U.S. patent application number 13/751514 was filed with the patent office on 2013-08-01 for spider silk based matrix.
This patent application is currently assigned to Allergan, Inc.. The applicant listed for this patent is Allergan, Inc.. Invention is credited to Gregory H. Altman, David Horan, Rebecca Horan, David Kaplan.
Application Number | 20130197194 13/751514 |
Document ID | / |
Family ID | 21734509 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130197194 |
Kind Code |
A1 |
Kaplan; David ; et
al. |
August 1, 2013 |
SPIDER SILK BASED MATRIX
Abstract
A silk fiber based matrix composition comprising spider silk
that can be biodegradable, from the spider species Nephila clavipes
(or from genetically engineered bacteria making Nephila clavipes
silk), and in the form of a mesh or film.
Inventors: |
Kaplan; David; (Concord,
MA) ; Altman; Gregory H.; (Arlington, MA) ;
Horan; Rebecca; (Arlington, MA) ; Horan; David;
(Westfield, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allergan, Inc.; |
Irvine |
CA |
US |
|
|
Assignee: |
Allergan, Inc.
Irvine
CA
|
Family ID: |
21734509 |
Appl. No.: |
13/751514 |
Filed: |
January 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10990199 |
Nov 16, 2004 |
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13751514 |
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10008924 |
Nov 16, 2001 |
6902932 |
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10990199 |
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Current U.S.
Class: |
530/353 |
Current CPC
Class: |
C12N 5/066 20130101;
C12N 2533/50 20130101; A61L 27/3834 20130101; A61P 9/00 20180101;
Y10T 428/249921 20150401; A61K 35/12 20130101; A61F 2/08 20130101;
A61L 27/48 20130101; C12N 5/0663 20130101; A61L 27/386 20130101;
A61L 27/3895 20130101; C07K 14/43518 20130101; A61P 19/04 20180101;
A61L 27/3804 20130101; C08L 89/00 20130101; A61L 27/227 20130101;
A61K 38/17 20130101; A61L 27/3821 20130101; A61F 2/00 20130101;
A61K 9/70 20130101; A61P 21/00 20180101; A61L 27/3608 20130101;
A61L 27/48 20130101; A61P 19/00 20180101; A61L 2430/10 20130101;
Y10T 442/2566 20150401; A61K 35/28 20130101; Y10T 428/298 20150115;
A61L 27/3662 20130101 |
Class at
Publication: |
530/353 |
International
Class: |
C07K 14/435 20060101
C07K014/435 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under
contract numbers DE13405 and AR46563 awarded by the National
Institutes of Health and under contract number R43 HD42352 awarded
by the National Institutes of Health through SBIR. The Government
has certain rights in this invention.
Claims
1. A silk-fiber-based matrix composition comprising spider
silk.
2. The matrix of claim 1, wherein the spider silk is sericin
extracted.
3. The matrix of claim 1, wherein the spider silk is
biodegradable.
4. The matrix of claim 1, wherein the spider silk is dragline silk
from an orb weaving spider.
5. The matrix of claim 1, wherein the spider silk is from the
spider species Nephila clavipes.
6. The matrix of claim 1, wherein the matrix is in the form of a
mesh.
7. The matrix of claim 1, wherein the matrix is in the form of a
film.
8. A silk-fiber-based matrix composition comprising spider silk,
wherein the spider silk is biodegradable, from the spider species
Nephila clavipes, and is in the form of a mesh or film.
9. A silk-fiber-based matrix composition comprising spider silk
made by genetically engineered bacteria, wherein the spider silk is
biodegradable, is spider species Nephila clavipes silk made by the
genetically engineered bacteria, and is in the form of a mesh or
film.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/990,199, filed Nov. 16, 2004, which is a
continuation of U.S. patent application Ser. No. 10/008,924, filed
Nov. 16, 2001, now U.S. Pat. No. 6,902,932, the entire of contents
of each of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention is directed to a matrix or scaffold
used in the production of bioengineered tissue, particularly
ligaments and tendons. More particularly, the invention relates to
a novel silk-fiber-based matrix upon which pluripotent cells may be
seeded ex vivo and which proliferate and differentiate thereon into
an anterior cruciate ligament for implantation into a recipient in
need thereof.
BACKGROUND OF THE INVENTION
[0004] Every year, hundreds of thousands of Americans sprain, tear,
or rupture ligaments and tendons of the knee, elbow, hand,
shoulder, wrist and jaw (Langer et al., Science 260: 920-926
(1993)). Of particular importance is the anterior cruciate ligament
of the knee. More than 200,000 people in the U.S. alone, will tear
or rupture their anterior cruciate ligament (ACL) each year
(Albright et al., 1999. Chapter 42-Knee and Leg:Soft-Tissue Trauma.
In Orthopaedic Knowledge Update 6. American Academy of Orthopaedic
Surgeons). The ACL serves as a primary stabilizer of anterior
tibial translation and as a secondary stabilizer of valgus-varus
knee angulation, and is often susceptible to rupture or tear
resulting from a flexion-rotation-valgus force associated with
sports injuries and traffic accidents. Ruptures or tears often
result in severe limitations in mobility, pain and discomfort, and
the loss of an ability to participate in sports and exercise.
Failures of the ACL are classified in three categories: (1)
ligamentous (ligament fibers pull apart due to tensile stress), (2)
failure at the bone-ligament interface without bone fracture, and
(3) failure at the bone-ligament interface with bone fracture at
the attachment site of bone and ligament. The most common type of
ACL failure is the first category, ligamentous.
[0005] It is widely known throughout the medical community that the
ACL has poor healing capabilities. Total surgical replacement and
reconstruction are required when injury to the ACL involves
significant tear or rupture. Four options have been utilized for
repair or replacement of a damaged ACL: (1) autografts, (2)
allografts, (3) xenografts, and (4) synthetic prostheses
(degradable and non-degradable). To date, no surgical repair
procedure has been shown to restore knee function completely, and
novel treatment options would likely benefit a large number of
patients.
[0006] The problems associated with the use of synthetic ACL
replacements, along with the limited availability of the donor
tissue, have motivated research towards the development of
functional and biocompatible equivalents of native tissues. This
shift from synthetic to biologically-based ACL replacements first
applied in early studies in which collagenous ACL prostheses were
prepared as composite structures consisting of reconstituted type I
collagen fibers in a collagen I matrix with polymethylmethacrylate
bone fixation plugs, and used as anterior cruciate ligament
replacement tissues in rabbits (Dunn et al., Am. J. Sports Medicine
20: 507-515 (1992)). Subsequent studies incorporated active
biological components into the process, such as ligament
fibroblasts seeded on cross-linked collagen fiber scaffolds that
were used as ligament analogs (Dunn et al., J. Biomedical Materials
Res. 29: 1363-1371 (1995); Dunn, M. G., Materials Res. Soc.
Bulletin, November: 43-46 (1996)), and suggested that structures
approximating native ligaments can be generated.
[0007] A tendon gap model, based on pre-stressed collagen sutures
seeded with mesenchymal stem cells provided improved repair of
large tendon defects (Young et al., 1998). Goulet et al. modified
the collagen-fibroblast system by using ligament fibroblasts in
non-cross-linked collagen, with bone anchors to pre-stress the
tissue and facilitate surgical implantation (Goulet et al., Tendons
and Ligaments. In Principles of Tissue Engineering, Ed. R. Lanza,
R. Langer, W. Chick. R. G. Landes Co. pp 633-643, R. G. Lanz Co.
and Academic Press, Inc., San Diego, Calif. (1997)). Passive
tension produced by growing the new ligament in a vertical position
induced fibroblast elongation and the alignment of the cells and
surrounding extracellular matrix.
[0008] Silk has been shown to offer new options for the design of
biomaterials and tissue-engineering scaffolds with a wide range of
mechanical properties (Sofia, S., et al., J. Biol. Mat. Res. 54:
139-148 (2001). For example, the dragline silk from the orb weaving
spider, Nephila clavipes, has the highest strength of any natural
fiber, and rivals the mechanical properties of synthetic high
performance fibers. Silks also resist failure in compression, are
stable at high physiological temperatures, and are insoluble in
aqueous and organic solvents. In recent years, silks have been
studied as a model for the study of structure-function
relationships of fibrous proteins. The manipulation of silk genes,
both native and artificial versions, has provided insight into silk
protein expression, assembly, and properties. Thus,
biocompatibility, the ability to engineer the materials with
specific and impressive mechanical properties, and a diverse range
of surface chemistries for modification or decoration suggests that
silk may provide an important class of biomaterial. Recent studies
by the inventors of the present invention have demonstrated the
successful attachment and growth of fibroblasts on silk films from
silkworm silk of Bombyx mori.
[0009] Tissue engineering can potentially provide improved clinical
options in orthopaedic medicine through the in vitro generation of
biologically based functional tissues for transplantation at the
time of injury or disease. Further, adult stem cells are becoming
increasingly recognized for their potential to generate different
cell types and thereby function effectively in vitro or in vivo in
tissue repair. (Sussman, M. Nature 410: 640 (2001). The knee joint
geometry and kinematics and the resultant effects on ACL structure
must be incorporated into the construct design if a tissue
engineered ACL generated in vivo is to successfully stabilize the
knee and function in vivo. A mismatch in the ACL structure-function
relationship would result in graft failure.
