approximately 200 MPa and
750 MPa, between approximately 250 MPa and 700 MPa, between
approximately 300 MPa and 650 MPa, between approximately 400 MPa
and 600 MPa, or between approximately 450 MPa and 550 MPa at 5%
strain.
[0098] In some embodiments, article of manufacture described herein
has a mean compressive elastic modulus of about 525 MPa. In some
embodiments, the article of manufacture described herein can
comprise a compressive elastic modulus of at least 100 MPa, at
least 150 MPa, at least 200 MPa, at least 250 MPa, at least 300
MPa, at least 350 MPa, at least 400 MPa, at least 450 MPa, at least
500 MPa, or at least 525 MPa.
[0099] Density of the article of manufacture can range from about 1
g/cm.sup.3 to about 10 g/cm.sup.3. For example, the density can be
between about 1.05 g/cm.sup.3 to about 5 g/cm.sup.3, between about
1.1 g/cm.sup.3 to about 2.5 g/cm.sup.3, between about 1.2
g/cm.sup.3 to about 2.0 g/cm.sup.3, between about 1.25 g/cm.sup.3
to about 1.5 g/cm.sup.3. In some embodiments, density of the
article of manufacture is about 1.32 g/cm.sup.3.
[0100] The article of manufacture can be used for medical
applications, e.g. medical devices, or the article can be for
non-medical applications.
[0101] As used herein, the term medical device is intended to
encompass all types of medical devices, including those used in
connection with either external or internal treatment of a mammal.
Medical devices used in the external treatment of a mammal include,
but are not limited to, wound dressings, burn dressings or other
skin coverings, and surgical thread. Medical devices used in the
internal treatment of a mammal include, but are not limited to,
vascular grafts, stents, catheters, valves, artificial joints,
artificial organs, surgical thread, and the like.
[0102] Exemplary medical devices include, but are not limited to,
an orthopedic implant, a facial implant, a nasal implant (e.g., for
nasal reconstruction), a suture anchor, a dental implant, a Swanson
prosthetic, and any combinations thereof. In some embodiments, the
article of manufacture is a continuous, one-phase suture
anchor.
[0103] As used herein, the term "orthopedic implant" includes
within its scope any device intended to be implanted into the body
of a vertebrate animal, in particular a mammal such as a human, for
preservation and restoration of the function of the musculoskeletal
system, particularly joints and bones, including the alleviation of
pain in these structures. Exemplary orthopedic implants include,
but are not limited to, orthopedic screws, orthopedic plates,
orthopedic rods, orthopedic tulips, or any combinations
thereof.
[0104] In one embodiments, the article of manufacture is a tapping
screw, e.g., self-tapping screw.
[0105] In some embodiments, the article of manufacture is a suture
anchor. Suture anchor are composed of an anchor, eyelet, and
suture. The anchor is inserted to the bone which can be a screw
mechanism or interference fit and the eyelet is the hole or loop in
the anchor through which the suture passes.
[0106] As used herein, the term "dental implant" includes within
its scope any device intended to be implanted into the oral cavity
of a vertebrate animal, in particular a mammal such as a human, in
tooth restoration procedures. Dental implants can also be denoted
as dental prosthetic devices. Generally, a dental implant is
composed of one or several implant parts. For instance, a dental
implant usually comprises a dental fixture coupled to secondary
implant parts, such as an abutment and/or a dental restoration such
as a crown, bridge or denture. However, any device, such as a
dental fixture, intended for implantation can alone be referred to
as an implant even if other parts are to be connected thereto.
Dental implants are presently preferred embodiments.
[0107] Bone screws consist of a thread portion and head used for
insertion and stabilization of associated equipment such as bone
plates.
[0108] The Swanson Finger Joint Implant is a flexible
intramedullary-stemmed one-piece implant that helps restore
function to hands and wrists disabled by rheumatoid, degenerative
or traumatic arthritis. It is composed of a silicone elastomer and
its primary function is to help maintain proper joint space and
alignment with good lateral stability and minimal
flexion-extensional restriction. These implants bear minimal load
as the majority of the compressive loads are distributed to the
bones.
[0109] A nasal reconstruction is performed in order to create an
aesthetically inconspicuous nose while maintaining function.
Structural grafts are often required to provide rigidity to the
sidewall and resist lateral collapse and establish nasal contour
and projection. Current materials include alloplasts such as
silicone and porous high density polyethylene as well has
homografts such as alloderm or rib cartilage.
[0110] Otoplasty is the process of reconstructing partial or total
ear defects typically resulting from congenital hypoplasia, trauma,
cancer ablation, and prominent ears. The ears can be reconstructed
by using cartilage from the rib cage or an artificial ear can be
created. The rib cartilage is carved and wired together using fine
stainless steel wire to create a very detailed framework.
[0111] In addition to the above-discussed specific medical devices
and implants, the method disclosed herein can be used for facial
implants (dermal fillers, cheek implants, eye socket),
occuloplasty, lip enhancement, reproductive organ plastic surgeries
(penile implant, vaginaplasty, sex conversion), buttock
augmentation, and other "plastys."
[0112] Non-medical applications include manufacturing of dice,
thumbtacks, bullets, children's toys (e.g., building blocks, Legos,
Checkers, etc. . . . ), and biodegradable plastic alternatives.
[0113] In some embodiments, the article of manufacture described
herein is osteoconductive. Osteoconductivity is generally defined
as the ability of a material to facilitate the migration of
osteogenic cells to the surfaces of a scaffold through the fibrin
clot established immediately after implantation the material. The
porosity of a material affects the osteoconductivity of that
material.
[0114] In some embodiments, the article of manufacture described
herein is osteoinductive. Osteoinductivity is generally defined as
the ability to induce non-differentiated stem cells or
osteoprogenitor cells (osteoblasts), which is a component of
osseous (bone) tissue, to differentiate into osteoblasts. The
simplest test of osteoinductivity is the ability to induce the
formation of hone in tissue locations such as muscle, which do not
normally form bone (ectopic bone growth). It is generally
understood that article of manufacture described herein can be made
osteoinductive by adding growth factors such as rhBMP-2
(recombinant human bone morphogenic protein-2) to them. The
mineralization and the addition of growth factors can affect the
osteoinducivity of a material.
[0115] In some embodiments, the article of manufacture described
herein is osteogenic and shows new bone formation after
implantation in vivo. Osteogenesis is the process of laying down
new bone material using osteoblasts. Osteoblasts build bone by
producing osteoid to form an osteoid matrix, which is composed
mainly of Type I collagen. Osseous tissue comprises the osteoid
matrix and minerals (mostly with calcium phosphate) that form the
chemical arrangement termed calcium hydroxyapatite. Osteoblasts are
typically responsible for mineralization of the osteoid matrix to
form osseous tissue. Without wishing to be bound by a theory, the
osteoconductivity and osteoinductivity of the material has an
impact on osteogenesis. The material can show new bone formation
within 6 months of implantation in vivo. In some embodiments, the
material shows new bone formation within 8 weeks of implantation in
vivo.
[0116] In some embodiments, the article of manufacture described
herein can comprise one or more supplementary material. The
supplementary material is selected based upon its compatibility
with one or more components of the silk composition and its ability
to impart properties (biological, chemical, physical, or
mechanical) to the composite, which are desirable for a particular
therapeutic purpose or for post-sterilization stability. For
example, the supplementary material can be selected to improve
tensile strength and hardness, increase fracture toughness, and
provide imaging capability of the paste after implantation,
hydration, and hardening. The supplementary materials are desirably
biocompatible.
[0117] The supplementary material can be present in the silk
composition in varying amounts and in a variety of physical forms,
dependent upon the anticipated therapeutic use. For example, the
supplementary material can be in the form of solid structures, such
as sponges, meshes, films, fibers, gels, filaments or particles,
including microparticles and nanoparticles. The supplementary
material itself can be a composite. The supplementary material can
be a particulate or liquid additive or doping agent.
[0118] In many instances, it is desirable that the supplementary
material be bioresorbable. Bioresorbable material for use as
supplementary material include, without limitation,
polysaccharides, nucleic acids, carbohydrates, proteins,
polypeptides, poly(.alpha.-hydroxy-acids), poly (lactones),
poly(amino acids), poly(anhydrides), poly (orthoesters), poly
(anhydride-co-imides), poly (orthocarbonates), poly(.alpha.-hydroxy
alkanoates), poly (dioxanones), and poly(phosphoesters).
