U.S. patent application number 10/602789 was filed with the patent office on 2008-08-21 for system and method for forming a connective tissue construct.
This patent application is currently assigned to The Regents of The University Of Michigan. Invention is credited to Ellen M. Arruda, Sarah C. Calve, Robert G. Dennis, Paul E. Kosnik.
Application Number | 20080199953 10/602789 |
Document ID | / |
Family ID | 39707018 |
Filed Date | 2008-08-21 |
United States Patent
Application |
20080199953 |
Kind Code |
A1 |
Kosnik; Paul E. ; et
al. |
August 21, 2008 |
SYSTEM AND METHOD FOR FORMING A CONNECTIVE TISSUE CONSTRUCT
Abstract
A system and method are provided for forming a connective tissue
construct, such as a tendon construct, in vitro. A substrate is
provided with at least two anchors secured thereto in spaced
relationship. Fibroblast cells are provided on the substrate in the
absence of a synthetic matrix, where at least some of the cells are
in contact with the anchors. The cells are cultured in vitro under
conditions to allow the cells to self-organize and become confluent
between the anchors, where the anchors are receptive to the cells
and allow the cells to attach thereto while permitting the cells to
detach from the substrate to form a three-dimensional connective
tissue construct.
Inventors: |
Kosnik; Paul E.; (Bay City,
MI) ; Dennis; Robert G.; (Ann Arbor, MI) ;
Calve; Sarah C.; (Darien, CT) ; Arruda; Ellen M.;
(Ann Arbor, MI) |
Correspondence
Address: |
BROOKS KUSHMAN P.C.
1000 TOWN CENTER, TWENTY-SECOND FLOOR
SOUTHFIELD
MI
48075
US
|
Assignee: |
The Regents of The University Of
Michigan
Ann Arbor
MI
|
Family ID: |
39707018 |
Appl. No.: |
10/602789 |
Filed: |
June 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09709890 |
Nov 9, 2000 |
6777234 |
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10602789 |
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09153721 |
Sep 15, 1998 |
6207451 |
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09709890 |
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Current U.S.
Class: |
435/325 ;
435/289.1; 435/366 |
Current CPC
Class: |
A61L 27/3895 20130101;
C12N 2533/52 20130101; A61L 27/3804 20130101; A61L 27/386 20130101;
C12N 2533/56 20130101; C12N 5/066 20130101; C12N 2533/18 20130101;
A61L 27/3604 20130101; C12N 2500/38 20130101; A61K 35/12
20130101 |
Class at
Publication: |
435/325 ;
435/366; 435/289.1 |
International
Class: |
C12N 5/06 20060101
C12N005/06; C12N 5/08 20060101 C12N005/08; C12M 1/00 20060101
C12M001/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The invention was made with Government support under
Contract No. N66001-02-C-8034 from DARPA (Contracting Agent:
SPAWAR) and under Contract No. CMS 9988693 from the National
Science Foundation (CMS Division).
Claims
1-18. (canceled)
19. A method for forming a connective tissue construct, comprising:
providing a substrate; securing at least two anchors to the
substrate in spaced relationship; providing fibroblast cells in a
growth medium on the substrate without disposing the cells within
an exogenous scaffold material, wherein at least some of the cells
are in contact with the anchors and attach thereto such that the
fibroblast cells grow to confluency; and replacing the growth
medium with a differentiation medium to induce construct formation,
the differentiation medium having a lower serum concentration than
the growth medium, such that culturing the fibroblast cells in
vitro causes the fibroblast cells to detach from the substrate and
form a three-dimensional connective tissue construct.
20. The method according to claim 19, wherein providing fibroblast
cells includes deriving the fibroblast cells from tendon
tissue.
21. (canceled)
22. The method according to claim 19, wherein providing the
fibroblast cells includes deriving the fibroblast cells from stem
cells.
23. The method according to claim 19, wherein culturing the
fibroblast cells allows the cells to self-organize to form the
three-dimensional connective tissue construct.
24. The method according to claim 19, wherein the anchors include
silk suture segments coated with cell adhesion molecules.
25. The method according to claim 24, wherein the cell adhesion
molecules include laminin.
26. The method according to claim 19, wherein the anchors include
at least one of hydroxyapatite and calcium phosphate.
27. The method according to claim 19, further comprising coating
the substrate with cell adhesion molecules.
28. The method according to claim 27, wherein the cell adhesion
molecules include laminin.
29. The method according to claim 28, wherein the concentration of
laminin is about 1.5 to 3.0 .mu.g/cm.sup.2.
30. The method according to claim 27, wherein the cell adhesion
molecules include thrombin.
31. The method according to claim 19, further comprising incubating
the substrate and anchors with a growth medium prior to providing
fibroblast cells on the substrate.
32. (canceled)
33. The method according to claim 19, further comprising
supplementing the fibroblast cells with ascorbic acid.