[0010] To date, no human clinical trials have been reported with
tissue culture bioengineered anterior cruciate ligaments. This is
due to the fact that each approach has failed to address one or
more of the following issues: (1) the lack of a readily available
cell or tissue source, (2) the unique structure (e.g., crimp
pattern, peripheral helical pattern and isometric fiber
organization) of an ACL, and (3) the necessary remodeling time in
vivo for progenitor cells to differentiate and/or autologous cells
to infiltrate the graft, thus extending the time a patient must
incur a destabilized knee and rehabilitation. The development of a
matrix for generating more fully functional bioengineered anterior
cruciate ligaments would greatly benefit the specific field of knee
reconstructive surgery, and would also provide wider benefits to
the overall field of in vitro tissue generation and replacement
surgery.
SUMMARY OF THE INVENTION
[0011] The present invention provides a novel silk-fiber-based
matrix for producing ligaments and tendons ex vivo. More
specifically, the present invention is directed to engineering
mechanically and biologically functional anterior cruciate ligament
using a novel silk-fiber-based matrix that may be seeded with
pluripotent cells, such as bone marrow stromal cells (BMSCs). The
mechanically and biologically autologous or allogenic anterior
cruciate ligament comprised of the novel matrix and pluripotent
cells may be prepared within a bioreactor environment to induce de
novo ligament tissue formation in vitro prior to implantation.
Surprisingly, it has now been found that the novel silk-fiber-based
matrix supports BMSC differentiation towards ligament lineage
without the need for directed mechanical stimulation during culture
within a bioreactor. The inventors believe that mechanical
stimulation will serve only to enhance the differentiation and
tissue development process.
[0012] The present invention also provides a method for the
generation of tissue engineered ACL ex vivo using the novel
silk-fiber-based matrix that comprises the steps of seeding
pluripotent stem cells in the silk-fiber-based matrix, anchoring
the seeded matrix by attachment to at least two anchors, and
culturing the cells within the matrix under conditions appropriate
for cell growth and regeneration. The culturing step may comprise
the additional step of subjecting the matrix to one or more
mechanical forces via movement of one or both of the attached
anchors. In a preferred embodiment for producing an ACL,
pluripotent cells, and more particularly bone marrow stromal cells,
are used. Suitable anchor materials comprise any materials to which
the matrix can attach (either temporarily or permanently), and
which supports ligament and tendon tissue growth or bone tissue
growth at the anchors. Preferred anchor materials include
hydroxyapatite, demineralized bone, and bone (allogenic or
autologous). Anchor materials may also include titanium, stainless
steel, high density polyethylene, Dacron and Teflon, amongst other
materials. Giniopora coral which has been treated to convert the
calcium carbonate to calcium phosphate has also been used as an
anchor material. In a preferred embodiment, the mechanical forces
to which the matrix may be subjected mimic mechanical stimuli
experienced by an anterior cruciate ligament in vivo. This is
accomplished by delivering the appropriate combination of tension,
compression, torsion and shear, to the matrix.
[0013] The bioengineered ligament which is produced according to
the present invention is advantageously characterized by a cellular
orientation and/or matrix crimp pattern in the direction of applied
mechanical forces, and also by the production of ligament and
tendon specific markers including collagen type I, collagen type
III, and fibronectin proteins along the axis of mechanical load
produced by the mechanical forces or stimulation, if such forces
are applied. In a preferred embodiment, the ligament or tendon is
characterized by the presence of fiber bundles which are arranged
into a helical organization.
[0014] Another aspect of the present invention is a method for
producing a wide range of ligament and tendon types ex vivo using
the novel silk-fiber-based matrix, and an adaptation of the method
for producing an anterior cruciate ligament by adapting the matrix
(e.g., geometry, organization, composition) (see Example 1) and
anchor size to reflect the size of the specific type of ligament or
tendon to be produced (e.g., posterior cruciate ligament, rotator
cuff tendons, medial collateral ligament of the elbow and knee,
flexor tendons of the hand, lateral ligaments of the ankle and
tendons and ligaments of the jaw or temporomandibular joint), and
also adapting the specific combination of forces applied, to mimic
the mechanical stimuli experienced in vivo by the specific type of
ligament or tendon to be produced. Similar adaptations of this
method, and which are considered to be a part of the invention, can
be made to produce other tissues ex vivo from pluripotent stem
cells, by adapting additional matrix compositions, geometries,
organizations, and the mechanical forces applied during cell
culture to mimic stresses experienced in vivo by the specific
tissue type to be produced. The methods of the present invention
can be further modified to incorporate other stimuli experienced in
vivo by the particular developing tissue. Some examples of other
stimuli include chemical stimuli and electro-magnetic stimuli.
[0015] As used herein, the term "tissue" is intended to take on its
generally recognized biological/medical definition to those of
skill in the art. As a non-limiting example, "tissue" is defined in
Stedman's Medical Dictionary as: [A] collection of similar cells
and the intercellular substances surrounding them. There are four
basic tissues in the body: 1) epithelium; 2) the connective
tissues, including blood, bone, and cartilage; 3) muscle tissue;
and 4) nerve tissue.
[0016] Another aspect of the present invention relates to the
specific ligaments or tendons which are produced by the methods of
the present invention. Some examples of ligaments or tendons that
can be produced include anterior cruciate ligament, posterior
cruciate ligament, rotator cuff tendons, medial collateral ligament
of the elbow and knee, flexor tendons of the hand, lateral
ligaments of the ankle and tendons and ligaments of the jaw or
temporomandibular joint. Other tissues that may be produced by
methods of the present invention include cartilage (both articular
and meniscal), bone, muscle, skin and blood vessels.
[0017] The above description sets forth rather broadly the more
important features of the present invention in order that the
detailed description thereof that follows may be understood, and in
order that the present contributions to the art may be better
appreciated. Other objects and features of the present invention
will become apparent from the following detailed description
considered in conjunction with the accompanying drawings. It is to
be understood, however, that the drawings are designed solely for
the purposes of illustration and not as a definition of the limits
of the invention, for which reference should be made to the
appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1A is a scanning electron microscopy (SEM) image of
single native silk fiber having a sericin coating;
[0019] FIG. 1B illustrates SEM of the silk fiber of FIG. 1A
extracted for 60 min at 37.degree. C.;
[0020] FIG. 1C illustrates SEM of the silk fiber of FIG. 1A
extracted for 60 min at 90.degree. C. and illustrating complete
removal of the sericin coating;
[0021] FIG. 1D is a chart showing ultimate tensile strength (UTS)
as a function of extraction conditions;
[0022] FIG. 2A illustrates a single cord of Matrix 1 having a
wire-rope geometry composed of two levels of twisting hierarchy.
When six cords are used in parallel (e.g., Matrix 1), the matrix
has mechanical properties similar to a native ACL.
[0023] FIG. 2B illustrates a single cord of Matrix 2 having a
wire-rope geometry composed of three levels of twisting hierarchy.
When six cords are used in parallel (e.g., Matrix 2), the matrix
has mechanical properties similar to a native ACL.
[0024] FIG. 3A illustrates load-elongation curves (N=5) for Matrix
1 formed from six parallel silk fibroin cords illustrated in FIG.
2A.
[0025] FIG. 3B is a chart of cycles to failure at UTS, 1680N, and
1200N loads (n=5 for each load) illustrating Matrix 1 fatigue data.
Regression analysis of Matrix 1 fatigue data, when extrapolated to
physiological load levels (400 N) to predicite number of cycles to
failure in vivo, indicates a matrix life of 3.3 million cycles;
[0026] FIG. 3C illustrates load-elongation curves for Matrix 2
(N=3) formed from six parallel silk fibroin cords as illustrated in
FIG. 2B;
[0027] FIG. 3D is a chart of cycles to failure at UTS, 2280N, 2100N
and 1800N loads (N=3 for each load) illustrating Matrix 2 fatigue
data. Regression analysis of Matrix 2 fatigue data, when
extrapolated to physiological load levels (400 N) to predicate
number of cycles to failure in vivo, indicates a matrix life of
greater than 10 million cycles;
[0028] FIG. 4A illustrates SEM of extracted silk fibroin prior to
seeding with cells;
[0029] FIG. 4B illustrates SEM of bone marrow stromal cells seeded
and attached on silk fibroin immediately post seeding;
[0030] FIG. 4C illustrates SEM of bone marrow cells attached and
spread on silk fibroin 1 day post seeding;
[0031] FIG. 4D illustrates SEM of bone marrow stromal cells seeded
on silk fibroin 14 days post seeding forming an intact
cell-extracellular matrix sheet;
[0032] FIG. 5A illustrates a 3 cm length of the silk fibroin cord
illustrated in FIG. 2A and seeded with bone marrow stromal cells,
cultured for 14 days in a static environment and stained with MTT
to show even cell coverage of the matrix following the growth
period;
[0033] FIG. 5B illustrates a control strand of silk fibroin cord 3
cm in length stained with MTT;
[0034] FIG. 6A is a chart illustrating bone marrow stromal cell
proliferation on silk fibroin Matrix 1 determined by total cellular
DNA over 21 day culture period indicating a significant increase in
cell proliferation after 21 days of culture;
[0035] FIG. 6B is a bar graph illustrating bone marrow stromal cell
proliferation on silk fibroin Matrix 2 determined by total cellular
DNA over 14 day culture period indicating a significant increase in
cell proliferation after 14 days of culture;
[0036] FIG. 7 illustrates the ultimate tensile strength of a 30
silk fiber extracted construct which is either seeded with bone
marrow stromal cells or non-seeded over 21 days of culture in
physiological growth conditions;
[0037] FIG. 8 illustrates gel eletrophoretic analysis of RT-PCR
amplification of selected markers over time. The gel shows
upregulation in both collagen types I and III expression levels
normalized to the housekeeping gene, GAPDH by bone marrow stromal
cell grown on Matrix 2 over 14 days in culture. Collagen type II
(as a marker for cartilage) and bone sialoprotein (as a marker of
bone tissue formation) were not detected indicating a ligament
specific differentiation response by the BMSCs when cultured with
Matrix 2.