Preferably, the bioresorbable polymer is a naturally occurring
polymer, such as collagen, glycogen, chilin, starch, keratins,
silk, demineralized bone matrix, and hyaluronic acid; or a
synthetic polymer, such as poly(L-lactide) (PLLA),
poly(D,L-lactide) (PDLLA), polyglycolide (PGA),
poly(lactide-co-glycolide (PLGA), poly(L-lactide-co-D,L-lactide),
poly(D,L-lactide-co-trimethylene carbonate), polyhydroxybutyrate
(PHB), poly(.epsilon.-caprolactone), poly(.gamma.-valerolactone),
poly(.gamma.-butyrolactone), poly(caprolaclone), or copolymers
thereof. Such polymers are known to bioerode and are suitable for
use in the article of manufacture described herein for bone grafts
and the like. In addition, bioresorbable inorganic supplementary
materials, such as compositions including SiO2, Na2O, SaO, P205,
Al2O3 and/a CaF2, can be used, as well as salts, e.g., NaCl, and
sugars, e.g., mannitol, and combinations thereof.
[0119] Supplementary materials can also be selected from
nonresorbable or poorly resorbable materials. Suitable
non-resorbable or poorly resorbable materials include, without
limitation, dextrans, cellulose and derivatives thereof (e.g.,
methylcellulose, carboxy methylcellulose, hydroxypropyl
methylcellulose, and hydroxyethyl cellulose), polyethylene,
polymethylmethacrylate (PMMA), carbon fibers, poly(ethylene
glycol), poly(ethylene oxide), poly(vinyl alcohol),
poly(vinylpyrrolidone), poly (ethyloxazoline), poly(ethylene
oxide)-co-poly(propylene oxide) block copolymers, poly(ethylene
terephthalate)polyamide, and lubricants, such as polymer waxes,
lipids and fatty acids.
[0120] The article of manufacture described herein is also useful
for the preparation of delivery vehicles for biologically active
agents. In general, the only requirement is that the substance
remain active within material during fabrication or be capable of
being subsequently activated or re-activated, or that the
biologically active agent be added to the material after at the
time of implantation of into a host or following fabrication of the
vehicle.
[0121] Biologically active agents that can be incorporated into the
article of manufacture described herein include, without
limitation, organic molecules, inorganic materials, proteins,
peptides, nucleic acids (e.g., genes, gene fragments, gene
regulatory sequences, and antisense molecules), nucleoproteins,
polysaccharides, glycoproteins, and lipoproteins. Classes of
biologically active compounds that can be incorporated into the
article of manufacture described herein include, without
limitation, anticancer agents, antibiotics, analgesics,
anti-inflammatory agents, immunosuppressants, enzyme inhibitors,
antihistamines, anti-convulsants, hormones, muscle relaxants,
antispasmodics, ophthalmic agents, prostaglandins,
anti-depressants, anti-psychotic substances, trophic factors,
osteoinductive proteins, growth factors, and vaccines.
[0122] In some cases the article of manufacture, e.g. an orthopedic
implant needs to be tuned to degrade in a shorter time frame to
allow for dynamic transfer of the load back to the healing bone.
This could be accomplished numerous different ways such as
autoclaving multiple times to degrade the silk fibroin or
incorporating enzymes into the constructs that activate upon
hydration [28, 33, 34]. Coating the silk devices with bioactive
compounds such as BMP-2 nd other pharmaceuticals could provide
benefits in bone fixation systems. The article of manufacture can
also incorporate bioactive compounds such as BMP-2 or antibiotics
to promote bone ingrowth [29-31]. Without wishing to be bound by a
theory, it is believed that such factors can be used to modulate
healing and promote remodeling of bone.
[0123] The combination of the silk with bioactive compounds such as
enzymes, bone morphogenctic protein 2 (BMP-2), and pharmaceuticals
is believed to provide multifunctional benefits not currently
utilized in bone fixation systems.
[0124] Generally, any therapeutic agent can be encapsulated in the
drug delivery vehicle or composition comprising the article of
manufacture described herein. As used herein, the term "therapeutic
agent" means a molecule, group of molecules, complex or substance
administered to an organism for diagnostic, therapeutic,
preventative medical, or veterinary purposes. As used herein, the
term "therapeutic agent" includes a "drug" or a "vaccine." This
term include externally and internally administered topical,
localized and systemic human and animal pharmaceuticals,
treatments, remedies, nutraceuticals, cosmeceuticals, biologicals,
devices, diagnostics and contraceptives, including preparations
useful in clinical and veterinary screening, prevention,
prophylaxis, healing, wellness, detection, imaging, diagnosis,
therapy, surgery, monitoring, cosmetics, prosthetics, forensics and
the like. This term can also be used in reference to agriceutical,
workplace, military, industrial and environmental therapeutics or
remedies comprising selected molecules or selected nucleic acid
sequences capable of recognizing cellular receptors, membrane
receptors, hormone receptors, therapeutic receptors, microbes,
viruses or selected targets comprising or capable of contacting
plants, animals and/or humans. This term can also specifically
include nucleic acids and compounds comprising nucleic acids that
produce a therapeutic effect, for example deoxyribonucleic acid
(DNA), ribonucleic acid (RNA), or mixtures or combinations thereof,
including, for example, DNA nanoplexes, siRNA, shRNA, aptamers,
ribozymes, decoy nucleic acids, antisense nucleic acids, RNA
activators, and the like.
[0125] The term "therapeutic agent" also includes an agent that is
capable of providing a local or systemic biological, physiological,
or therapeutic effect in the biological system to which it is
applied. For example, the therapeutic agent can act to control
infection or inflammation, enhance cell growth and tissue
regeneration, control tumor growth, act as an analgesic, promote
anti-cell attachment, and enhance bone growth, among other
functions. Other suitable therapeutic agents can include anti-viral
agents, hormones, antibodies, or therapeutic proteins. Other
therapeutic agents include prodrugs, which are agents that are not
biologically active when administered but, upon administration to a
subject are converted to biologically active agents through
metabolism or some other mechanism. Additionally, a silk-based drug
delivery composition can contain combinations of two or more
therapeutic agents.
[0126] A therapeutic agent can include a wide variety of different
compounds, including chemical compounds and mixtures of chemical
compounds, e.g., small organic or inorganic molecules; saccharines;
oligosaccharides; polysaccharides; biological macromolecules, e.g.,
peptides, proteins, and peptide analogs and derivatives;
peptidomimetics; antibodies and antigen binding fragments thereof;
nucleic acids; nucleic acid analogs and derivatives; an extract
made from biological materials such as bacteria, plants, fungi, or
animal cells; animal tissues; naturally occurring or synthetic
compositions; and any combinations thereof, in some embodiments,
the therapeutic agent is a small molecule.
[0127] As used herein, the term "small molecule" can refer to
compounds that are "natural product-like," however, the term "small
molecule" is not limited to "natural product-like" compounds.
Rather, a small molecule is typically characterized in that it
contains several carbon-carbon bonds, and has a molecular weight of
less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still
more preferably less than 2 kDa, and most preferably less than 1
kDa. In some cases it is preferred that a small molecule have a
molecular weight equal to or less than 700 Daltons.
[0128] Exemplary therapeutic agents include, but are not limited
to, those found in Harrison's Principles of Internal Medicine,
13.sup.th Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y.,
N.Y., Physicians' Desk Reference, 50.sup.th Edition, 1997, Oradell
N.J., Medical Economics Co.; Pharmacological Basis of Therapeutics,
8.sup.th Edition, Goodman and Gilman, 1990; United States
Pharmacopeia, The National Formulary, USP XII NF XVII, 1990, the
complete contents of all of which are incorporated herein by
reference.