34. The method according to claim 33, wherein the ascorbic acid
includes approximately 100 .mu.g/ml of L-ascorbic acid
2-phosphate.
35. The method according to claim 19, further comprising measuring
a functional property of the connective tissue construct and using
the measured property as feedback to control the formation of the
connective tissue construct.
36. The method according to claim 35, wherein the functional
property includes a tensile strength of the connective tissue
construct.
37. The method according to claim 19, further comprising culturing
myogenic precursor cells in combination with the fibroblast
cells.
38. The method according to claim 19, further comprising harvesting
the fibroblast cells from mammalian tissue.
39. The method according to claim 19, further including implanting
the connective tissue construct in a suitable recipient.
40. A method for forming a tendon construct, comprising: providing
a substrate; securing at least two anchors to the substrate in
spaced relationship; providing a growth medium including fibroblast
cells and ascorbic acid on the substrate without disposing the
cells within an exogenous scaffold material, wherein at least some
of the cells are in contact with the anchors and attach thereto
such that the fibroblast cells grow to confluency; and replacing
the growth medium with a differentiation medium to induce construct
formation, the differentiation medium having a lower serum
concentration than the growth medium, such that culturing the
fibroblast cells in vitro causes the fibroblast cells to detach
from the substrate and form a three-dimensional tendon
construct.
41. The method according to claim 40, wherein culturing the
fibroblast cells allows the cells to self-organize to form the
three-dimensional tendon construct.
42. The method according to claim 40, wherein the anchors include
silk suture segments coated with cell adhesion molecules.
43. The method according to claim 42, wherein the cell adhesion
molecules include laminin.
44. The method according to claim 40, wherein the anchors include
at least one of hydroxyapatite and calcium phosphate.
45. The method according to claim 40, further comprising coating
the substrate with cell adhesion molecules.
46. The method according to claim 45, wherein the cell adhesion
molecules include laminin.
47. The method according to claim 46, wherein the concentration of
laminin is about 1.5 to 3.0 .mu.g/cm.sup.2.
48. The method according to claim 45, wherein the cell adhesion
molecules include thrombin.
49. The method according to claim 40, further comprising incubating
the substrate and anchors with a growth medium prior to providing
fibroblast cells on the substrate.
50. The method according to claim 40, wherein the ascorbic acid
includes approximately 100 .mu.g/ml of L-ascorbic acid
2-phosphate.
51. The method according to claim 40, further comprising measuring
a functional property of the connective tissue construct and using
the measured property as feedback to control the formation of the
tendon construct.
52. The method according to claim 51, wherein the functional
property includes a tensile strength of the tendon construct.
53. The method according to claim 40, further comprising culturing
myogenic precursor cells in combination with the fibroblast
cells.
54. The method according to claim 40, further comprising harvesting
the fibroblast cells from mammalian tissue.
55. The method according to claim 40, further including implanting
the tendon construct in a suitable recipient.
56. The method according to claim 40, wherein replacing the growth
medium with the differentiation medium occurs after about 5
days.
57. The method according to claim 19, wherein replacing the growth
medium with the differentiation medium occurs after about 5 days.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/709,890 filed Nov. 9, 2000, which is a
divisional of U.S. application Ser. No. 09/153,721 filed Sept. 15,
1998, now U.S. Pat. No. 6,207,451.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates to the field of tissue engineering,
and more particularly to a system and method for producing a
connective tissue construct, such as a tendon construct, in
vitro.
[0005] 2. Background Art
[0006] There are approximately 33 million musculoskeletal injuries
each year in the United States. The associated soft tissues, which
include tendons, comprise almost 50% of these injuries. In some
cases, the tendon is damaged beyond repair, and partial or whole
replacement of the tendon is necessary. The ideal replacement would
be autologous tendon, but transplantation is limited by the
availability of viable autograft tissue. As a result, clinical
practice has turned to the use of synthetic materials (see
Goldstein et al., Journal of Bone and Joint Surgery-American
Volume, 71A, p. 1183, 1989), where current synthetic replacements
include DACRON.RTM. grafts, carbon fibers, and silastic sheets.
Unfortunately, these materials are unable to adequately restore
function for the long term due to their inherent mechanical
incompatibility with the in vivo environment as well as their
tendency to degrade (see lannace et al., Biomaterials, 16, p. 675,
1995).
[0007] Tendons are densely packed connective tissues that transmit
the forces between muscle and bone. They are stiff in tension, yet
flexible enough to conform to their anatomical environment. The
material properties of tendon tissue can be attributed to the
parallel fibrils of collagen which make up approximately 75% of the
dry weight of adult tendons. In the resting state, the fibrils
display a periodic wavy pattern, defined as the crimp. As a tendon
is stretched, the crimped collagen fibrils begin to straighten out
and may cause the tendon to become stiffer with increasing
mechanical strain. Tendons have a low cell density, around 20% of
the tissue volume, but fibroblasts are integral in the development
and maintenance of the tissue. The distinct spatial orientation of
tendon fibroblasts is associated with the organization of collagen
fibers into the hierarchical tendon structure.