[0038] FIGS. 9A-9B illustrate a single cord of Matrix 1 (not seeded
at the time of implantation) following six weeks of implantation in
vivo and used to reconstruct the medial collateral ligament (MCL)
in a rabbit model. (A) shows Matrix 1 fibroin fibers surrounded by
progenitor host cells and tissue ingrowth into the matrix and
around the individual fibroin fibers visualized by hematoxylin and
eosin staining; (B) shows collagenous tissue ingrowth into the
matrix and around the individual fibroin fibers visualized by
trichrome staining; and
[0039] FIGS. 10A-10C illustrate bone marrow stromal cells seeded
and grown on collagen fibers for (A) 1 day and (B) 21 days; (C)
RT-PCR and gel electrophoretic analysis of collagen I and III
expression vs. the housekeeping gene GAPDH: a=Collagen I, day 14;
b=Collagen I, day 18; c=Collagen III, day 14; d=Collagen III, day
18; e=GAPDH, day 14; f=GAPDH, day 18. Collagen type II (as a marker
for cartilage) and bone sialoprotein (as a marker of bone tissue
formation) were not detected indicating a ligament specific
differentiation response; and
[0040] FIG. 11 illustrates real-time quantitative RT-PCR at 14 days
which yielded a transcript ratio of collagen I to collagen III,
normalized to GAPDH, of 8.9:1.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention is directed to a novel
silk-fiber-based matrix upon which pluripotent cells may be seeded
and which proliferate and differentiate into ligament and tendon
fibroblasts resulting in the formation of an anterior cruciate
ligament (ACL), or other ligaments, tendons or tissues. The novel
silk-fiber-based matrix is designed having fibers in a wire-rope
(twisted or braided-like) geometry, which exhibits mechanical
properties identical to a natural anterior cruciate ligament (see
Table 1, infra) and where simple variations in matrix organization
and geometry can result in the formation of any desired ligament or
tendon tissue (see Table 2, infra).
[0042] The present invention is also based on the finding that
pluripotent bone marrow stromal cells (BMSCs) isolated and cultured
as described in Example 1, seeded on the silk-fiber-based matrix,
and cultured in a bioreactor under static conditions will undergo
ligament and tendon specific differentiation forming viable tissue.
In addition, the histomorphological properties of a bioengineered
tissue produced in vitro generated from pluripotent cells within a
matrix are affected by the direct application of mechanical force
to the matrix during tissue generation. This discovery provides
important new insights into the relationship between mechanical
stress, biochemical and cell immobilization methods and cell
differentiation, and has applications in producing a wide variety
of ligaments, tendons and tissues in vitro from pluripotent
cells.
[0043] One aspect of the present invention relates to a matrix
comprised of silk fibers having a wire-rope (twisted or
braided-like) geometry, as illustrated in FIGS. 2A and 2B.
[0044] As described in the Examples below, mechanical properties of
the silk fibroin (as illustrated in FIGS. 1A-C) were characterized
and geometries for forming applicable matrices for ACL engineering
were derived using a theoretical computational model (see FIG. 1D).
A six-cord construct was chosen for use as an ACL replacement to
increase matrix surface area and to enhance support for tissue
in-growth. Two of several optimal construct geometrical hierarchies
have been determined to comprise the following: Matrix 1:1 ACL
prosthesis=6 parallel cords; 1 cord=3 twisted strands (3
twists/cm); 1 strand=6 twisted bundles (3 twists/cm); 1 bundle=30
parallel washed fibers; and Matrix 2: 1 ACL matrix=6 parallel
cords; 1 cord=3 twisted strands (2 twists/cm); 1 strand=3 twisted
bundles (2.5 twists/cm); 1 bundle=3 groups (3 twists/cm); 1
group=15 parallel extracted silk fibroin fibers. The number of
fibers and geometries for Matrix 1 and Matrix 2 were selected such
that the silk prostheses are similar to the ACL biomechanical
properties in ultimate tensile strength, linear stiffness, yield
point and % elongation at break, serving as a solid starting point
for the development of a tissue engineered ACL. The ability to
generate two matrices with differing geometries both resulting in
mechanical properties which mimic properties of the ACL indicates
that an infinite number of geometrical configurations exist to
achieve the desired mechanical properties. One skilled in the art
will recognize that an alternative geometry for any desired
ligament or tendon tissue may comprise any number, combination or
organization of cords, strands, bundles, groups and fibers (see
Table 2, infra) that result in a matrix construct with applicable
mechanical properties that mimic those of the ligament or tendon
desired. For example, one (1) ACL prosthesis may have any number of
cords in parallel ranging from a single cord to an infinite number
of cords provided there is a means of anchoring the final matrix in
vitro or in vivo. One skilled in the art would also recognize that
no limit exists to the number of twisting levels (where a single
level is defined as either a group, bundle, strand or cord) for a
given geometry providing the matrix results in the desired
mechanical properties. Furthermore, one skilled in the art would
realize the large degree of freedom in designing the matrix
geometry and organization in engineering an ACL prosthesis, and
will therefore understand the utility of the developed theoretical
computational model to predict the matrix design of a desired
ligament or tendon tissue (see Example 1). One skilled in the art
would, therefore, recognize that a variation in geometry (i.e., the
number of cords used to make a prosthesis or number of fibers in a
group) could be used to generate matrices applicable to most
ligaments and tendons. For example, for smaller ligaments or
tendons of the hand, the geometry and organization used to generate
a single cord of Matrix 1 (or two cords or three cords, etc.) may
be appropriate given the matrix's organization results in
mechanical properties suitable for the particular physiological
environment.
[0045] The invention is not, however, limited with respect to the
wire-rope geometry as described, and any geometry or combinations
of geometries (e.g., parallel, twisted, braided, mesh-like) can be
used that results in matrix mechanical properties similar to the
ACL (i.e., >2000 N ultimate tensile strength, between 100-600
N/mm linear stiffness for a native ACL or commonly used replacement
graft such as the patellar tendon with length between 26-30 mm) or
to the desired ligament and tendon that is to be produced. The
number of fibers and geometry of both Matrix 1 and Matrix 2 were
selected to generate mechanically appropriate ACL matrices, or
other desired ligament or tendon matrices (e.g., posterior cruciate
ligament (PCL)). For example, a single cord of the six-cord Matrix
1 construct was used to reconstruct the medial collateral ligament
(MC) in a rabbit (see FIG. 9). The mechanical properties of the
silk six-cord constructs of Matrix 1 and Matrix 2 are described in
Table 1 and in FIG. 3. Additional geometries and their relating
mechanical properties are listed in Table 2 as an example of the
large degree of design freedom that would result in a matrix
applicable in ACL tissue engineering in accordance with the present
invention.
TABLE-US-00001 TABLE 1 UTS Stiffness Yield Pt. Elongation (N)
(N/mm) (N) (%) Silk matrix 1 2337 +/- 72 354 +/- 26 1262 +/- 36
38.6 +/- 2.4 Silk Matrix 2 3407 +/- 63 580 +/- 40 1647 +/- 214 29
+/- 4 Human ACL 2160 +/- 242 +/- 28.sup.4 ~1200 ~26-32% 157.sup.4
Mechanical properties for two different cords based on a cord
length of 3 cm.