[0129] Therapeutic agents include the herein disclosed categories
and specific examples. It is not intended that the category be
limited by the specific examples. Those of ordinary skill in the
art will recognize also numerous other compounds that fall within
the categories and that are useful according to the present
disclosure. Examples include a radiosensitizer, a steroid,
xanthine, a beta-2-agonist bronchodilator, an anti-inflammatory
agent, an analgesic agent, a calcium antagonist, an
angiotensin-converting enzyme inhibitors, a beta-blocker, a
centrally active alpha-agonist, an alpha-1-antagonist, an
anticholinergic/antispasmodic agent, a vasopressin analogue, an
antiarrhythmic agent, an antiparkinsonian agent, an
antiangina/antihypertensive agent, an anticoagulant agent, an
antiplatelet agent, a sedative, an ansiolytic agent, a peptidic
agent, a biopolymeric agent, an antineoplastic agent, a laxative,
an antidiarrheal agent, an antimicrobial agent, an antifungal
agent, a vaccine, a protein, or a nucleic acid. In a further
aspect, the pharmaceutically active agent can be coumarin, albumin,
steroids such as betamethasone, dexamethasone, methylprednisolone,
prednisolone, prednisone, triamcinolone, budesonide,
hydrocortisone, and pharmaceutically acceptable hydrocortisone
derivatives; xanthines such as theophylline and doxophylline;
beta-2-agonist bronchodilators such as salbutamol, fenterol,
clenbuterol, bambuterol, salmeterol, fenoterol; antiinflammatory
agents, including antiasthmatic anti-inflammatory agents,
antiarthritis antiinflammatory agents, and non-steroidal
antiinflammatory agents, examples of which include but are not
limited to sulfides, mesalamine, budesonide, salazopyrin,
diclofenac, pharmaceutically acceptable diclofenac salts,
nimesulide, naproxene, acetaminophen, ibuprofen, ketoprofen and
piroxicam; analgesic agents such as salicylates; calcium channel
blockers such as nifedipine, amlodipine, and nicardipine;
angiotensin-converting enzyme inhibitors such as captopril,
benazepril hydrochloride, fosinopril sodium, trandolapril,
ramipril, lisinopril, enalapril, quinapril hydrochloride, and
moexipril hydrochloride; beta-blockers (i.e., beta adrenergic
blocking agents) such as sotalol hydrochloride, timolol maleate,
esmolol hydrochloride, carteolol, propanolol hydrochloride,
betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate,
metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol,
and bisoprolol fumarate; centrally active alpha-2-agonists such as
clonidine; alpha-1-antagonists such as doxazosin and prazosin;
anticholinergic/antispasmodic agents such as dicyclomine
hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium
bromide, flavoxate, and oxybutynin; vasopressin analogues such as
vasopressin and desmopressin; antiarrhythmic agents such as
quinidine, lidocaine, tocainide hydrochloride, mexiletine
hydrochloride, digoxin, verapamil hydrochloride, propafenone
hydrochloride, flecainide acetate, procainamide hydrochloride,
moricizine hydrochloride, and disopyramide phosphate;
antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa,
selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine,
and bromocryptine; antiangina agents and antihypertensive agents
such as isosorbide mononitrate, isosorbide dinitrate, propranolol,
atenolol and verapamil; anticoagulant and antiplatelet agents such
as Coumadin, warfarin, acetylsalicylic acid, and ticlopidine;
sedatives such as benzodiazapines and barbiturates; ansiolytic
agents such as lorazepam, bromazepam, and diazepam; peptidic and
biopolymeric agents such as calcitonin, leuprolide and other LHRH
agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin,
interferon, desmopressin, somatotropin, thymopentin, pidotimod,
erythropoietin, interleukins, melatonin,
granulocyte/macrophage-CSF, and heparin; antineoplastic agents such
as etoposide, etoposide phosphate, cyclophosphamide, methotrexate,
5-fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea,
leucovorin calcium, tamoxifen, flutamide, asparaginase,
altretamine, mitotane, and procarbazine hydrochloride; laxatives
such as senna concentrate, casanthranol, bisacodyl, and sodium
picosulphate; antidiarrheal agents such as difenoxine
hydrochloride, loperamide hydrochloride, furazolidone,
diphenoxylate hydrochloride, and microorganisms; vaccines such as
bacterial and viral vaccines; antimicrobial agents such as
penicillins, cephalosporins, and macrolides, antifungal agents such
as imidazolic and triazolic derivatives; and nucleic acids such as
DNA sequences encoding for biological proteins, and antisense
oligonucleotides.
[0130] Anti-cancer agents include alkylating agents, platinum
agents, antimetabolites, topoisomerase inhibitors, antitumor
antibiotics, antimitotic agents, aromatase inhibitors, thymidylate
synthase inhibitors, DNA antagonists, farnesyltransferase
inhibitors, pump inhibitors, histone acetyltransferase inhibitors,
metalloproteinase inhibitors, ribonucleoside reductase inhibitors,
TNP alpha agonists/antagonists, endothelinA receptor antagonists,
retinoic acid receptor agonists, immuno-modulators, hormonal and
antihormonal agents, photodynamic agents, and tyrosine kinase
inhibitors.
[0131] Antibiotics include aminoglycosides (e.g., gentamicin,
tobramycin, netilmicin, streptomycin, amikacin, neomycin),
bacitracin, corbapenems (e.g., imipenem/cislastatin),
cephalosporins, colistin, methenamine, monobactams (e.g.,
aztreonam), penicillins (e.g., penicillin G, penicillin V,
methicillin, natcillin, oxacillin, cloxacillin, dicloxacillon,
ampicillin, amoxicillin, carbenicllin, ticarcillin, piperacillin,
mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin;
and bacteriostatic agents such as chloramphenicol, clindanyan,
macrolides (e.g., erythromycin, azithromycin, clarithromycin),
lincomyan, nitrofurantoin, sulfonamides, tetracyclines (e.g.,
tetracycline, doxycycline, minocycline, demeclocyline), and
trimethoprim. Also included are metronidazole, fluoroquinolones,
and ritampin.
[0132] Enzyme inhibitors are substances which inhibit an enzymatic
reaction. Examples of enzyme inhibitors include edrophonium
chloride. N-methylphysostigmine, neostigmine bromide, physostigmine
sulfate, tacrine, tacrine, 1-hydroxy maleate, iodotubercidin,
p-bromotetramiisole, 10-(alpha-diethylaminopropionyl)-phenothiaxine
hydrochloride, calmidazolium chloride,
hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase
inhibitor I, diacylglycerol kinase inhibitor II,
3-phenylpropargylmaine, N.degree.-monomethyl-Larginine acetate,
carbidopa, 3-hydroxybenzylhydrazine, hydralazine, clorgyline,
deprenyl, hydroxylamine, iproniazid phosphate,
6-MeO-tetrahydro-9H-pyrido-indol, nialamide, parglyline,
quinacrine, semicarbazide, tranylcypromise,
N,N-diethyalminoethyl-2,2-diphenylvalerate hydrochloride,
3-isobutyl-1-methylxanthne, papaverine, indomethacind,
2-cyclooctyl-2-hydroxyethylamine hydrochloride,
2,3-dichloro-a-methylbenzylamine (DCMB),
8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride;
p-amino glutethimide, p-aminoglutethimide tartrate, 3-iodotyrosine,
alpha-methyltyrosine, acetazolamide, dichlorphenamide,
6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.
[0133] Antihistamines include pyrilamine, chlorpheniramine, and
tetrahydrazoline, among others.
[0134] Anti-inflammatory agents include corticosteroids,
nonsteroidal anti-inflammatory drugs (e.g., aspirin,
phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen,
piroxicam, and fenamates), acetaminophen, phenacetin, gold salts,
chloroquine, D-Penicillamine, methotrexate colchicine, allopurinol,
probenecid, and sulfinpyrazone.
[0135] Muscle relaxants include mephenesin, methocarbomal,
cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride,
levodopa/carbidopa, and biperiden.
[0136] Anti-spasmodics include atropine, scopolamine, oxyphenonium,
and papaverine.
[0137] Analgesics include aspirin, phenylbutazone, idomethacin,
sulindac, tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen,
phenacetin, morphine sulfate, codeine sulfate, meperidine,
nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate,
hydrocodone bitartrate, loperamide, morphine sulfate, noscapine,
norcodeine, normorphine, thebaine, norbinaltorphimine,
buprenorphine, chlomaltrexamine, funaltrexamione, nalbuphine,
nalorphine, naloxone, naloxonazine, naltrexone, and naltrindole),
procaine, lidocain, tetracaine and dibucaine.
[0138] Ophthalmic agents include sodium fluorescein, rose bengal,
methacholine, adrenaline, cocaine, atropine, alpha-chymotrypsin,
hyaluronidase, betaxalol, pilocarpine, timolol, timolol salts, and
combinations thereof
[0139] Prostaglandins are art recongized and are a class of
naturally occurring chemically related, long-chain hydroxy fatty
acids that have a variety of biological effects.