[0008] Because of its relatively avascular nature, tendon is a
prime candidate for engineered tissue replacement. Previous
attempts have been made to create biologically based tendons in
vitro, but these have met with limited success due to the
difficulty in creating an in vitro tissue, or "construct", that is
both mechanically and biologically compatible with the in vivo
environment (see Butler and Awad, Clinical Orthopaedics and Related
Research, 367, p. S324, 1999; Goldstein et al., Journal of Bone and
Joint Surgery-American Volume, 71A, p. 1183, 1989; Torres et al.,
Biomaterials, 21, p. 1607, 2000; Cao et al., Plastic and
Reconstructive Surgery, 110, p. 1280, 2002; Koob and Hernandez,
Biomaterials, 23; p. 203, 2002).
[0009] Mechanical difficulties can arise from the reliance on
artificial scaffolds when attempting to engineer tendon. Type I
collagen is the most widely used scaffold material since it was
observed that fibroblasts will contract a collagen gel to form a
tissue-like structure (see Bell et al., Proceedings of the National
Academy of Sciences of the United States of America, 76, p. 1274,
1979). Collagen would appear to be the ideal foundation for an
artificial tendon, but presently the mechanical properties of in
vitro fibroblast-collagen constructs are inferior to those of
native tissues (see Huang et al., Annals of Biomedical Engineering,
21, p. 289, 1993; Wakatsuki et al., Biophysical Journal, 79, p.
2353, 2000; Seliktar et al., Annals of Biomedical Engineering, 28,
p. 351, 2000; Brown et al., Journal of Cellular Physiology, 175, p.
323, 1998; Cacou et al., Medical Engineering & Physics, 22, p.
327, 2000).
[0010] An explanation for this discrepancy is that gelled collagen
is generally disorganized and only forms fibrils of physiological
thickness under stringent conditions (see Holmes et al., Journal of
Biological Macromolecules, 8, p. 161, 1986). Furthermore, native
tendons possess an extracellular matrix (ECM) composed of many
proteins, glycosaminoglycans, and proteoglycans which control the
assembly of the collagen fibril, the load bearing unit, and
contribute to the formation of the tissue hierarchy. Fibroblasts
rely on cell-matrix signaling pathways during development to
properly assemble the fibrils and maintain form and function after
maturation. Koob and Hernandez created a mechanically relevant
construct by cross-linking extruded collagen fibers with NGDA, a
plant derived anti-oxidant, for which only ultimate strengths were
reported and not the entire elastic response (see Koob and
Hernandez, Biomaterials, 23; p. 203, 2002). Goldstein et al. used
the same idea of creating a fiber composite to create artificial
prostheses, but relied upon cross-linking methods that were
cytotoxic and/or non-biodegradable (see Goldstein et al., Journal
of Bone and Joint Surgery-American Volume, 71A, p. 1183, 1989).
[0011] Accordingly, a need exists for a tendon construct that
incorporates as many of the native properties of tendon as possible
in order to sufficiently restore function.
SUMMARY OF THE INVENTION
[0012] It is an object according to the present invention to
provide a system and method for producing a connective tissue
construct in vitro.
[0013] It is another object according to the present invention to
provide a system and method for inducing tendon fibroblasts to
self-assemble into a three-dimensional tendon construct.
[0014] It is another object according to the present invention to
provide a system and method for producing a tendon construct that
self-organizes without the need for exogeneous scaffolding.
[0015] Accordingly, a system for forming a connective tissue
construct is provided which includes a substrate and at least two
anchors secured to the substrate in spaced relationship. Fibroblast
cells are provided on the substrate in the absence of a synthetic
matrix, where at least some of the cells are in contact with the
anchors. The cells are cultured in vitro under conditions to allow
the cells to become confluent between the anchors, where the
anchors are receptive to the cells and allow the cells to attach
thereto while permitting the cells to detach from the substrate to
form a three-dimensional connective tissue construct.
[0016] According to the present invention, the fibroblast cells can
be derived from tendon tissue, or alternatively could be derived
from ligament tissue or other connective tissue. The fibroblast
cells can also be derived from stem cells. In another embodiment,
myogenic precursor cells can be cultured in combination with the
fibroblast cells. The fibroblast cells self-organize to form the
three-dimensional tissue construct.