TABLE-US-00002 TABLE 2 Twisting Level (# of twists/cm) Matrix 1
Matrix 2 Matrix 3 Matrix 4 Matrix 5 Matrix 6 Matrix 7 # fibers per
group 30 15 1300 180 20 10 15 (0) (0) (0) (0) (0) (0) (0) # groups
per bundle 6 3 3 3 6 6 3 (3) (3) (2) (3.5) (3) (3) (3) # bundles
per strand 3 6 1 3 3 3 3 (3) (2.5) (0) (2) (2) (2.5) (2.5) #
strands per cord 6 3 -- 2 3 3 3 (0) (2.0) (0) (1) (2) (2) # cords
per ACL -- 6 -- -- 3 6 12 (0) (0) (0) (0) UTS (N) 2337 3407 2780
2300 2500 2300 3400 Stiffness (N/mm) 354 580 300 350 550 500 550
Examples of several geometry hierarchies that would result in
suitable mechanical properties for replacement of the ACL. Note:
Matrix 1 and 2 have been developed as shown in example 1; Matrix 3
would yield a single bundle prosthesis, Matrix 4 would yield a 2
strand prosthesis, Matrix 5 would yield a 3 cord prosthesis, Matrix
6 is another variation of a 6 cord prosthesis, and Matrix 7 will
yield a 12 cord prosthesis.
[0046] Advantageously, the silk-fiber based matrix is comprised
solely of silk. Types and sources of silk include the following:
silks from silkworms, such as Bombyx mori and related species;
silks from spiders, such as Nephila clavipes; silks from
genetically engineered bacteria, yeast mammalian cells, insect
cells, and transgenic plants and animals; silks obtained from
cultured cells from silkworms or spiders; native silks; cloned full
or partial sequences of native silks; and silks obtained from
synthetic genes encoding silk or silk-like sequences. In their raw
form, the native silk fibroin obtained from the Bombyx mori
silkworms are coated with a glue-like protein called sericin, which
typically is extracted from the fibers before the fibers which make
up the matrix are seeded with cells.
[0047] In an alternative embodiment, the matrix may be comprised of
a composite of: (1) silk and collagen fibers (2) silk and collagen
foams, meshes, or sponges, or a composite of (3) silk fibroin
fibers and silk foams, meshes, or sponges, (4) silk and
biodegradable polymers (e.g., cellulose, cotton, gelatin, poly
lactide, poly glycolic, poly(lactide-co-glycolide), poly
caprolactone, polyamides, polyanhydrides, polyaminoacids, polyortho
esters, poly acetals, proteins, degradable polyurethanes,
polysaccharides, polycyanoacrylates, Glycosamino glycans--e.g.,
chondroitin sulfate, heparin, etc., Polysaccharides--native,
reprocessed or genetically engineered versions--e.g. hyaluronic
acid, alginates, xanthans, pectin, chitosan, chitin, and the like,
Elastin--native, reprocessed or genetically engineered and chemical
versions, Collagens--native, reprocessed or genetically engineered
versions), or (5) silk and non-biodegradable polymers (e.g.,
polyamide, polyester, polystyrene, polypropylene, polyacrylate,
polyvinyl, polycarbonate, polytetrafluorethylene, or nitrocellulose
material. The composite generally enhances the matrix properties
such as porosity, degradability, and also enhances cell seeding,
proliferation, differentiation or tissue development. FIG. 10
illustrates the ability of collagen fibers to support BMSC growth
and ligament specific differentiation.
[0048] The matrix of the present invention may also be treated to
enhance cell proliferation and/or tissue differentiation thereon.
Exemplary matrix treatments for enhancing cell proliferation and
tissue differentiation include, but are not limited to, metals,
irradiation, crosslinking, chemical surface modifications (e.g. RGD
(arg-gly-asp) peptide coating, fibronectin coating, coupling growth
factors), and physical surface modifications.
[0049] A second aspect of the present invention relates to a
mechanically and biologically functional ACL formed from a novel
silk-fiber-based matrix and autologous or allogenic (depending on
the recipient of the tissue) bone marrow stromal cells (BMSCs)
seeded on the matrix. The silk-fiber-based matrix induces stromal
cell differentiation towards ligament lineage without the need for
any mechanical stimulation during bioreactor cultivation. BMSCs
seeded on the silk-fiber-based matrix and grown in a petri dish
begin to attach and spread (see FIG. 4), proliferate covering the
matrix (see FIGS. 5 and 6) and differentiate as shown by the
expression of ligament specific markers (see FIG. 8). Markers for
cartilage (collagen type II) and for bone (bone sialoprotein) were
not expressed (see FIG. 8). Data illustrating the expression of
ligament specific markers is set forth in Example 2.
[0050] In another aspect, the present invention relates to a method
for producing an ACL ex vivo. Cells capable of differentiating into
ligament cells are grown under conditions which simulate the
movements and forces experienced by an ACL in vivo through the
course of embryonic development into mature ligament function. This
is accomplished by the following steps: under sterile conditions,
pluripotent cells are seeded within a three dimensional
silk-fiber-based matrix to which cells can adhere and which is
advantageously of cylindrical shape. The three dimensional
silk-fiber based matrix used in the method serves as a preliminary
matrix, which is supplemented and possibly even replaced by
extracellular matrix components produced by the differentiating
cells. Use of the novel silk-fiber-based matrix may enhance or
accelerate the development of the ACL. For instance, the novel
silk-fiber-based matrix which has specific mechanical properties
(e.g., increased tensile strength) that can withstand strong forces
prior to reinforcement from cellular extracellular matrix
components. Other advantageous properties of the novel silk-fiber
based preliminary matrix include, without limitation,
biocompatibility and susceptibility to biodegradation.
[0051] The pluripotent cells may be seeded within the preliminary
matrix either pre- or post-matrix formation, depending upon the
particular matrix used and the method of matrix formation. Uniform
seeding is preferable. In theory, the number of cells seeded does
not limit the final ligament produced, however optimal seeding may
increase the rate of generation. Optimal seeding amounts will
depend on the specific culture conditions. In one embodiment, the
matrix is seeded with from about 0.05 to 5 times the physiological
cell density of a native ligament.
[0052] One or more types of pluripotent cells are used in the
method. Such cells have the ability to differentiate into a wide
variety of cell types in response to the proper differentiation
signals and to express ligament specific markers. More
specifically, the method requires the use of cells that have the
ability to differentiate into cells of ligament and tendon tissue.
In a preferred embodiment, bone marrow stromal cells are used. If
the resulting bioengineered ligament is to be transplanted into a
patient, the cells should be derived from a source that is
compatible with the intended recipient. Although the recipient will
generally be a human, applications in veterinary medicine also
exist. In one embodiment, the cells are obtained from the recipient
(autologous), although compatible donor cells may also be used to
make allogenic ligaments. For example, when making allogenic
ligaments (e.g., using cells from another human such as bone marrow
stromal cells isolated from donated bone marrow or ACL fibroblasts
isolated from donated ACL tissue), human anterior cruciate ligament
fibroblast cells isolated from intact donor ACL tissue (e.g.
cadaveric or from total knee transplantations), ruptured ACL tissue
(e.g., harvested at the time of surgery from a patient undergoing
ACL reconstruction) or bone marrow stromal cells may be used. The
determination of compatibility is within the means of the skilled
practitioner.
[0053] In alternative embodiments of the present invention,
ligaments or tendons including, but not limited to, the posterior
cruciate ligament, rotator cuff tendons, medial collateral ligament
of the elbow and knee, flexor tendons of the hand, lateral
ligaments of the ankle and tendons and ligaments of the jaw or
temporomandibular joint other than ACL, cartilage, bone and other
tissues may be engineered with the matrix in accordance with the
method of the present invention. In this manner, the cells to be
seeded on the matrix are selected in accordance with the tissue to
be produced (e.g., pluripotent or of the desired tissue type).
Cells seeded on the matrix in accordance with the present invention
may be autologous or allogenic. The use of autologous cells
effectively creates an allograft or autograft for implantation in a
recipient.
[0054] As recited, to form an ACL, cells, which are advantageously
bone marrow stromal cells, are seeded on the matrix. Bone marrow
stromal cells are a type of pluripotent cell, and are also referred
to in the art as mesenchymal stem cells or simply as stromal cells.
As recited, the source of these cells can be autologous or
allogenic. The present invention also contemplates the use of adult
or embryonic stem or pluripotent cells, in so long as the proper
environment (either in vivo or in vitro), seeded on the silk-fiber
based matrix, can recapitulate an ACL or any other desired ligament
or tissue in extracellular matrix composition (e.g., protein,
glycoprotein content) organization, structure or function.
[0055] Fibroblast cells are also contemplated by the present
invention for seeding on the inventive matrix. Since fibroblast
cells are often not referred to as pluripotent cells, fibroblasts
are intended to include mature human ACL fibroblasts (autologous or
allogenic) isolated from ACL tissue, fibroblasts from other
ligament tissue, fibroblasts from tendon tissue, from neonatal
foreskin, from umbilical cord blood, or from any cell, whether
mature or pluripotent, mature dedifferentiated, or genetically
engineered, such that when cultured in the proper environment
(either in vivo or in vitro), and seeded on the silk-fiber based
matrix, can recapitulate an ACL or any other desired ligament or
tissue in extracellular matrix composition (e.g., protein,
glycoprotein content), organization, structure or function.