[0140] Anti-depressants are substances capable of preventing or
relieving depression. Examples of anti-depressants include
imipramine, amitriplyline, nortriptyline, protriptyline,
desipramine, amoxapine, doxepin, maprotiline, tranyleypromine,
phenelzine, and isocarboxazide.
[0141] Trophic factors are factors whose continued presence
improves the viability or longevity of a cell. Trophic factors
include, Without limitation, platelet-derived growth factor (PDGP),
neutrophil-activating protein, monocyte chemoattractant protein,
macrophage-inflammatory protein, platelet factor, platelet basic
protein, and melanoma growth stimulating activity; epidermal growth
factor, transforming growth factor (alpha), fibroblast growth
factor, platelet-derived endothelial cell growth factor,
insulin-like growth factor, glial derived growth neurotrophic
factor, ciliary neurotrophic factor, nerve growth factor, bone
growth/cartilage-inducing factor (alpha and beta), bone
morphogenetic proteins, interleukins (e.g., interleukine inhibitors
or interleukine receptors, including interleukin 1 through
interleukin 10), interferons (e.g., interferon alpha, beta and
gamma), hematopoietic factors, including erythropoietin,
granulocyte colony stimulating factor, macrophage colony
stimulating factor and granulocyte-macrophage colony stimulating
factor; tumor necrosis factors, and transforming growth factors
(beta), including beta-1, beta-2, beta-3, and activin.
[0142] Hormones include estrogens (e.g, estradiol, estrone,
estriol, diethylstibestrol, quinestrol, chlorotrianisene, ethinyl
estradiol, mestranol), anti-estrogens (e.g., clomiphene,
tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone,
hydroxyprogesterone, norgestrel), antiprogestin (mifepristone),
androgens (e.g. testosterone cypionate, fluoxymesterone, danazol,
testolactone), anti-androgens (e.g., cyproterone acetate,
flutamide), thyroid hormones (e.g, triodothyronne, thyroxine,
propylthiouracil, methimazole, and iodixode), and pituary pituitary
hormones (e.g., corticotropin, sumutotropin, oxytocin, and and
vasopressin). Hormones are commonly employed in hormone replacement
therapy and or for purposes of birth control. Steroid hormones,
such as prednisone, are also used as immunosuppressants and
anti-inflammatories.
[0143] The biologically active can be an osteogenic protein.
Accordingly, in some embodiments, the biologically active agent is
desirably selected from the family of proteins known as the
transforming growth factors beta (TGF-[3) superfamily of proteins,
which includes the activins, inhibins and bone morphogenetic
proteins (BMPs). Most preferably, the active agent includes at
least one protein selected from the subclass of proteins known
generally as BMPs, which have been disclosed to have osteogenic
activity, and other growth and differentiation type activities.
These BMPs include BMP proteins BMP-2, BMP-3, BMP-4, BMP5, BMP-6
and BMP-7, disclosed for instance in U.S. Pat. Nos. 5,108,922;
5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8,
disclosed in PCT publication WO91/18098; and BMP-9, disclosed in
PCT publication WO93/00432, BMP-10, disclosed in PCT application
WO94/26893; BMP-11, disclosed in PCT application WO94/26892, or
BMP-12 or BMP-13, disclosed in PCT application WO 95/16035; BMP-14;
BMP-15, disclosed in U.S. Pat. No. 5,635,372; or BMP-16, disclosed
in U.S. Pat. No. 5,965,403. Other TGF-.beta. proteins, which can be
used include Vgr-2, Jones et al., Mol. Endocrinol. 611961 (1992),
and any of the growth and differentiation factors (GDFs), including
those described in PCT applications WO94/15965; WO94/15949;
WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539;
WO96/01845; WO96/02559 and others. Also useful in the invention can
be BIP, disclosed in WO94/01557; HP00269, disclosed in JP
Publication number: 7-250688; and BMP-14 (also known as MP52,
CDMP1, and GDF5), disclosed in PCT application WO93/16099. The
disclosures of all of the above applications are incorporated
herein by reference. Subsets of BMPs which can be used include
BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,
BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, and
BMP18. Other osteogenic agents known in the art can also be used,
such as teriparatide (FORTEO.TM.), CHRYSALIN.RTM., prostaglandin
E2, or LIM protein, among others.
[0144] The biologically active agent can be recombinantly produced,
or purified from a protein composition. The active agent, if a
TGF-.beta. such as a BMP, or other dimeric protein, can be
homodimeric, or can be heterodimeric with other BMPs (e.g., a
heterodimer composed of one monomer each of BMP-2 and BMP-6) or
With other members of the TGF-.beta. superfamily, such as activins,
inhibins, and TGF-.beta.1 (e.g., a heterodimer composed of one
monomer each of a BMP and a related member of the TGF-.beta.
superfamily). Examples of such heterodimeric proteins are described
for example in Published PCT Patent Application WO 93/09229, the
content of which is incorporated herein by reference.
[0145] The active agent can further include additional agents such
as the Hedgehog, Frazzled, Chordin, Noggin, Cerberus and
Follislatin proteins. These families of proteins are generally
described in Sasai et al., Cell 791779-790 (1994) (Chordin); PCT
Patent Publication WO94/05800 (Noggin); and Fukui et al., Devel.
Biol. 159: 1 31 (1993) (Follistatin). Hedgehog proteins are
described in WO96/16668; WO96/17924; and WO95/18856. The Frazzled
family of proteins is a recently discovered family of proteins With
high homology to the extracellular binding domain of the receptor
protein family known as Frizzled. The Frizzled family of genes and
proteins is described in Wang et al., J. Biol. Chem. 271 :44684476
(1996). The active agent can also include other soluble receptors,
such as the truncated soluble receptors disclosed in PCT patent
publication WO95/07982. From the teaching of WO95/07982, one
skilled in the art will recognize that truncated soluble receptors
can be prepared for numerous other receptor proteins. The above
publications are hereby incorporated by reference herein.
[0146] The amount of osteogenic protein effective to stimulate
increased osteogenic activity of present or infiltrating progenitor
or other cells will depend upon the size and nature of the defect
being treated, as well as the carrier being employed. Generally,
the amount of protein to be delivered is in a range of from about
0.1 to about 100 mg; preferably about 1 to about 100 mg; most
preferably about 10 to about 80 mg.
[0147] Biologically active agents can be introduced into the
article of manufacture described herein during or after its
formation. Agents can conveniently be mixed into the starting
solution prior to fabrication of the article of manufacture
described herein. Alternatively, the article of manufacture
described herein can be fabricated, shaped into a desired shape,
and then exposed to the biologically active agent in solution. This
particular approach is particularly well suited for proteins, which
are known to have an affinity for apatitic materials. A buffer
solution containing the biologically active agent can be employed,
instead of water, as the aqueous solution in which the article of
manufacture described herein is, or example, irrigated prior to
use. Buffers can be used in any pH range, but most often will be
used in the range of 5.0 to 8.0 in preferred embodiments the pH
will be compatible with prolonged stability and efficacy of the
desired biologically active agent and, in most preferred
embodiments, will be in the range of 5.5 to 7.4. Suitable buffers
include, but are not limited to, carbonates, phosphates (e.g.,
phosphate buffered saline), and organic buffers such as Tris,
HEPES, and MOPS. Most often, the buffer will be selected for its
biocompatibilily with the host tissues and its compatibility with
the biologically active agent. For most applications of nucleic
acids, peptides or antibiotics a simple phosphate buffered saline
can suffice.
[0148] Standard protocols and regimens for delivery of the above
listed agents are known in the art. Typically, these protocols are
based on oral or intravenous delivery. Biologically active agents
are introduced into the vehicle in amounts that allow delivery of
an appropriate dosage of the agent to the implant site. In most
cases, dosages are determined using guidelines known to
practitioners and applicable to the particular agent in question.