[0017] The anchors preferably include silk suture segments coated
with cell adhesion molecules, where the cell adhesion molecules can
include laminin. The anchors can also include a bone-like
substrate. The substrate is coated with cell adhesion molecules,
such as laminin. In a preferred embodiment, the concentration of
laminin is about 1.5 to 3.0 .mu.g/cm.sup.2. In addition, the cell
adhesion molecules can include thrombin. Preferably, the substrate
and anchors are incubated with a growth medium prior to providing
fibroblast cells on the substrate.
[0018] According to the present invention, the fibroblast cells are
preferably disposed in a growth medium prior to becoming confluent,
and are disposed in a differentiation medium after becoming
confluent. The fibroblast cells are also preferably supplemented
with ascorbic acid, most preferably approximately 100 .mu.g/ml of
L-ascorbic acid 2-phosphate.
[0019] In further accordance with the present invention, a system
for forming a tendon construct is provided. The system includes a
substrate and at least two anchors secured to the substrate in
spaced relationship. The system further includes a medium including
fibroblast cells and ascorbic acid provided on the substrate, where
at least some of the cells are in contact with the anchors. The
cells are cultured in vitro under conditions to allow the cells to
self-organize and become confluent between the anchors, and the
anchors are receptive to the cells and allow the cells to attach
thereto while permitting the cells to detach from the substrate to
form a three-dimensional tendon construct.
[0020] Correspondingly, a method for forming a connective tissue
construct is provided. The method includes providing a substrate
and securing at least two anchors to the substrate in spaced
relationship. The method further includes providing fibroblast
cells on the substrate in the absence of a synthetic matrix, where
at least some of the cells are in contact with the anchors. Still
further, the method includes culturing the fibroblast cells in
vitro under conditions to allow the cells to become confluent
between the anchors, where the anchors are receptive to the cells
and allow the cells to attach thereto while permitting the cells to
detach from the substrate and form a three-dimensional connective
tissue construct.
[0021] According to the method of the present invention, providing
fibroblast cells includes deriving the fibroblast cells from tendon
tissue, or alternatively from ligament tissue, other connective
tissue, or stem cells. The fibroblast cells can be harvested from
mammalian tissue, and the resulting construct can be implanted in a
suitable donor. The fibroblast cells can also be cultured in
combination with myogenic precursor cells. Culturing the fibroblast
cells allows for self-organization of the into the
three-dimensional connective tissue construct.
[0022] The anchors preferably include silk suture segments coated
with cell adhesion molecules, such as laminin, and the anchors can
also include a bone-like substrate. The method of the present
invention further includes coating the substrate with cell adhesion
molecules, such as laminin or thrombin. For laminin, the preferred
concentration is about 1.5 to 3.0 .mu.g/cm.sup.2. The method
preferably further includes incubating the substrate and anchors
with a growth medium prior to providing fibroblast cells on the
substrate.
[0023] Still further, the method according to the present invention
includes disposing the fibroblast cells in a growth medium prior to
becoming confluent, and disposing the fibroblast cells in a
differentiation medium after becoming confluent. Preferably, the
method also includes supplementing the fibroblast cells with
ascorbic acid, most preferably approximately 100 .mu.g/ml of
L-ascorbic acid 2-phosphate. The method can further include
measuring a functional property of the connective tissue construct,
such as tensile strength, and using the measured property as
feedback to control the formation of the connective tissue
construct.
[0024] In further accordance with the present invention, a method
is provided for forming a tendon construct, including providing a
substrate and securing at least two anchors to the substrate in
spaced relationship. The method further includes providing a medium
including fibroblast cells and ascorbic acid on the substrate,
where at least some of the cells are in contact with the anchors.
Still further, the method includes culturing the fibroblast cells
in vitro under conditions to allow the cells to self-organize and
become confluent between the anchors, where the anchors are
receptive to the cells and allow the cells to attach thereto while
permitting the cells to detach from the substrate and form a
three-dimensional tendon construct.
[0025] The above objects and other objects, features, and
advantages of the present invention are readily apparent from the
following detailed description of the best mode for carrying out
the invention when taken in connection with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-1D show a tendon construct according to the present
invention in the process of forming, wherein the construct is shown
at (A) 9 days, (B) 12 days, (C) 15 days, and (D) 3 months after
plating of fibroblast cells;
[0027] FIGS. 2A and 2B are light micrographs of longitudinal
sections of a tendon construct according to the present invention
and neonatal tendon, respectively;
[0028] FIGS. 3A and 3B are electron micrographs of longitudinal
sections of a tendon construct according to the present invention
and neonatal tendon, respectively; and
[0029] FIG. 4 is a graph of the stress-strain response of tendon
constructs according to the present invention compared with the
stress-strain response of embryonic tendon.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
[0030] The present invention provides a three-dimensional
connective tissue construct and a system and method for producing
the construct from primary cell culture. A method for the culture
of primary skeletal muscle myogenic precursor cells is disclosed in
commonly assigned U.S. Pat. No. 6,207,451 which is incorporated by
reference herein. According to the present invention, this method
has been modified to promote the self-organization of fibroblast
cells in vitro to form connective tissue, in particular tendon
tissue.