[0056] The faces of the matrix cylinder are each attached to
anchors, through which a range of forces are to be applied to the
matrix. To facilitate force delivery to the matrix, it is
preferable that the entire surface of each respective face of the
matrix contact the face of the respective anchors. Anchors with a
shape which reflects the site of attachment (e.g., cylindrical) are
best suited for use in this method. Once assembled, the cells in
the anchored matrix are cultured under conditions appropriate for
cell growth and regeneration. The matrix is subjected to one or
more mechanical forces applied through the attached anchors (e.g.,
via movement of one or both of the attached anchors) during the
course of culture. The mechanical forces are applied over the
period of culture to mimic conditions experienced by the native
ACL, or other tissues in vivo.
[0057] The anchors must be made of a material suitable for matrix
attachment, and the resulting attachment should be strong enough to
endure the stress of the mechanical forces applied. In addition, it
is preferable that the anchors be of a material which is suitable
for the attachment of extracellular matrix which is produced by the
differentiating cells. The anchors support bony tissue in-growth
(either in vitro or in vivo) while anchoring the developing
ligament. Some examples of suitable anchor material include,
without limitation, hydroxyappatite, Goniopora coral, demineralized
bone, bone (allogenic or autologous). Anchor materials may also
include titanium, stainless steel, high density polyethylene,
Dacron and Teflon.
[0058] Alternatively, anchor material may be created or further
enhanced by infusing a selected material with a factor which
promotes either ligament matrix binding or bone matrix binding or
both. The term infuse is considered to include any method of
application which appropriately distributes the factor onto the
anchor (e.g., coating, permeating, contacting). Examples of such
factors include without limitation, laminin, fibronectin, any
extracellular matrix protein that promotes adhesion, silk, factors
which contain arginine-glycine-aspartate (RGD) peptide binding
regions or the RGD peptides themselves. Growth factors or bone
morphogenic protein can also be used to enhance anchor attachment.
In addition, anchors may be pre-seeded with cells (e.g., stem
cells, ligament cells, osteoblasts, osteogenic progenitor cells)
which adhere to the anchors and bind the matrix, to produce
enhanced matrix attachment both in vitro and in vivo.
[0059] An exemplary anchor system is disclosed in applicant's
co-pending application U.S. Ser. No. 09/950,561, filed Sep. 10,
2001. The matrix is attached to the anchors via contact with the
anchor face or alternatively by actual penetration of the matrix
material through the anchor material. Because the force applied to
the matrix via the anchors dictates the final ligament produced,
the size of the final ligament produced is, in part, dictated by
the size of the attachment site of the anchor. One of skill in the
art will appreciate that an anchor of appropriate size to the
desired final ligament should be used. A preferred anchor shape for
the formation of an ACL is a cylinder. However, one of skill in the
art will appreciate that other anchor shapes and sizes will also
function adequately. In a preferred embodiment, anchors have an
appropriate size and composition for direct insertion into bone
tunnels in the femur and tibia of a recipient of the bioengineered
ligament.
[0060] In an alternative embodiment of the present invention,
anchors may be used only temporarily during in vitro culture, and
then removed when the matrix alone is implanted in vivo.
[0061] In another embodiment, the novel silk-fiber-based matrix is
seeded with BMSCs and cultured in a bioreactor. Two types of growth
environments currently exist that may be used in accordance with
this invention: (1) the in vitro bioreactor apparatus system, and
(2) the in vivo knee joint, which serves as a "bioreactor" as it
provides the physiologic environment including progenitor cells and
stimuli (both chemical and physical) necessary for the development
of a viable ACL given a matrix with proper biocompatible and
mechanical properties. The bioreactor apparatus provides optimal
culture conditions for the formation of a ligament in terms of
differentiation and extracellular matrix (ECM) production, and
which thus provides the ligament with optimal mechanical and
biological properties prior to implantation in a recipient.
Additionally, when the silk-fiber based matrix is seeded and
cultured with cells in vitro, a petri dish may be considered to be
the bioreactor within which conditions appropriate for cell growth
and regeneration exist, i.e., a static environment.
[0062] In accordance with the one embodiment of present invention,
cells may also be cultured on the matrix without the application of
any mechanical forces, i.e., in a static environment. For example,
the silk-fiber based matrix alone, with no in vitro applied
mechanical forces or stimulation, when seeded and cultured with
BMSCs, induces the cells to proliferate and express ligament and
tendon specific markers (See Example 2 and FIG. 8). The knee joint
may serve as a physiological growth and development environment
that can provide the cells and the correct environmental signals
(chemical and physical) to the matrix such that an ACL technically
develops. Therefore, the knee joint (as its own form of bioreactor)
plus the matrix (either non-seeded, seeded and not differentiated
in vitro, or seeded and differentiated in vitro prior to
implantation) will result in the development of an ACL, or other
desired tissue depending upon the cell type seeded on the matrix
and the anatomical location of matrix implantation. FIG. 9
illustrates the effects of the medial collateral knee joint
environment on medial collateral ligament (MCL) development when
only a non-seeded silk-based matrix with appropriate MCL mechanical
properties is implanted for 6 weeks in vivo. Whether the cells are
cultured in a static environment with no mechanical stimulation
applied, or in a dynamic environment, such as in a bioreactor
apparatus, conditions appropriate for cell growth and regeneration
are advantageously present for the engineering of the desired
ligament or tissue.
[0063] In the experiments described in the Examples section below,
the applied mechanical stimulation was shown to influence the
morphology, and cellular organization of the progenitor cells
within the resulting tissue. The extracellular matrix components
secreted by the cells and organization of the extracellular matrix
throughout the tissue was also significantly influenced by the
forces applied to the matrix during tissue generation. During in
vitro tissue generation, the cells and extracellular matrix aligned
along the axis of load, reflecting the in vivo organization of a
native ACL which is also along the various load axes produced from
natural knee joint movement and function. These results suggest
that the physical stimuli experienced in nature by cells of
developing tissue, such as the ACL, play a significant role in
progenitor cell differentiation and tissue formation. They further
indicate that this role can be effectively duplicated in vitro by
mechanical manipulation to produce a similar tissue. The more
closely the forces produced by mechanical manipulation resemble the
forces experienced by an ACL in vivo, the more closely the
resultant tissue will resemble a native ACL.
[0064] When mechanical stimulation is applied in vitro to the
matrix via a bioreactor, there exists independent but concurrent
control over both cyclic and rotation strains as applied to one
anchor with respect to the other anchor. In an alternative
embodiment, the matrix alone may be implanted in vivo, seeded with
ACL cells from the patient and exposed in vivo to mechanical
signaling via the patient.
[0065] When the matrix is seeded with cells prior to implantation,
the cells are cultured within the matrix under conditions
appropriate for cell growth and differentiation. During the culture
process, the matrix may be subjected to one or more mechanical
forces via movement of one or both of the attached anchors. The
mechanical forces of tension, compression, torsion and shear, and
combinations thereof, are applied in the appropriate combinations,
magnitudes, and frequencies to mimic the mechanical stimuli
experienced by an ACL in vivo.
[0066] Various factors will influence the amount of force which can
be tolerated by the matrix (e.g., matrix composition, cell
density). Matrix strength is expected to change through the course
of tissue development. Therefore, mechanical forces or strains
applied will increase, decrease or remain constant in magnitude,
duration, frequency and variety over the period of ligament
generation, to appropriately correspond to matrix strength at the
time of application.
[0067] When producing an ACL, the more accurate the intensity and
combination of stimuli applied to the matrix during tissue
development, the more the resulting ligament will resemble a native
ACL. Two issues must be considered regarding the natural function
of the ACL when devising the in vitro mechanical force regimen that
closely mimics the in vivo environment: (1) the different types of
motion experienced by the ACL and the responses of the ACL to knee
joint movements and (2) the extent of the mechanical stresses
experienced by the ligament. Specific combinations of mechanical
stimuli are generated from the natural motions of the knee
structure and transmitted to the native ACL.
[0068] To briefly describe the motions of the knee, the connection
of the tibia and femur by the ACL between provides six degrees of
freedom when considering the motions of the two bones relative to
each other: the tibia can move in the three directions and can
rotate relative to the axes for each of these three directions. The
knee is restricted from achieving the full ranges of these six
degrees of freedom due to the presence of ligaments and capsular
fibers and the knee surfaces themselves (Biden et al., Experimental
methods used to evaluate knee ligament function. In Knee Ligaments:
Structure, Function, Injury and Repair, Ed. D. Daniel et al. Raven
Press, pp. 135-151 (1990)). Small translational movements are also
possible. The attachment sites of the ACL are responsible for its
stabilizing roles in the knee joint. The ACL functions as a primary
stabilizer of anterior-tibial translation, and as a secondary
stabilizer of valgus-varus angulation, and tibial rotation
(Shoemaker et al., The limits of knee motion. In Knee Ligaments:
Structure, Function, Injury and Repair, Ed. D. Daniel et al. Raven
Press, pp. 1534-161 (1990)). Therefore, the ACL is responsible for
stabilizing the knee in three of the six possible degrees of
freedom. As a result, the ACL has developed a specific fiber
organization and overall structure to perform these stabilizing
functions. The present invention simulates these conditions in
vitro to produce a tissue with similar structure and fiber
organization.