The exemplary amount of biologically active agent to be included in
the the article of manufacture described herein is likely to depend
on such variables as the type and extent of the condition, the
overall health status of the particular patient, the formulation of
the active agent, and the bioresorbability of the delivery vehicle
used. Standard clinical trials may be used to optimize the dose and
dosing frequency for any particular biologically active agent
[0149] Generally, any amount of the supplementary material, such as
a biocompatible polymer, biologically active agent, and therapeutic
agent can be loaded into the article of manufacture described
herein, for example, from about 0.1 ng to about 1000 mg of the
therapeutic agent can be loaded in the article of manufacture
described herein. In some embodiment, amount of the supplementary
in the silk solution, silk composition or the article of
manufacture is selected from the range about from 0.001% (w/w) up
to 95% (w/w), preferably, from about 5% (w/w) to about 75% (w/w),
and most preferably from about 10% (w/w) to about 60% (w/w) of the
total composition. In some embodiments, amount of amount of the
supplementary in the article of manufacture described herein is
from about 0.01% to about 95% (w/v), from about 0.1% to about 90%
(w/w), from about 1% to about 85% (w/w), from about 5% to about 75%
(w/w), from about 10% to about 65% (w/w), or from about 10% to
about 50% (w/w), of the total composition.
[0150] In some embodiments, amount of the supplementary in the
article of manufacture described herein is from about 1% to about
99% (w/w), from about 0.05% to about 99% (w/w), from about 0.1% to
about 90% (w/w), from about 0.5% to about 85% (w/w), from about 5%
to about 80% (w/w), from about 10% to about 60% (w/w) of the total
composition. In some embodiments, amount of the supplementary in
the silk solution, the silk composition or the article of
manufacture is from about 0.1% to about 99% (w/w), from about 1% to
about 90% (w/w), from about 2% to about 80% (w/w), from about 5% to
about 75% (w/w), from about 5% to about 50% (w/w), from about
0.055% to about 0.1% (w/w) of the total composition.
[0151] After preparation, the article of manufacture described
herein can be sterilized using conventional sterilization process
such as radiation-based sterilization (i.e. gamma-ray), chemical
based sterilization (ethylene oxide), autoclaving, or other
appropriate procedures. In some embodiments, sterilization process
can be with ethylene oxide at a temperature between from about
52.degree. C. to about 55.degree. C. for a time of 8 or less hours.
The the article of manufacture described herein can also be
processed aseptically. Sterile article of manufacture described
herein can be packaged in an appropriate sterilize moisture
resistant package for shipment.
[0152] Without wishing to be bound by a theory, the article of
manufacture described herein provides a number of advantages. The
material can withstand physiological loading forces; can initiate
new bone formation and stimulate healing through direct bone-silk
interface; can promote osteogenesis by local delivery of bone
morphogenic growth factors; and can achieve complete graft
resorption and non-union closure. The methods and articles of
manufacture prepared using the same provide a number advantages
over the prior art.
[0153] Embodiments of the invention can be described by any of the
following paragraphs: [0154] 1. A method comprising: [0155] (i)
providing a composition comprising silk particles; and [0156] (ii)
compacting the composition by application of pressure into a solid
state. [0157] 2. The method of paragraph 1, wherein the silk
particles are nanoparticles or microparticles. [0158] 3. The method
of paragraph 1 or 2, wherein the composition comprises silk in an
amount of about 25% (w/w) or higher. [0159] 4. The method of any of
paragraphs 1-3, wherein said pressure is at least 0.05 bar. [0160]
5. The method of any of paragraphs 1-4, wherein said compacting is
at an elevated temperature. [0161] 6. The method of paragraph 5,
wherein the elevated temperature is at least 30.degree. C. [0162]
7. The method of any of paragraphs 1-6, wherein the composition
further comprises a binder. [0163] 8. The method of paragraph 7,
wherein the binder is a liquid. [0164] 9. The method of paragraph 7
or 8, wherein the binder is water. [0165] 10. The method of any of
paragraphs 7-9, wherein the composition comprises from about 0.1%
(w/vv) to about 50% (w/w) of the binder. [0166] 11. The method of
any of paragraphs 1-10, wherein the silk particles comprise
degummed silk. [0167] 12. The method of any of paragraphs 1-11,
wherein the silk particles comprise non-degummed silk. [0168] 13.
The method of any of paragraphs 1-12, wherein the composition
comprises a mixture of silk particles comprising degummed silk and
silk particles comprising non-degummed silk. [0169] 14. The method
of paragraph 13, wherein ratio of dcgumnicd silk to non-degummed
silk is from about 50:1 to about 1:50 (w/w). [0170] 15. The method
of paragraph 13 or 14, wherein the ratio of degummed silk to
non-degummed silk is from about 1:1 to about 1:20. [0171] 16. The
method of any of paragraphs 1-15, wherein the composition further
comprises an additive. [0172] 17. The method of paragraph 16,
wherein the additive is selected from the group consisting of small
organic or inorganic molecules; saccharines; oligosaccharides;
polysaccharides; biological macromolecules, e.g., peptides,
proteins, and peptide analogs and derivatives; peptidomimetics;
antibodies and antigen binding fragments thereof; nucleic acids;
nucleic acid analogs and derivatives; glycogens or other sugars;
immunogens; antigens; an extract made from biological materials
such as bacteria, plants, fungi, or animal cells; animal tissues;
naturally occurring or synthetic compositions; and any combinations
thereof. [0173] 18. The method of paragraph 16 or 17, wherein the
additive is in a form selected from the group consisting of a
particle, a fiber, a film, a gel, a hydrogel, a mesh, a mat, a
non-woven mat, a powder, a fabric, a scaffold, a tube, a slab or
block, a fiber, a foam or a sponge, a needle, a lyophilized
article, and any combinations thereof. [0174] 19. The method of
paragraph 18, wherein the additive particle is a nanoparticle or a
microparticle. [0175] 20. The method of any of paragraphs 16-19,
wherein the additive is a silk-based material. [0176] 21. The
method of paragraph 20, wherein the silk-based material is selected
from the group consisting of silk particles, silk fibers,
micro-sized silk fibers, unprocessed silk fibers, and any
combinations thereof. [0177] 22. The method of any of paragraphs
16-21, wherein the composition comprises from about 0.1% to (w/w)
to about 99% (w/w) of the additive. [0178] 23. The method of any of
paragraphs 16-22, wherein ratio of silk to the additive is from
about 10:1 to about 1:10 (w/w). [0179] 24. The method of any of
paragraphs 16-23, wherein the additive is an active agent. [0180]
25. The method of paragraph 24, wherein the active agent is a
therapeutic agent. [0181] 26. The method of any of paragraphs 1-25,
wherein the composition is in a mold. [0182] 27. The method of any
of paragraphs 1-26, further comprising processing the composition
to a desired shape after said compacting step. [0183] 28. The
method paragraph 27, wherein said processing is machining, turning
(lathe), rolling, thread rolling, drilling, milling, sanding,
punching, die cutting, blanking, broaching, and any combinations
thereof. [0184] 29. The method of any of paragraphs 1-28, further
comprising inducing a conformational change in silk fibroin to a
beta-sheet conformation. [0185] 30. The method of paragraph 29,
wherein said inducing a conformational change comprises solvent
immersion, water annealing, water vapor annealing, sonication, pH
reduction, exposure to an electric field, controlled slow drying,
freeze-drying, compressing, heating, application of shear stress,
and any combinations thereof. [0186] 31. An article of manufacture
comprising silk obtained by the method of any of paragraphs 1-30.
[0187] 32. The article of manufacture of paragraph 31, wherein the
article of manufacture is a medical device. [0188] 33. The article
of manufacture of paragraph 32, wherein the medical device is
selected from the group consisting of an orthopedic implant, a
facial implant, a nasal implant, a suture anchor, a dental implant,
a Swanson prosthetic, and any combinations thereof. [0189] 34. The
article of manufacture of paragraph 33, wherein said orthopedic
implant is selected from the group consisting of an orthopedic
screw, an orthopedic plate, an orthopedic rod, an orthopedic tulip,
and any combinations thereof. [0190] 35. The article of manufacture
of any of paragraphs 31-34, wherein the article of manufacture is a
tapping screw. [0191] 36. The article of manufacture of any of
paragraphs 31-35, wherein the article of manufacture is
osteocondcutive, osteoinductive, osteogenic, or any combinations
thereof. [0192] 37. The article of manufacture of any of paragraphs
31-36, wherein the article of manufacture is bioresorbable. [0193]
38. An article of manufacture comprising silk, wherein the article
has a compressive strength of at least 5 MPa; a compressive elastic
modulus (Young's modulus) of at least 100 MPa; a shear strength of
at least 104 MPa; or a density of at least 1.1 g/cm.sup.3. [0194]
39. The article of manufacture of paragraph 38, wherein the article
of manufacture is obtained by a method of any of paragraphs 1-30.