[0031] As described herein, precursor cells are defined as any cell
which can be used to develop a particular tissue of interest. In
the case of tendons, precursor cells include, but are not limited
to, fibroblasts. In addition, stem cells, such as bone marrow
cells, can be induced to differentiate into tendon precursors as is
known in the art.
[0032] By way of example, the connective tissue construct and
method for producing the construct of the present invention are
described with reference to the use of tendon tissue originating
from rats. However, the construct and method of the present
invention are not intended to be limited to one particular cell
origin or age, construct shape, time frame, component
concentration, or culture condition. For example, it is fully
contemplated that tendon tissue from any mammal, including human
beings, could be similarly utilized according to the method
described herein. Furthermore, it is contemplated that fibroblast
cells could be obtained from body tissues other than tendon, such
as ligaments, lung tissue, skin, corneal tissue, or others. One
skilled in the art can readily appreciate that various
modifications can be made to the method described herein without
departing from the scope of the invention disclosed.
[0033] As is apparent to those skilled in the art, the culture of
cells as described below must be carried out in accordance with
commonly practiced cell culture techniques. For example, all
materials and media which will be placed in contact with living
cells must be appropriately sterilized and handled. In addition,
the cells and tendon constructs must be maintained in an otherwise
aseptic environment. Of course, it is understood that all reagent
measurements and submersion times described herein are approximate,
and can be varied slightly without affecting the resulting
method.
[0034] In the description and data that follow, primary rat tendon
fibroblasts were obtained from Achilles tendons harvested from
Fischer 344 retired breeder rats (Charles River Laboratories, MA).
According to the method of the present invention, Achilles tendon
cells are dissociated by placement in a 0.25% trypsin-EDTA solution
containing 200 units/ml type I collagenase. The solution is placed
in a reciprocal shaking bath at 37.degree. C. for approximately 6
hours to facilitate breakdown of the ECM. After the tissue is
dissociated, the cells are pelleted by centrifugation at 100 g for
approximately 15 minutes and the supernatant is then removed by
aspiration. The cells are resuspended with growth medium (400 ml
Ham F-12, 100 ml fetal bovine serum (FBS), 100 units/ml
antibiotic-antimycotic) and then expanded in tissue culture flasks.
Cells are passaged at .about.60% confluence and stored in liquid
nitrogen until needed.
[0035] An amenable substrate on which to form the tendon constructs
is preferably created by coating 35 mm culture dishes with
approximately 1.5 ml SYLGARD.RTM. (Dow Chemical Corp., Midland,
Minn., type 184 silicone elastomer). Of course, another elastomeric
polymer having similar non-porous, hydrophobic properties could
also be used. After curing for .about.2 weeks, the dishes are
rinsed with Dulbecco's Phosphate-Buffered Saline (DPBS) or another
suitable balanced salt solution, and approximately 2.0
.mu.g/cm.sup.2 natural mouse laminin is applied as a solution of
9.6 mg/ml laminin in DPBS. The laminin concentration on the
substrate is used to control both the rate of cell growth and the
time of cell monolayer delamination for the formation of the tendon
construct of the present invention. In particular, the laminin
disappears from the substrate within a couple of weeks, which
facilitates the detachment process to allow tendon construct
formation. The optimal value for use in the present invention
appear to be in the range of about 1.5 .mu.g/cm.sup.2 to 3.0
.mu.g/cm.sup.2. Of course, another cell adhesion protein could also
be used in accordance with the present invention. For example,
thrombin may also be used to stimulate contractility and therefore
facilitate detachment of the cell layer from the substrate. The
DPBS is allowed to evaporate overnight in a biological safety
cabinet, leaving a layer of laminin-coated SYLGARD.RTM.. Dishes are
subsequently rinsed with DPBS.
[0036] According to a preferred embodiment of the invention, silk
suture segments coated with cell adhesion molecules are utilized as
anchors, functioning as constraints or guides around which the
delaminating cell layer forms the tendon construct. Preferably, the
cell adhesion molecules are extracellular matrix (ECM) attachment
molecules, most preferably laminin. The anchors are produced by
cutting silk suture, preferably size 0, to a convenient length.
Lengths of 6 to 8 mm are easily pinned in place, but the length can
be varied without limit as dictated by the specific circumstances.
The segments of suture are dipped in a solution of 50 .mu.g/ml
laminin, preferably natural mouse laminin (Gibco), in DPBS with
care taken to thoroughly wet the suture. The suture segments are
then allowed to dry before use.