[0069] The extent of mechanical stresses experienced by the ACL can
be similarly summarized. The ACL undergoes cyclic loads of about
400 N between one and two million cycles per year (Chen et al., J.
Biomed. Mat. Res. 14: 567-586 (1980). It is also critical to
consider linear stiffness (.about.182 N/mm), ultimate deformation
(100% of ACL) and energy absorbed at failure (12.8 N-m) (Woo et
al., The tensile properties of human anterior cruciate ligament
(ACL) and ACL graft tissues. In Knee Ligaments: Structure,
Function, Injury and Repair, Ed. D. Daniel et al. Raven Press, pp.
279-289 (1990)) when developing an ACL surgical replacement.
[0070] The Examples section below details the production of a
prototype bioengineered anterior cruciate ligament (ACL) ex vivo.
Mechanical forces mimicking a subset of the mechanical stimuli
experienced by a native ACL in vivo (rotational deformation and
linear deformation) were applied in combination, and the resulting
ligament which was formed was studied to determine the effects of
the applied forces on tissue development. Exposure of the
developing ligament to physiological loading during in vitro
formation induced the cells to adopt a defined orientation along
the axes of load, and to generate extracellular matrices along the
axes as well. These results indicate that the incorporation of
complex multi-dimensional mechanical forces into the regime to
produce a more complex network of load axes that mimics the
environment of the native ACL, will produce a bioengineered
ligament which more closely resembles a native ACL.
[0071] The different mechanical forces that may be applied include,
without limitation, tension, compression, torsion, and shear. These
forces are applied in combinations which simulate forces
experienced by an ACL in the course of natural knee joint movements
and function. These movements include, without limitation, knee
joint extension and flexion as defined in the coronal and sagittal
planes, and knee joint flexion. Optimally, the combination of
forces applied mimics the mechanical stimuli experienced by an
anterior cruciate ligament in vivo as accurately as is
experimentally possible. Varying the specific regimen of force
application through the course of ligament generation is expected
to influence the rate and outcome of tissue development, with
optimal conditions to be determined empirically. Potential
variables in the regimen include, without limitation: (1) strain
rate, (2) percent strain, (3) type of strain, e.g. translation and
rotation, (4) frequency, (5) number of cycles within a given
regime, (6) number of different regimes, (7) duration at extreme
points of ligament deformation, (8) force levels, and (9) different
force combinations. It will be recognized by one of skill in the
art that a potentially unlimited number of variations exist. In a
preferred embodiment the regimen of mechanical forces applied
produces helically organized fibers similar to those of the native
ligament, described below.
[0072] The fiber bundles of a native ligament are arranged into a
helical organization. The mode of attachment and the need for the
knee joint to rotate .about.140.degree. of flexion has resulted in
the native ACL inheriting a 90.degree. twist and with the
peripheral fiber bundles developing a helical organization. This
unique biomechanical feature allows the ACL to sustain extremely
high loading. In the functional ACL, this helical organization of
fibers allows anterior-posterior and posterior-anterior fibers to
remain relatively isometric in respect to one another for all
degrees of flexion, thus load can be equally distributed to all
fiber bundles at any degree of knee joint flexion stabilizing the
knee throughout all ranges of joint motion. In a preferred
embodiment of the invention, mechanical forces that simulate a
combination of knee joint flexion and knee joint extension are
applied to the developing ligament to produce an engineered ACL
which possesses this same helical organization. The mechanical
apparatus used in the experiments presented in the Examples below
provides control over strain and strain rates (both translational
and rotational). The mechanical apparatus will monitor the actual
load experienced by the growing ligaments, serving to `teach` the
ligaments over time through monitoring and increasing the loading
regimes.
[0073] Another aspect of the present invention relates to the
bioengineered anterior cruciate ligament produced by the above
described methods. The bioengineered ligament produced by these
methods is characterized by cellular orientation and/or a matrix
crimp pattern in the direction of the mechanical forces applied
during generation. The ligament is also characterized by the
production/presence of extracellular matrix components (e.g.,
collagen type I, and type III, fibronectin, and tenascin-C
proteins) along the axis of mechanical load experienced during
culture. In a preferred embodiment, the ligament fiber bundles are
arranged into a helical organization, as discussed above.
[0074] The above methods using the novel silk-fiber-based matrix
are not limited to the production of an ACL, but can also be used
to produce other ligaments and tendons found in the knee (e.g.,
posterior cruciate ligament) or other parts of the body (e.g.,
hand, wrist, ankle, elbow, jaw and shoulder), such as for example,
but not limited to posterior cruciate ligament, rotator cuff
tendons, medial collateral ligament of the elbow and knee, flexor
tendons of the hand, lateral ligaments of the ankle and tendons and
ligaments of the jaw or temporomandibular joint. All moveable
joints in a human body have specialized ligaments which connect the
articular extremities of the bones in the joint. Each ligament in
the body has a specific structure and organization which is
dictated by its function and environment. The various ligaments of
the body, their locations and functions are listed in Anatomy,
Descriptive and Surgical (Gray, H., Eds. Pick, T. P., Howden, R.,
Bounty Books, New York (1977)), the pertinent contents of which are
incorporated herein by reference. By determining the physical
stimuli experienced by a given ligament or tendon, and
incorporating forces which mimic these stimuli, the above described
method for producing an ACL ex vivo can be adapted to produce
bioengineered ligaments and tendons ex vivo which simulates any
ligament or tendon in the body.
[0075] The specific type of ligament or tendon to be produced is
predetermined prior to tissue generation since several aspects of
the method vary with the specific conditions experienced in vivo by
the native ligament or tendon. The mechanical forces to which the
developing ligament or tendon is subjected during cell culture are
determined for the particular ligament or tendon type being
cultivated. The specific conditions can be determined by those
skilled in the art by studying the native ligament or tendon and
its environment and function. One or more mechanical forces
experienced by the ligament or tendon in vivo are applied to the
matrix during culture of the cells in the matrix. The skilled
practitioner will recognize that a ligament or tendon which is
superior to those currently available can be produced by the
application of a subset of forces experienced by the native
ligament or tendon. However, optimally, the full range of in vivo
forces will be applied to the matrix in the appropriate magnitudes
and combinations to produce a final product which most closely
resembles the native ligament or tendon. These forces include,
without limitation, the forces described above for the production
of an ACL. Because the mechanical forces applied vary with ligament
or tendon type, and the final size of the ligament or tendon will
be influenced by the anchors used, optimal anchor composition, size
and matrix attachment sites are to be determined for each type of
ligament or tendon by the skilled practitioner. The type of cells
seeded on the matrix is obviously determined based on the type of
ligament or tendon to be produced.
[0076] Another aspect of the present invention relates to the
production of other tissue types ex vivo using methods similar to
those described above for the generation of ligaments or tendons ex
vivo. The above described methods can also be applied to produce a
range of engineered tissue products which involve mechanical
deformation as a major part of their function, such as muscle
(e.g., smooth muscle, skeletal muscle, cardiac muscle), bone,
cartilage, vertebral discs, and some types of blood vessels. Bone
marrow stromal cells possess the ability to differentiate into
these as well as other tissues. The geometry of the silk-based
matrix or composite matrix can easily be adapted to the correct
anatomical geometrical configuration of the desired tissue type.
For example, silk fibroin fibers can be reformed in a cylindrical
tube to recreate arteries.
[0077] The results presented in the Examples below indicate that
growth in an environment which mimics the specific mechanical
environment of a given tissue type will induce the appropriate cell
differentiation to produce a bioengineered tissue which
significantly resembles native tissue. The ranges and types of
mechanical deformation of the matrix can be extended to produce a
wide range of tissue structural organization. Preferably, the cell
culture environment reflects the in vivo environment experienced by
the native tissue and the cells it contains, throughout the course
of embryonic development to mature function of the cells within the
native tissue, as accurately as possible. Factors to consider when
designing specific culture conditions to produce a given tissue
include, without limitation, the matrix composition, the method of
cell immobilization, the anchoring method of the matrix or tissue,
the specific forces applied, and the cell culture medium. The
specific regimen of mechanical stimulation depends upon the tissue
type to be produced, and is established by varying the application
of mechanical forces (e.g., tension only, torsion only, combination
of tension and torsion, with and without shear, etc.), the force
amplitude (e.g., angle or elongation), the frequency and duration
of the application, and the duration of the periods of stimulation
and rest.
[0078] The method for producing the specific tissue type ex vivo is
an adaptation of the above described method for producing an ACL.
Components involved include pluripotent cells, a three-dimensional
matrix to which cells can adhere, and a plurality of anchors which
have a face suitable for matrix attachment. The pluripotent cells
(preferably bone marrow stromal cells) are seeded in the three
dimensional matrix by means to uniformly immobilize the cells
within the matrix. The number of cells seeded is also not viewed as
limiting, however, seeding the matrix with a high density of cells
may accelerate tissue generation.