[0195] 40. A method for increasing compressive strength, elastic
modulus, flexural stiffness, or shear stiffness of a silk-based
material, the method comprising: [0196] (i) providing a composition
comprising silk particles; and [0197] (ii) compacting the
composition by application of pressure into a solid state.
SOME SELECTED DEFINITIONS
[0198] For convenience, certain icons employed herein, in the
specification, examples and appended claims are collected herein.
Unless stated otherwise, or implicit from context, the following
terms and phrases include the meanings provided below. Unless
explicitly stated otherwise, or apparent from context, the terms
and phrases below do not exclude the meaning that the term or
phrase has acquired in the an to which it pertains. The definitions
are provided to aid in describing particular embodiments, and are
not intended to limit the claimed invention, because the scope of
the invention is limited only by the claims. Further, unless
otherwise required by context, singular terms shall include
pluralities and plural terms shall include the singular.
[0199] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as those commonly understood to
one of ordinary skill in the art to which this invention pertains.
Although any known methods, devices, and materials may be used in
the practice or testing of the invention, the methods, devices, and
materials in this regard are described herein.
[0200] The term "herein" is meant to include all of the disclosure
and is not intended to be limited to a subsection of the
disclosure.
[0201] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0202] 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.
[0203] 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 .+-.5% of the value being
referred to. For example, about 100 means from 95 to 105.
[0204] 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 term "comprises" means "includes." 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."
[0205] The terms "decrease", "reduced", "reduction", "decrease" or
"inhibit" are all used herein generally to mean a decrease by a
statistically significant amount. However, for avoidance of doubt,
"reduced", "reduction" or "decrease" or "inhibit" means a decrease
by at least 10% as compared to a reference level, for example a
decrease by at least about 20%, or at least about 30%, or at least
about 40%, or at least about 50%, or at least about 60%, or at
least about 70%, or at least about 80%, or at least about 90% or up
to and including a 100% decrease (e.g. absent level as compared to
a reference sample), or any decrease between 10-100% as compared to
a reference level.
[0206] The terms "increased", "increase" or "enhance" or "activate"
are all used herein to generally mean an increase by a statically
significant amount; for the avoidance of any doubt, the terms
"increased", "increase" or "enhance" or "activate" means an
increase of at least 10% as compared to a reference level, for
example an increase of at least about 20%, or at least about 30%,
or at least about 40%, or at least about 50%, or at least about
60%, or at least about 70%, or at least about 80%, or at least
about 90% or up to and including a 100% increase or any increase
between 10-100% as compared to a reference level, or at least about
a 2-fold, or at least about a 3-fold, or at least about a 4-fold,
or at least about a 5-fold or at least about a 10-fold increase, or
any increase between 2-fold and 10-fold or greater as compared to a
reference level.
[0207] The term "statistically significant" or "significantly"
refers to statistical significance and generally means at least two
standard deviation (2SD) away from 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.
[0208] The term "blank" as used herein means an unfinished part of
simple geometry that can later be modified by various machining
methods to create the desired shape of the article of
manufacture.
[0209] The term "drying" means removal of at least a portion of any
liquid carrier.
[0210] The term "bone repair" refers to any procedure for repairing
bone, including those which use a material as a substitute for bone
grafts.
[0211] The term "bone augmentation" refers to the use of any
procedure for adding or building bone.
[0212] The term "bone replacement" refers to the use of any
procedure for replacing existing bone.
[0213] As used herein, the term "microparticle" refers to a
particle having a particle size of about 0.01 .mu.m to about 1000
.mu.m.
[0214] As used herein, the term "nanoparticle" refers to particle
having a particle size of about 0.1 nm to about 1000 nm.
[0215] It will be understood by one of ordinary skill in the art
that particles usually exhibit a distribution of particle sizes
around the indicated "size." Unless otherwise stated, the term
"particle size" as used herein refers to the mode of a size
distribution of 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 theology, and light or electron microscopy).
[0216] In some embodiments, the particles can be substantially
spherical. What is meant by "substantially spherical" is that the
ratio of the lengths of the longest to the shortest perpendicular
axes of the particle cross section is less than or equal to about
1.5. Substantially spherical does not require a line of symmetry.
Further, the particles can have surface texturing, such as lines or
indentations or protuberances that are small in scale when compared
to the overall size of the particle and still be substantially
spherical. In some embodiments, the ratio of lengths between the
longest and shortest axes of the particle is less than of equal to
about 1.5, less than or equal to about 1.45, less than or equal to
about 1.4, less than or equal to about 1.35, less than or equal to
about 1.30, less than or equal to about 1.25, less than or equal to
about 1.20, less than or equal to about 1.15 less than or equal to
about 1.1. Without wishing to be bound by a theory, surface contact
is minimized in particles that are substantially spherical, which
minimizes the undesirable agglomeration of the particles upon
storage. Many crystals or flakes have flat surfaces that can allow
large surface contact areas where agglomeration can occur by ionic
or non-ionic interactions. A sphere permits contact over a much
smaller area.
[0217] In some embodiments, the particles have substantially the
same particle size. Particles having a broad size distribution
where there are both relatively big and small particles allow for
the smaller particles to fill in the gaps between the larger
particles, thereby creating new contact surfaces. A broad size
distribution can result in larger spheres by creating many contact
opportunities for binding agglomeration. The particles described
herein are within a narrow size distribution, thereby minimizing
opportunities for contact agglomeration. What is meant by a "narrow
size distribution" is a particle size distribution that has a ratio
of the volume diameter of the 90th percentile of the small
spherical particles to the volume diameter of the 10th percentile
less than or equal to 5. In some embodiments, the volume diameter
of the 90th percentile of the small spherical particles to the
volume diameter of the 10th percentile is less than or equal 4.5,
less than or equal to 4, less than or equal to 3.5, less than or
equal to 3, less than or equal to 2.5, less than or equal to 2,
less than or equal to 1.5, less than or equal to 1.45, less than or
equal to 1.40, less than or equal to 1.35, less than or equal to
1.3, less than or equal to 1.25, less than or equal to 1.20, less
than or equal to 1.15, or less than or equal to 1.1.
[0218] Geometric Standard Deviation (GSD) can also be used to
indicate the narrow size distribution. GSD calculations involved
determining the effective cutoff diameter (ECD) at the cumulative
less than percentages of 15.9% and 84.1%. GSD is equal to the
square root of the ratio of the ECD less than 84.17% to ECD less
than 15.9%. The GSD has a narrow size distribution when GSD<2.5.
In some embodiments, GSD is less than 2, less than 1.75, or less
than 1.5. In one embodiment, GSD is less than 1.8.
[0219] The disclosure is further illustrated by the following
examples, which should not be construed as limiting. The examples
are illustrative only, and are not intended to limit, in any
manner, any of the aspects described herein. The following examples
do not in any way limit the invention.
EXAMPLES
Example 1. Exemplary Methods Used for Making Compacted Silk
Articles
[0220] Fabrication of aluminum powder compaction press and first
sample. The compaction press was designed in Solidworks and
fabricated from aluminum alloys, except for the piston, which was
made from steel. The entire assembly is held together with 1/4''-20
screws, which also are used to apply requisite pressure to the
piston/sample. The initial sample was made using silk powder that
had been pulverized and ball milled. A total of 25% of the powder
was generated from degummed silk fibroin, while the remaining 75%
was from a non-degumming fibroin source. A total of about 4 grams
of powder was mixed with 2 ml of distilled water. The assembled
compaction press was stoted in an oven for 48 hours at 60.degree.
C. The first sample had an excellent disk shape, with smooth
surfaces, a dull surface finish, and relatively light coloration.
Upon cooling, the sample could not be broken by hand, indicating
excellent toughness and strength properties.
[0221] Fabrication of an acrylic compaction press. In order to
improve the process and make fabrication easier, a new compaction
press was designed to be fabricated out of acrylic. In addition,
the acrylic material could be cut on a Trotec Speedy 300 laser
etcher, allowing for rapid reproduction of future press components.
Given the acrylic was not heat resistance, only room temperature
testing was appropriate.
[0222] Sample fabrication I using acrylic compaction press. A silk
powder mixture consisting of 10% degummed fibroin and 90%
non-degummed fibroin was utilized. Approximately 5 grams of powder
was mixed with 2.5 ml of distilled water and silk solution. The
press was then clamped lightly in a vise for 3 days at ambient
temperature. When released from the press, the silk construct was
not completely dry, and therefore it cracked and flaked. Material
that had not flaked away was relatively stiff, but extremely
brittle (and easily crumbled under pressure).