[0037] As described in U.S. Pat. No. 6,207,451 incorporated herein
by reference, the anchors can alternatively be produced from small
acellularized fragments of skeletal muscle, wherein the
acellularized fragments have cell adhesion molecules associated
therewith. Preferably, the fragments contain ECM attachment
molecules, such as laminin, collagen, and pronectin. As still
another alternative, a bone-like substrate, synthetic and/or
natural (for example, hydroxyapatite or calcium phosphate), could
also be used as a possible anchor material, thereby mimicking
developmental conditions.
[0038] The anchors are preferably pinned approximately 12 mm apart
in the prepared culture dish with stainless steel minutien pins.
The dishes are then filled with approximately 1 ml growth medium,
enough to cover the top of the sutures. The plates are sterilized
via ultraviolet irradiation in a biological safety cabinet for
approximately 90 minutes, and then placed in an incubator (5%
CO.sub.2, 37.degree. C.) for 5-8 days prior to seeding with
fibroblasts. It is believed that during incubation, the proteins in
FBS adhere to the substrate and anchors, thereby enhancing cell
adhesion. Although two anchors are used to create the preferred
tendon construct shape of the present invention, more anchors may
be used to form any desired size or shape of tendon construct.
[0039] After incubation, the growth medium is aspirated and
preferably approximately 5+10.sup.5 cells suspended in 2 ml growth
medium are seeded onto each culture dish. The plated cells are
supplemented with ascorbic acid, preferably approximately 100
.mu.g/ml of L-ascorbic acid 2-phosphate, a stable derivative of
ascorbic acid. Ascorbic acid is necessary for the synthesis of
collagen, the main constituent of tendons, and promotes the
maintenance of a confluent cell layer in this system. Fresh
ascorbic acid is added each time the growth medium is changed,
every .about.2-3 days. When the cells become confluent, after
approximately 5 days, differentiation medium (465 ml DMEM, 35 ml
FBS, 100 units/ml antibiotic-antimycotic) is substituted for growth
medium to induce construct formation. The differentiation medium is
changed every .about.2-3 days and the culture dishes maintained in
an incubator (37.degree. C., 5% CO.sub.2) until the constructs are
used for testing.
[0040] The integrity of the tendon constructs according to the
present invention appears to be dependent on the time of media
exchange and the ascorbic acid concentration. If the medium is
switched to differentiation medium prior to 5 days, viable
constructs may not form reliably. This is likely the result of a
decrease in the production of ECM and/or the proliferation of the
fibroblasts when placed in the low serum differentiation medium, as
the concentration of serum has been shown to affect both quantities
(see Kang and Kang, Yonsei Medical Journal, 40, p. 26, 1999).
Furthermore, in the absence of ascorbic acid, a confluent layer of
fibroblasts may not be maintained. This is attributed to a
decreased ability to form a stable ECM, most importantly type I
collagen, which is dependent on the inclusion of ascorbic acid (see
Kurata and Hata, Journal of Biological Chemistry, 266, P. 9997,
1991).
[0041] FIGS. 1A-1D depict the progressive delamination of the
two-dimensional cell layer and gradual self-organization into the
three-dimensional tendon construct according to the present
invention. In accordance with the present invention, the formation
of a tendon construct typically begins with peripheral delamination
of the edges of tendon tissue from the substrate, as shown in FIGS.
1A and 1B. The delamination process typically commences at 14 to 21
days, corresponding with the disappearance of laminin from the
substrate, and progresses radially inward until the entire cell
monolayer has peeled away from the substrate material, as shown in
FIG. 1C. The constructs are then supported between the anchors
above the SYLGARD.RTM. substrate under self-mediated tension. When
probed with tweezers, the tendon constructs are noticeably taut,
and when released from one anchor, the constructs contract
slightly. The delaminating monolayer will eventually roll up and
lift off of the substrate to form a three-dimensional structure
between the two anchors, as shown in FIG. 1D. The self-assembling
process of the tendon constructs of the present invention is highly
repeatable.
[0042] Following the switch from growth to differentiation medium,
the tendon constructs form in about two weeks. No external
influence is necessary for the cell layer to detach from the sides
of the culture dish and SYLGARD.RTM.. The tendon constructs of the
present invention can be maintained in culture for greater than 15
weeks, wherein the time depends upon such conditions as the density
at which the cells are plated, the anchor material and spacing, the
frequency of feeding, and the type and density of the substrate
cell adhesion molecules, such as laminin. There should be no
limitations on the length or diameter of the tendon constructs that
can be created.