[0079] The specific forces applied are to be determined for each
tissue type produced through examination of native tissue and the
mechanical stimuli experienced in vivo. A given tissue type
experiences characteristic forces which are dictated by location
and function of the tissue within the body. For instance, cartilage
is known to experience a combination of shear and
compression/tension in vivo, bone experiences compression.
Determination of the specific mechanical stimuli experienced in
vivo by a given tissue is within the means of one of skill in the
art.
[0080] Additional stimuli (e.g., chemical stimuli, electro-magnetic
stimuli) can also be incorporated into the above described methods
for producing bioengineered ligaments, tendons and other tissues.
Cell differentiation is known to be influenced by chemical stimuli
from the environment, often produced by surrounding cells, such as
secreted growth or differentiation factors, cell-cell contact,
chemical gradients, and specific pH levels, to name a few. Other
more unique stimuli are experienced by more specialized types of
tissues (e.g., the electrical stimulation of cardiac muscle). The
application of such tissue specific stimuli (e.g., 1-10 ng/ml
transforming growth factor beta-1 (TGF-1) independently or in
concert with the appropriate mechanical forces is expected to
facilitate differentiation of the cells into a tissue which more
closely approximates the specific natural tissue.
[0081] Tissues produced by the above described methods provide an
unlimited pool of tissue equivalents for surgical implantation into
a compatible recipient, particularly for replacement or repair of
damaged tissue. Engineered tissues may also be utilized for in
vitro studies of normal or pathological tissue function, e.g., for
in vitro testing of cell- and tissue-level responses to molecular,
mechanical, or genetic manipulations. For example, tissues based on
normal or transfected cells can be used to assess tissue responses
to biochemical or mechanical stimuli, identify the functions of
specific genes or gene products that can be either over-expressed
or knocked-out, or to study the effects of pharmacological agents.
Such studies will likely provide more insight into ligament, tendon
and tissue development, normal and pathological function, and
eventually lead toward fully functional tissue engineered
replacements, based in part on already established tissue
engineering approaches, new insights into cell differentiation and
tissue development, and the use of mechanical regulatory signals in
conjunction with cell-derived and exogenous biochemical factors to
improve structural and functional tissue properties.
[0082] The production of engineered tissues such as ligaments and
tendons also has the potential for applications such as harvesting
bone marrow stromal cells from individuals at high risk for tissue
injury (e.g., ACL rupture) prior to injury. These cells could be
either stored until needed or seeded into the appropriate matrix
and cultured and differentiated in vitro under mechanical stimuli
to produce a variety of bioengineered prosthetic tissues to be held
in reserve until needed by the donor. The use of bioengineered
living tissue prosthetics that better match the biological
environment in vivo, provide the required physiological loading to
sustain for example, the dynamic equilibrium of a normal, fully
functional ligament, should reduce rehabilitation time for a
recipient of a prosthesis from months to weeks, particularly if the
tissue is pre-grown and stored. Benefits include a more rapid
regain of functional activity, shorter hospital stays, and fewer
problems with tissue rejections and failures.
[0083] It is to be understood that the present invention is not
intended to be limited to a silk-fiber-based matrix for producing
ACL, and other ligaments and tendons, as well as other tissues,
such as cartilage, bone, skin and blood vessels are contemplated by
the present invention by utilizing the novel silk-fiber based
matrix seeded with the appropriate cells and exposed to the
appropriate mechanical stimulation if necessary, for proliferating
and differentiating into the desired ligament, tendon or
tissue.
[0084] Additionally, the present invention is not limited to using
bone marrow stromal cells for seeding on the matrix, and other
progenitor, pluripotent and stem cells, such as those in bone,
muscle and skin for example, may also be used to differentiate into
ligaments and other tissues.
[0085] The invention is further defined by reference to the
following examples. It will be apparent to those skilled in the art
that many modifications, both to the materials and methods, may be
practiced without departing from the purpose and interest of the
invention.
EXAMPLES
Example 1
a. Preparation of Silk Films
[0086] Raw Bombyx mori silkworm fibers, shown in FIG. 1A, were
extracted to remove sericin, the glue-like protein coating the
native silk fibroin (see FIG. 1A-C). The appropriate number of
fibers per group were arranged in parallel and extracted in an
aqueous solution of 0.02M Na.sub.2CO.sub.3 and 0.3% (w/v) Ivory
soap solution for 60 min at 90 C, then rinsed thoroughly with water
to extract the glue-like sericin proteins.
b. Preparation of and Properties of the Silk-Fiber Based Matrix
Construct
[0087] Costello's equation for a three-strand wire rope was derived
to predict mechanical properties of the silk-fiber-based matrix.
The derived model is a series of equations that when combined, take
into account extracted silk fiber material properties and desired
matrix geometrical hierarchy to compute the overall strength and
stiffness of the matrix as a function of pitch angle for a given
level of geometrical hierarchy.
[0088] The material properties of a single silk fiber include fiber
diameter, modulus of elasticity, Poisson's ratio, and the ultimate
tensile strength (UTS). Geometrical hierarchy may be defined as the
number of twisting levels in a given matrix level. Each level
(e.g., group, bundle, strand, cord, ligament) is further defined by
the number of groups of fibers twisted about each other and the
number of fibers in each group of the first level twisted where the
first level is define as a group, the second level as a bundle, the
third as a strand and the fourth as a cord, the fifth as the
ligament).
[0089] The model assumes that each group of multiple fibers act as
a single fiber with an effective radius determined by the number of
individual fibers and their inherent radius, i.e., the model
discounts friction between the individual fibers due to its limited
role in given a relatively high pitch angle.
[0090] Two applicable geometries (Matrix 1 and Matrix 2) of the
many matrix geometrical configurations (see Table 2, supra)
computed to yield mechanical properties mimicking those of a native
ACL were derived for more detailed analysis. A six-cord construct
was selected for use as the ACL replacement. Matrix configurations
are as follows: Matrix 1:1 ACL prosthesis=6 parallel cords; 1
cord=3 twisted strands (3 twists/cm); 1 strand=6 twisted bundles (3
twists/cm); 1 bundle=30 parallel washed fibers; and Matrix 2: 1 ACL
matrix=6 parallel cords; 1 cord=3 twisted strands (2 twists/cm); 1
strand=3 twisted bundles (2.5 twists/cm); 1 bundle=3 groups (3
twists/cm); 1 group=15 parallel extracted silk fibroin fibers. The
number of fibers and geometries were selected such that the silk
prostheses are similar to the ACL biomechanical properties in UTS,
linear stiffness, yield point and % elongation at break (see Table
2, supra), thus serving as a solid starting point for the
development of a tissue engineered ACL.
[0091] Mechanical properties of the silk fibroin were characterized
using a servohydraulic Instron 8511 tension/compression system with
Fast-Track software (see FIG. 1D). Single pull-to-failure and
fatigue analysis were performed on single silk fibers, extracted
fibroin and organized cords. Fibers and fibroin were organized in
both parallel and wire-rope geometries (Matrix 1 (see FIG. 2A) and
Matrix 2 (see FIG. 2B)) for characterization. Single pull to
failure testing was performed at a strain rate of 100%/sec; force
elongation histograms were generated and data analyzed using
Instron Series IX software. Both Matrix 1 and Matrix 2 yielded
similar mechanical and fatigue properties to the ACL in UTS, linear
stiffness, yield point and % elongation at break (see Table 2 and
FIG. 3).
[0092] Fatigue analyses were performed using a servohydraulic
Instron 8511 tension/compression system with Wavemaker software on
single cords of both Matrix 1 and Matrix 2. Data was extrapolated
to represent the 6-cord ACL prostheses, which is shown in FIGS. 3B
and 3D. Cord ends were embedded in an epoxy mold to generate a 3 cm
long construct between anchors. Cycles to failure at UTS, 1,680N
and 1,200N (N=5 for each load) for Matrix 1 (see FIG. 3B) and at
UTS, 2280N, 2100N and 1800N loads (n=3 for each load) for Matrix 2
(see FIG. 3D) were determined using a H-sine wave function at 1 Hz
generated by Wavemaker 32 version 6.6 (Instron, Canton, Mass.).
Fatigue testing was conducted in a neutral phosphate buffered
saline (PBS) solution at room temperature.
Results
[0093] Complete sericin removal was observed after 60 min at
90.degree. C. as determined by SEM (see FIGS. 1A-C). Removal of
sericin from silk fibers altered the ultrastructure of the fibers,
resulting in a smoother fiber surface and the underlying silk
fibroin was revealed (shown in FIGS. 1A-C), with average diameter
ranging between 20-40 m. The fibroin exhibited a significant 15.2%
decrease in ultimate tensile strength (1.033+/-0.042 N/fiber to
0.876+/-0.1 N/fiber) (p<0.05, paired Students t-test) (see FIG.