[0223] Sample fabrication II using acrylic compaction press. A
second sample was created using the acrylic press. Instead of ball
milled powder, however, commercial pure silk powder was used. This
powder was fabricated using hydrolysis, not milling. This powder
was very easy to solubulize in distilled water and had a much
whiter color than the milled powder. As in the prior experiment,
silk powder made of 10% degummed silk fibroin and 90% non-degummed
fibroin was utilized. Combining approximately 5 grams of powder
with 2.5 ml distilled water, the mixture was clamped in the acrylic
press for 4 days. The final silk construct was whiter than the
construct fabricated above in Sample fabrication I using acrylic
compaction press and the mechanical properties seemed to be worse,
with the construct having a chalky feel and the characteristic of
crumbling very easily.
[0224] Sample at higher temperature using aluminum compaction
press. Using the aluminum compaction press, and a powder mixture of
10% degummed silk fibroin/90% non-degummed fibroin, approximately 3
grams of powder were mixed with 2 ml of distilled water. The
sample/press was stored in an oven at 120.degree. C. for 48 hours.
The resulting sample was darker and appeared to be stiffer and
somewhat translucent. The hardness qualitatively seemed higher than
with previous samples. The darker color can be attributed to the
much higher heat that potentially caused some burning of silk
fibroin.
[0225] Compaction sample I using water and silk solution. Using the
aluminum compaction press and a powder mixture of 25% degummed silk
fibroin and 75% non-degummed silk fibroin, approximately 3 grams of
powder was mixed with 2.5 ml of distilled water and silk solution.
Given the silk solution concentration was approximately 7% w/v, the
powder was essentially mixed with a 3.5% w/v silk solution (the
distilled water acts to dilute the silk fibroin concentration). The
sample and press were stored in an oven at 120.degree. C. for 48
hours. The resulting construct was somewhat burned and cracked. The
darker coloration is attributed to the higher temperature (earlier
samples using no heating evidenced no color change). Without
wishing to be bound by theory. The crack may be developed as a
result of the addition of silk solution.
[0226] Compaction sample II using water and silk solution. A silk
sample was produced using the aluminum compaction press, a powder
mixture of 25% degummed silk fibroin and 75% non-degummed silk
fibroin, and a dilute silk solution (distilled water added to silk
solution). Approximately 3 grams of powder was combined wilh a
total of 2.5 ml dilute silk solution. After storing in an oven at
120.degree. C. oven for 48 hours, the resulting construct was
removed. The construct had a glassy surface, was somewhat
translucent, and contained many serious cracks. It is likely that
the glass-like appearance is due to the temperatures exceeding the
material's glass transition temperature. Without wishing to be
bound by theory, the cracking can be linked to the addition of silk
solution instead of pure water as a binding agent.
[0227] New test protocol to develop silk powder compaction. To
better understand the effect of various processing parameters on
the properties of the resulting silk samples, a test protocol was
developed. The two parameters investigated are the specific powder
blends of degummed and non-degummed silk fibroin, as well as the
amount of distilled water used (no silk solution was to be added in
this series of experiments). The first sample created with the test
protocol utilized a powder blend of 10% degummed and 90%
non-degummed silk fibroin. The powder was mixed with 1 ml of
distilled water and the sample/press was stored in on oven at
60.degree. C. oven for 24 hours. One side of the resulting
construct was black while slightly translucent and lighter in color
on the other. The discoloration was attributed to corrosion that
had occurred on the steel piston. The sample was observed to be
fairly homogeneous, exhibited high strength and did not contain
cracks or flakes.
[0228] The second sample created with the test protocol utilized a
powder blend of 15% degummed and 85% non-degummed silk fibroin was
utilized. Approximately 3 grams of powder was mixed with 2 ml of
distilled water and the sample/press was stored in an oven at
90.degree. C. oven for 24 hours. The time in the oven was
insufficient to allow the sample to completely dry. It warped and
flaked once it started drying outside of the press in ambient
conditions. Once dry, the remaining construct exhibited good
properties (could not be fractured using hand pressure).
[0229] The third sample created with the test protocol utilized a
powder blend of 15% degummed and 85% non-degummed silk fibroin was
utilized. Approximately 3 grams of powder was mixed with 2 ml of
distilled water and the sample/press was stored in an oven at
90.degree. C. oven for 48 hours. The resulting construct was
excellent, with smooth surfaces and light color. Due to its
excellent geometric stability, stiffness, and toughness, the sample
was subjected to machinability rests. A Trotec Speedy 300 Laser
Engraver was used to cut some small squares from the sample.
Several cutting experiments were run, using varying laser movement
speed and power level (the slower the speed and higher the power,
the more energy is put into the sample being cut). Although laser
culling could penetrate the construct and provide the desired
geometric shape, the cut edges were burned during the process. The
silk fibroin also created an unpleasant odor during laser cutting.
In a second machining operation, a drill press was used to create
small holes through the construct. The drill moved easily through
the material, without cracking or chipping the sample. A final
machining operation was performed using a DeWalt rotozip tool
(handheld milling device). The operation ran smoothly, although the
higher rotational speeds of the rotozip tool produced some burning
of the machined surfaces.
[0230] The fourth sample created with the test protocol utilized a
powder blend of 20% degummed and 80% non-degummed silk fibroin.
Approximately 3 grams of powder was mixed with 2 ml of distilled
water and the sample press was stored in an oven at 90.degree. C.
oven for 48 hours. The resulting construct was excellent, with
smooth surfaces and light color. Due to its excellent geometric
stability, stiffness, and toughness, this sample was also used to
test machinability.
[0231] The fifth sample created with the test protocol utilized a
powder blend of 25% degummed and 75% non-degummed silk fibroin.
Approximately 3 grams of powder was mixed with 2 ml of distilled
water and the sample/press was stored in an oven at 90.degree. C.
oven for 48 hours. The silk/water mixture had not been evenly
distributed in the press, so the resulting construct cracked during
the release stage of the process. This is attributed to human
error; the sample generally exhibited good mechanical properties
otherwise.
[0232] New acrylic buffer plate under piston and new sample. To
prevent discoloration of samples due to corrosion of the compaction
press piston, a thin acrylic disk was laser cut and placed between
the piston and sample for subsequent experiments. This design
improvement was shown to be effective at preventing further piston
corrosion due to exposure to sample moisture and reducing
discoloration. A powder blend of 10% degummed and 90% non-degummed
silk fibroin was utilized. Approximately 3 grams of powder was
mixed with 2 ml of distilled water and the sample/press was stored
in an oven at 60.degree. C. oven for 24 hours. For this experiment,
an increase in piston pressure was applied (by lightening the
hold-down screws described above). Given the rough morphology of
the sample, it is assumed that the silk/water combination was
poorly mixed. The resulting construct was not homogeneous, although
the mechanical performance (stiffness and toughness) appeared to be
good.
[0233] Fabrication of new aluminum compaction press. In order to
fabricate rectangular silk constructs for mechanical testing using
powder compaction processing, a new aluminum compaction press was
fabricated. The desired geometry was a thin strip with a
length-to-width aspect ratio of at least 4. The mechanical testing
protocol initially involved 3-point bend testing. In this test, a
sample strip is supported underneath by two supports on either end
of the sample, while a single upper support in the center places
the sample under a bending load. To minimize corrosion issues, the
entire press was made from aluminum (no steel parts to generate a
brown corrosion product that could discolor the samples). The new
press was designed to produce four samples simultaneously,
including 4 rectangular wells where the silk powder/binder mixture
is placed, "pistons" that transfer pressure from the top plate to
the samples and hold-down screws.
[0234] Powder compaction with patterned die. The regular geometries
described in the prior experiments are useful for a variety of
applications. An additional range of applications could be
envisioned if embedded features could be created at the surfaces of
the silk construct. To explore this potential, a special patterned
die insert was created from acrylic (using the single-sample
aluminum compaction press). Using an image of an elephant (the
Tufts University mascot), the die was created by laser etching on a
Trotec Speedy 300 laser engraver. A powder blend of 25% degummed
and 75% non-degummed silk fibroin was utilized. Approximately 3
grams of powder was mixed with 2 ml of distilled water which was
added to the well, on top of the acrylic die insert. The sample and
press were stored in an oven at 90.degree. C. oven for 48 hours.