[0043] For morphological analyses, the resulting tendon constructs
of the present invention, along with native neonatal Achilles
tendons harvested from 2 day old Fisher 344 pups, were fixed in a
3% formaldehyde/glutaraldehyde in 0.1 M sodium cacodylate buffer
solution pH=7.4 (Electron Microscopy Sciences, Fort Washington, PA)
at 4.degree. C. and embedded in EPON (Ted Pella Inc., Redding,
Calif., Eponate 12 resin). For light microscopy, semi-thick
sections, 1 .mu.m, were cut with an ultramicrotome, mounted on
glass microscope slides, and stained with 1% (w/v) Toluidine Blue
solution (FIG. 2). Ultrathin slices, 50 nm, were cut for electron
microscopy and were mounted on uncoated copper grids and stained
with aqueous uranyl acetate and lead citrate. The ultrastructure of
the constructs was investigated using a transmission electron
microscope at 60 kV (FIG. 3).
[0044] FIGS. 2 and 3 provide a morphological comparison of a
representative tendon construct according to the present invention
and a neonatal rat tendon. As one skilled in the art will readily
appreciate, the longitudinal sections shown in FIGS. 2A and 3A
clearly display an ultrastructural morphology similar to that of
the neonatal rat Achilles tendon shown in FIGS. 2B and 3B.
Comparing FIGS. 2A and 2B, it is noted that both the tendon
construct of the present invention and the neonatal tendon are
highly cellular, and include some disorganized ECM surrounding the
collagen fibrils. At higher magnification (FIGS. 3A and 3B), it is
evident that the fibril diameter is similar (.about.50 nm, as
indicated by arrows) in both the tendon construct and the neonatal
tendon, wherein the fibrils display the characteristic periodic
striations of type I collagen.
[0045] For mechanical testing, tendon construct diameter was first
measured at several positions along the length using an inverted
microscope, and an average diameter was calculated using all
measured values. Tensile testing was performed using an 810
Material Testing System (MTS Systems Corp., Eden Prairie, Minn.)
outfitted with a custom optical 200 mN load cell (see Dennis and
Kosnik, In Vitro Cellular Developmental Biology-Animal, 36, p. 327,
2000) and grips machined from DELRIN.RTM. (Acetal). The grips
clamped the specimens via an adjustable set screw. Data acquisition
and control were performed using LabVIEW software (National
Instruments, Austin, Tex.) on a computer. The load cell was zeroed
before the attachment of each sample. Samples were moistened by
regularly applying drops of DPBS with a Pasteur pipette, as drying
out of specimens has been shown to significantly alter the
mechanical properties of tendons and other soft tissues. The gauge
length was taken to be the length of the construct between the
grips which was measured with digital calipers after inserting and
clamping the sample into both grips and applying a prestress of
approximately 4 kPa. The force associated with this prestress, 1
mN, was 0.4% of the full range of the load cell, and roughly twice
its resolution limit. The samples were tested at a constant true
strain rate of 0.05 s.sup.-1.
[0046] As depicted in FIG. 4, the stress-strain response of the
tendon constructs of the present invention closely resembles that
of immature tendon. The initial response is compliant and resembles
the well-known toe region of soft tissue response. At a nominal
strain of 0.05, the tangent modulus, or the slope of the tangent to
the stress-strain curve, markedly increases, and the stress-strain
response is approximately linear in the strain range of 0.11 to
0.19 at which point the construct failed by breaking in one of the
grips. The tangent modulus measured at a strain of 0.16, within the
linear region of the response, is 20 MPa. The tendon constructs of
the present invention are thus mechanically similar to embryonic
chicken extensor tendons which have a tangent modulus of 27 MPa
(see McBride et al., International Journal of Biological
Macromolecules, 10, p. 194, 1988). In fact, the ultimate tensile
strength of the tendon constructs may be larger than reported here
since the majority of the constructs failed at either the upper or
lower grip. This is attributed to the pressure applied by the grip
due to manual tightening that could result in localized lateral
compression of the construct at that interface, and the
introduction of a stress concentrator.
[0047] Therefore, the tendon constructs according to the present
invention not only display the non-linear response characteristic
of soft tissues, but they have similar mechanical parameters to
embryonic tendon. The ultimate tensile strength and tangent modulus
of the tendon constructs and embryonic chick tendon are very
similar (FIG. 4). One difference between the tissues is the strain
at failure, 0.12 for chick tendon and 0.19 for the constructs, but
this may be attributed to the testing procedure rather than the
intrinsic material properties of the tissues. It is unknown whether
or not McBride et al. preloaded their specimens which would
decrease the length of the toe region, and consequentially the
strain at failure. Another cause of this discrepancy may result
from the drying out of the specimens. While both procedures
entailed the periodic wetting of the tissues, the degree of
hydration may have been different in each of the tissues. As
tendons dry out, the stiffness increases and the toe region becomes
shorter.
[0048] The present invention demonstrates the ability to induce
primary tendon fibroblasts to secrete and organize their own ECM,
and under the right conditions to self-assemble into
three-dimensional constructs without the aid of exogenous
scaffolding. The resulting tendon constructs are both structurally
and functionally similar to embryonic tendons. The ECM gives the
construct mechanical properties close to that of the replaced
tendon so that mobilization can resume as soon as possible,
accelerating the process of healing.