1D). The mechanical properties of the optimized silk matrices (see
FIG. 2) are summarized in Table 1 above and in FIG. 3A (for Matrix
1) and in FIG. 3C (for Matrix 2). It is evident from these results
that the optimized silk matrices exhibited values comparable to
those of native ACL, which have been reported to have an average
ultimate tensile strength (UTS) of 2100 N, stiffness of 250 N/nm,
yield point 2100 N and 33% elongation at break (See Woo, SL-Y, et
al., The Tensile Properties of Human Anterior Cruciate Ligament
(ACL) and ACL Graft Tissue in Knee Ligaments: Structure, Function,
Injury and Repair, 279-289, Ed. D. Daniel et al., Raven Press
1990).
[0094] Regression analysis of matrix fatigue data, shown in FIG. 3B
for Matrix 1 and in FIG. 3D for Matrix 2, when extrapolated to
physiological load levels (400 N) predict the number of cycles to
failure in vivo, indicate a matrix life of 3.3 million cycles for
Matrix 1 and a life of >10 million cycles for Matrix 2. The wire
rope matrix design utilizing washed silk fibers resulted in a
matrix with physiologically equivalent structural properties,
confirming its suitability as a scaffold for ligament tissue
engineering.
Example 2
Cell Isolation and Culture
[0095] Bone Marrow Stromal Cells (BMSC), pluripotent cells capable
of differentiating into osteogenic, chondrogenic, tendonogenic,
adipogenic and myogenic lineages, were chosen since the formation
of the appropriate conditions can direct their differentiation into
the desired ligament fibroblast cell line (Markolf et al., J. Bone
Joint Surg. 71A: 887-893 (1989); Caplan et al., Mesenchymal stem
cells and tissue repair. In The Anterior Cruciate Ligament: Current
and Future Concepts, Ed. D. W. Jackson et al., Raven Press, Ltd,
New York (1993); Young et al., J. Orthopaedic Res. 16: 406-413
(1998)).
[0096] Human BMSCs were isolated from bone marrow from the iliac
crest of consenting donors .ltoreq.25 years of age by a commercial
vendor (Cambrex, Walkersville, Md.). Twenty-two milliliters of
human marrow was aseptically aspirated into a 25 ml syringe
containing three milliliters of heparinized (1000 units per
milliliter) saline solution. The heparinized marrow solution was
shipped overnight on ice to the laboratory for bone marrow stromal
cells isolation and culture. Upon arrival from the vendor, the
twenty-five milliliter aspirates were resuspended in Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 0.1 mM nonessential amino acids, 100 U/ml penicillin,
100 mg/L streptomycin (P/S), and 1 ng/ml basic fibroblast growth
factor (bFGF) (Life Technologies, Rockville, Md.) and plated at
8-10 microliters of aspirate/cm.sup.2 in tissue culture flasks.
Fresh medium was added to the marrow aspirates twice a week for up
to nine days of culture. BMSCs were selected based on their ability
to adhere to the tissue culture plastic; non-adherent hematopoietic
cells were removed during medium replacement after 9-12 days in
culture. Medium was changed twice per week thereafter. When primary
BMSC became near confluent (12-14 days), they were detached using
0.25% trypsin/1 mM EDTA and replated at 5.times.10.sup.3
cells/cm.sup.2. First passage (P1) hBMSCs were trypsinized and
frozen in 8% DMSO/10% FBS/DMEM for future use.
[0097] Silk Matrix Cell Seeding
[0098] Frozen P1 hBMSCs were defrosted, replated at
5.times.10.sup.3 cells/cm.sup.2 (P2), trypsinized when near
confluency, and used for matrix seeding. Sterilized (ethylene
oxide) silk matrices (specifically single cords of Matrix 1&2,
bundles of 30 parallel extracted silk fibers and wire-ropes of
collage fibers) were seeded with cells in customized seeding
chambers (1 ml total volume) machined in Teflon blocks to minimize
cell-medium volume and increase cell-matrix contact. Seeded
matrices, following a 4 hour incubation period with the cell slurry
(3.3.times.10.sup.6 BMSCs/ml) were transferred into a petri dish
contain an appropriate amount of cell culture medium for the
duration of the experiments.
[0099] To determine the degradation rate of the silk fibroin,
ultimate tensile strength (UTS) was measured as a function of
cultivation period in physiological growth conditions, i.e., in
cell culture medium. Groups of 30 parallel silk fibers 3 cm in
length were extracted, seeded with hBMSCs, and cultured on the
fibroin over 21 days at 37.degree. C. and 5% CO.sub.2. Non-seeded
control groups were cultured in parallel. Silk fibroin UTS was
determined as a function of culture duration for seeded and
non-seeded groups.
[0100] Results
[0101] The response of bone marrow stromal cells to the silk matrix
was also examined.
[0102] BMSCs readily attached and grew on the silk and collagen
matrices after 1 day in culture (See FIGS. 4A-C and FIG. 10A), and
formed cellular extensions to bridge neighboring fibers. As shown
in FIG. 4D and FIG. 10B, a uniform cells sheet covering the
construct was observed at 14 and 21 days of culture, respectively.
MTT analysis confirmed complete matrix coverage by seeded BMSCs
after 14 days in culture (see FIG. 5). Total DNA quantification of
cells grown on Matrix 1 (see FIG. 6A) and Matrix 2 (see FIG. 6B)
confirmed that BMSCs proliferated and grew on the silk construct
with the highest amount of DNA measured after 21 and 14 days,
respectively, in culture.
[0103] Both BMSC seeded or non-seeded extracted control silk
fibroin groups of 30 fibers, maintained their mechanical integrity
as a function of culture period over 21 days (see FIG. 7).
[0104] RT-PCR analysis of BMSCs seeded on cords of Matrix 2
indicated that both collagen I & III were upregulated over 14
days in culture (FIG. 8). Collagen type II and bone sialoprotein
(as indicators of cartilage and bone specific differentiation,
respectively) were either not detectable or negligibly expressed
over the cultivation period. Real-time quantitative RT-PCR at 14
days yielded a transcript ratio of collagen I to collagen III,
normalized to GAPDH, of 8.9:1 (see FIG. 11). The high ratio of
collagen I to collagen III indicates that the response is not wound
healing or scar tissue formation (as is observed with high levels
of collagen type III), but rather ligament specific; the relative
ratio of collagen I to collagen III in a native ACL is .about.6.6:1
(Amiel et al., In Knee Ligaments: Structure, Function, Injury, and
Repair. 1990).
Example 3
[0105] Studies are conducted to provide insight into the influence
of directed multi-dimensional mechanical stimulation on ligament
formation from bone marrow stromal cells in the bioreactor system.
The bioreactor is capable of applying independent but concurrent
cyclic multi-dimensional strains (e.g., translation, rotation) to
the developing ligaments. After a 7 to 14 day static rest period
(time post seeding), the rotational and translation strain rates
and linear and rotational deformation are kept constant for 1 to 4
weeks. Translational strain (3.3%-10%, 1-3 mm) and rotational
strain (25%, 90.degree.) are concurrently applied at a frequency of
0.0167 Hz (one full cycle of stress and relaxation per minute) to
the silk-based matrices seeded with BMSCs; an otherwise identical
set of bioreactors with seeded matrices without mechanical loading
serve as controls. The ligaments are exposed to the constant cyclic
strains for the duration of the experiment days.
[0106] Following the culture period, ligament samples, both the
mechanically challenged as well as the controls (static) are
characterized for: (1) general histomorphological appearance (by
visual inspection); (2) cell distribution (image processing of
histological and MTT stained sections); (3) cell morphology and
orientation (histological analysis); and (4) the production of
tissue specific markers (RT-PCR, immunostaining).
[0107] Mechanical stimulation markedly affects the morphology and
organization of the BMSCs and newly developed extracellular matrix,
the distribution of cells along the matrix, and the upregulation of
a ligament-specific differentiation cascade; BMSCs align along the
long axis of the fiber, take on a spheroid morphology similar to
ligament/tendon fibroblasts and upregulate ligament/tendon specific
markers. Newly formed extracellular matrix is expected to align
along the lines of load as well as the long axis of the matrix.
Directed mechanical stimulation is expected to enhance ligament
development and formation in vitro in a bioreactor resulting from
BMSCs seeded on the novel silk-based matrix. The longitudinal
orientation of cells and newly formed matrix is similar to ligament
fibroblasts found within an ACL in vivo (Woods et al. Amer. J.
Sports Med. 19: 48-55 (1991)). Furthermore, mechanical stimulation
maintains the correct expression ratio between collagen I
transcripts and collagen type III transcripts (e.g., >7:1)
indicating the presence of newly formed ligament tissue versus scar
tissue formation. The above results will indicate that the
mechanical apparatus and bioreactor system provide a suitable
environment (e.g., multi-dimensional strains) for in vitro
formation of tissue engineered ligaments starting from bone marrow
stromal cells and the novel silk-based matrix.
[0108] The culture conditions used in these preliminary experiments
can be further expanded to more accurately reflect the
physiological environment of a ligament (e.g. increasing the
different types of mechanical forces) for the in vitro creation of
functional equivalents of native ACL for potential clinical use.
These methods are not limited to the generation of a bioengineered
ACL. By applying the appropriate magnitude and variety of forces
experienced in vivo, any type of ligament in the body can be
produced ex vivo by the methods of the present invention.
* * * * *