This first patterned die was created with low resolution on the
laser engraver, so the silk construct did not contain a clean image
of the elephant. It was also determined that the powder was not
mixed thoroughly before the addition of water, so material in
homogeneity resulted (making the elephant image difficult to
see).
[0235] Powder compaction with patterned die II. This experiment
utilized the same single-sample aluminum compaction press as
described above in Powder compaction with patterned die. However, a
higher-resolution image of an elephant was used to create a
patterned die. To assist with visibility, a permanent marker was
used to color the area surrounding the elephant image on the die. A
powder blend of 25% degummed and 75% non-degummcd silk fibroin was
utilized. Approximately 3 grams of powder was mixed with 2 ml of
distilled water which was added to the well, on top of the acrylic
die insert. The sample press was-stored in an oven at 90.degree. C.
oven for 48 hours. The resulting construct, shown in FIG. 1A (next
to the high-resolution acrylic die insert), had excellent geometric
stability, with a finely detailed version of the elephant. Using a
stereomicroscope (FIG. 1B), the laser etcher's raftering and pulsed
response are both visible in the silk construct. FIG. 1D shows
another silk construct created using powder compaction and a coin
(FIG. 1C) as a die insert. The resulting detail replicated in the
silk is excellent.
[0236] Samples produced using new multi-sample aluminum compaction
press. A series of samples were produced using the newly designed
and fabrication multi-sample compaction press. The press has the
capability for producing up to 4 strip constructs simultaneously. A
powder blend of 25% degummed and 75% non-degummed silk fibroin was
utilized for all samples. Approximately 3 grams of powder was mixed
with 2 ml of distilled water and the sample/press was stored in an
oven at 90.degree. C. oven for 48 hours. Because of insufficient
filling of the wells with the powder/water mixture, the first
samples were thin and cracked in multiple locations during heating.
Doubling the volume of material per compaction well, a second set
of 4 samples were produced. While improved, these samples also
exhibited cracking during the heating phase of the process. Using
the same volume of material, significantly higher pressure was
applied in a third round of sample fabrication. These samples were
more robust, although some cracking did occur, resulting in
fracturing during sample removal from the press.
Example 2. Exemplary Methods Used for Making All-Silk Shoes
[0237] The ability to create three-dimensional constructs and/or to
perform post-processing operations enables the creation of complex
geometries. An exemplary method can relate to the development of an
all-silk shoe. This method can form a functional and complete
product by bringing many different types of silk-based materials
together--foams, fiber/solution composites, the hard constructs
described herein, cloth and eletrogelated silk. An exemplary design
of the shoe is shown in FIG. 2. In some embodiments, it can support
approximately 150 lbs without deforming significantly, and can be a
basic high heel design. In other embodiments, the capacity of the
shoe can increase to approximately 300 lbs.
[0238] Composition of the shoe. The shoe can be composed of several
different forms of silk. Each form has a different processing
protocol, and each material can be optimized for this specific use.
The heel and front platform of the shoe can be made from a solid
form of silk processed using HFIP (hexafluoro-2-propanol), as can
the bottom portion of the sole. The top portion of the sole can be
made from silk foam. This foam can be processed differently to
achieve different stiffnesses, and can be used for both the upper
part of the sole, and the padding. The upper part of the shoe can
be made from a composition of silk libers and silk solution;
combined to form a silk-silk composite with tailored, directional
properties.
[0239] Solid HFIP-processed silk for shoe heels, front platforms
and bottoms. FIG. 3 outlines the steps to make shoe heels, front
platforms, bottoms or any hard parts of the shoe utilizing
HFIP-processed silk. To make this form of silk, there are several
steps. First, e.g., silk cocoons are boiled to separate the
proteins (degumming). This degummed silk is then dried and
dissolved using, e.g., lithium bromide. This dissolved silk can
then be dialyzed with water to remove all traces of lithium
bromide. At this stage, the silk is referred to as silk solution.
The silk solution can then be freeze dried (lyophilized), at which
point it becomes silk foam. This foam can then be broken up into
small, uniform pieces (pulverized), and packed into a mold of a
desired shape. HFIP can be poured on top of the pulverized silk and
the moid can be covered. Once this new form of silk is cured, it
can be placed in a methanol bath, which washes out the HFIP. The
methanol is slowly replaced with water, until all or most methanol
has been removed. After this, the piece can be dried. Drying can
take any time, as long as several months, depending on the size of
the molded piece. As the piece dries, it can shrink.
[0240] The HFIP processed silk has been previously used to make
small, simple shapes, however it is possible to scale up the
production without significant changes in processing. Mold size and
shape can be altered to achieve the desired shape, and a new method
of drying the piece can be developed to ensure that even drying
takes place in all sections, especially in the heel, since the
volume to surface area ratio is larger than that of the sole.
[0241] For use as the sole and heel of a shoe, this new silk
material is preferred to be able to withstand enough force to
support a certain amount of weight, as well as the maximum possible
impact on the heel due to walking or running. In one embodiment,
the shoe can be designed to hold the weight of a 150 lb person. In
another embodiment, a maximum weight of 300 lbs can be allowable.
In order to show that this material is strong enough, the material
properties are characterized. One of the essential properties for
this application is the mechanical properties: compressive modulus
and bending modulus (stiffness). These values can be found by
performing various mechanical tests. A finite element model of the
heel can then be made and the required forces simulated to ensure
the material will not fail in any way under the maximum load.
Chemical testing can also be done in order to ensure the chemicals
used during processing are gone from the final product. Other tests
can be done on the material in order for the product to be sold
commercially, flammability and burn tests can be performed, as well
as tests for solvent resistance. Each of these tests is done on a
statistically significant sample size.
[0242] When the material is shown to have mechanical properties
acceptable for this application, a shoe prototype is constructed.
In order to do this, a mold can first be designed and manufactured.
The shoe prototype can take significantly longer to cure and dry
than the test samples. Once molded, the piece can also be machined,
as the shrinkage and warping that occurs during drying can distort
the original shape. Any art-recognized methods that can attach the
finished pieces made From this material to the rest of the shoe can
be used.
[0243] Silk foam. The material to be used in the upper part of the
sole (the silk foam) can sesrve as both the support material and
the padding in the sole of the shoe. FIG. 1 outlines the steps for
this process. Silk foam can be made by preparing silk solution as
previously described, then freezing it in a mold and lyophilizing
it (freeze drying). Depending on the concentration of the silk
solution used, the freezing time, settings of the lyophilizer, and
foams of different qualities can be produced. The properties of the
formed silk foam can be tuned to match with the padding currently
used in shoe manufacture. Compressive modulus, torsional modulus,
re-expansion rate after compression, and mode of failure can be
taken into account. These properties can then be reproduced in the
silk foam by altering variables in the processing. Post processing
steps, such as water annealing and methanol treatments, can also be
used to tweak certain properties, which a fleet the crystallinity
of the silk. Other qualities to test in this material include the
ease of adhesion and sewabilily of the foam, as well as the
mechanical properties in a hydrated environment (due to rain or
sweat).
[0244] Fiber solution composite. The material to be used as the
upper portion of the shoe can be composed of silk fibers woven
together and coated in silk solution, as prepared by the protocol
explained previously. There are different alterations to this
material, including, e.g., the ratio of fiber to solution, the
concentration of the solution, and the formation of the woven
fiber. Each of these variables can be optimized for the strength
and stiffness desired in the final product. FIG. 5 shows the steps
for this process.
[0245] Attachments and connections. Each of the pieces of the shoe
can be produced separately before the assembly of the final
product. The heel, upper, platform, padding and lining can be
attached to the sole of the shoe. Silk or nonsilk materials can be
used to achieve secure attachments. Some of the exemplary
attachment methods include, e.g., silk screws made from solid
HFIP-processed silk, embedding fibers into foams and solid silk
forms, and sewing with silk threads.
[0246] All patents and other publications identified in the
specification and examples are expressly incorporated herein by
reference for all purposes. These publications are provided solely
for their disclosure prior to the filing date of the present
application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention or for any other reason.
All statements as to the date or representation as to the contents
of these documents is based on the information available to the
applicants and does not constitute any admission as to the
correctness of the dates or contents of those documents.
[0247] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant ail that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow. Further, to the extent not already indicated, it will be
understood by those of ordinary skill in the art that any one of
the various embodiments herein described and illustrated can be
further modified to incorporate features shown in any of the other
embodiments disclosed herein.
* * * * *