[0049] The findings herein suggest that under the right conditions,
tendon fibroblasts rebuild tendon morphology by recapitulating the
embryonic state. This return to a primitive state has also been
shown in studies of tendon repair and may provide a mechanism to
increase tendon plasticity (see Postacchini and Demartino,
Connective Tissue Research, 8, p. 41, 1980). The work of
Postacchini and Demartino on the repair of tendon after partial
tenotomy demonstrated that, during repair, tendon morphology
progresses from developmental to mature in .about.16 weeks. The
first collagen fibrils produced have small diameters, 20-80 nm. The
fibrils continue to mature, and after sixteen weeks the fibrils
possess a diameter of 200-300 nm. The regenerative pattern of
tendon in vivo admits the hypothesis that the tendon constructs of
the present invention have the capacity to mature in vitro when
incubated in the proper environment. Interestingly, fibroblasts
would not necessarily have been expected to self-organize in the
same manner that a culture of myotubes would self-organize,
principally because fibroblasts do not generate the same magnitude
of forces in vitro as myotubes.
[0050] A tendon construct as described herein could be engineered,
transected, and the two halves used as artificial tendon anchors
for the formation of a muscle construct formed as described in U.S.
Pat. No. 6,207,451. Alternatively, a tendon construct and a muscle
construct can each be severed and placed in close proximity to one
another to form a myotendinous junction. Cross- links can be
removed locally at the severed ends of the constructs, for example
by using microfluidics or solutions containing reducing agents. The
end of the tendon construct can then be seeded with myoblasts and
the end of the muscle construct seeded with fibroblasts along a
controlled gradient. Still further, a co-culture of fibroblasts and
myoblasts could be created, for example, by temporal plating of
cells, spatial plating cells, or adhesion protein patterning on the
substrate.
[0051] The connective tissue constructs according to the present
invention have a variety of foreseeable applications, ranging from
transplantation in vivo to functional and pharmacological testing
in vitro. These constructs are potentially useful for studying the
developmental biology of tendon as well as for clinical use in
tendon repair and screening for disease. The functional capability
of the tendon constructs shown in FIG. 4 demonstrates their ability
to be used as an in vitro model of tendon tissue, allowing the
comparison of functional data from the tendon constructs with the
pool of published data for tendon tissue in the scientific and
clinical literature. Furthermore, the constructs could be used in
the engineering of tissue-to-tissue or tissue-to-machine interfaces
to provide the necessary mechanical impedance matching and
functional interface to allow efficient and biologically
appropriate integration.
[0052] With reference to commonly assigned U.S. Pat. Nos. 6,114,164
and 6,303,286, both incorporated by reference herein, the formation
of the connective tissue constructs of the present invention can be
guided by measuring a functional property of the construct and
subsequently using the measured property as feedback. For example,
the tensile strength of the tendon constructs could be determined
prior to failure, and this functional measure could then be used in
a feedback control loop to guide the tendon construct to a desired
phenotypic outcome.
[0053] The connective tissue constructs also have the potential to
be used in studies of ligament function and replacement. Tendons
and ligaments are generally grouped together in the literature
since these tissues possess similar structural and mechanical
characteristics, but there are also subtle differences that have
not been thoroughly investigated. There is generally a larger and
more variable amount of elastin in ligaments, and the development
and biochemical content of tendons and ligaments have been shown to
differ. These differences may be the result of phenotypically
different fibroblasts, loading environment, or extrinsic signals.
The formation of analogous constructs using ligament fibroblasts
may help to identify the phenotypic and mechanical differences
between these tissues. As described herein, the constructs have
been defined as tendon-like structures since the fibroblasts used
are derived from tendons. However, it is understood that the method
of the present invention can be utilized to produce other
connective tissue constructs, such as a ligament construct, in
addition to the tendon construct shown and described herein.
[0054] The tendon constructs described herein have the potential to
aid in further elucidating the factors that influence tendon
development. Using the tendon constructs, the role of specific
growth factors, genes, or mechanical forces in tendon development
can be studied in isolation, eliminating the confounding variables
present in vivo. For example, it is generally accepted that
mechanical interventions influence tendon development, but there
have been few studies that explicitly address this issue. The
greatest difficulty in determining the role of force on tendon
development has been the isolation of the effect that force has on
the tissue, independent of the influence of concurrent alterations
to adjacent tissues and the overall hormonal environment. The
tendon constructs of the present invention may provide the
necessary isolated environment needed to decipher the role these
factors play.
[0055] While embodiments of the invention have been illustrated and
described, it is not intended that these embodiments illustrate and
describe all possible forms of the invention. Rather, the words
used in the specification are words of description rather than
limitation, and it is understood that various changes may be made
without departing from the spirit and scope of the invention.
* * * * *