U.S. patent application number 10/842086 was filed with the patent office on 2004-12-02 for tissue-engineered ligament.
Invention is credited to Chan, Kwan-Ho, Goh, James Cho Hong.
Application Number | 20040243235 10/842086 |
Document ID | / |
Family ID | 32302150 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040243235 |
Kind Code |
A1 |
Goh, James Cho Hong ; et
al. |
December 2, 2004 |
Tissue-engineered ligament
Abstract
An apparatus and method for the reconstruction of a previously
torn ligament using a tissue-engineered ligament. The
tissue-engineered ligament includes a scaffold of biocompatible
material having at least one layer and forming a sheet. The
scaffold is placed in a cultured medium for seeding with fibrocyte
forming cells. The seeded scaffold is then placed in an incubator
to increase the number of cells. The seeded scaffold is then formed
into a slender structure suitable for implantation. The method of
making a tissue-engineered ligament includes forming a scaffold of
biocompatible material having at least one layer forming a sheet.
Next, the scaffold sheet is seeded with fibrocyte forming cells.
The method further includes increasing the number of cells on the
seeded scaffold and forming a slender structure suitable for
implantation from the scaffold.
Inventors: |
Goh, James Cho Hong;
(Singapore, SG) ; Chan, Kwan-Ho; (Lubbock,
TX) |
Correspondence
Address: |
Mark J. Pandiscio
Pandiscio & Pandiscio
470 Totten Pond Road
Waltham
MA
02154
US
|
Family ID: |
32302150 |
Appl. No.: |
10/842086 |
Filed: |
May 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10842086 |
May 10, 2004 |
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09710435 |
Nov 10, 2000 |
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6737053 |
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60165331 |
Nov 12, 1999 |
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Current U.S.
Class: |
623/13.17 ;
435/401 |
Current CPC
Class: |
A61F 2/08 20130101 |
Class at
Publication: |
623/013.17 ;
435/401 |
International
Class: |
A61F 002/08; C12N
005/08 |
Claims
1. An apparatus for reconstruction of a previously torn ligament,
said apparatus comprising: a scaffold of biocompatible material,
the scaffold having at least one layer forming a scaffold sheet;
means for seeding the scaffold sheet with fibrocyte forming cells
to form a seeded scaffold; means for increasing the number of the
fibrocyte forming cells seeded on the scaffold to create a
tissue-engineered scaffold; and means for forming a slender
structure from the tissue-engineered scaffold, the slender
structure being a tissue-engineered ligament suitable for
implantation into a patient for reconstruction of a ligament.
2-10. (Canceled)
11. The apparatus of claim 45 wherein the means for attaching is an
interference screw for fixation of the end of each ligament at the
implantation site.
12-17. (Canceled)
18. The apparatus of claim 1 wherein the fibrocyte forming cells
are fibroblast cells.
19-23. (Canceled)
24. A method of making a tissue-engineered ligament, said method
comprising: forming a scaffold sheet of biocompatible material
having at least one layer; seeding the scaffold sheet with
fibrocyte forming cells to form a seeded scaffold of at least one
sheet; increasing the number of the fibrocyte forming cells on the
seeded scaffold to create a tissue-engineered scaffold; and forming
a slender structure from the tissue-engineered scaffold, the
slender structure being a tissue-engineered ligament suitable for
implantation into a patient for reconstruction of a ligament.
25. The method of claim 24 further comprising attaching each end of
the tissue-engineered ligament to implantation sites within a
patient.
26. The method of claim 24 wherein the scaffold is a single
sheet.
27. The method of claim 24 wherein the scaffold is slit at least
once, the slit is made in the direction of a first end of the
scaffold to a second end of the scaffold, and the slit terminates
prior to the first end and the second end.
28. The method of claim 24 wherein seeding the scaffold with
fibrocyte forming cells includes placing the scaffold into a
cultured medium containing the fibrocyte forming cells.
29. The method of claim 24 wherein increasing the number of
fibrocyte cells includes incubating the scaffold sheet after
seeding.
30. The method of claim 25 wherein increasing the number of
fibrocyte cells further includes incubating the tissue-engineered
ligament after forming the slender structure.
31. The method of claim 24 wherein increasing the number of
fibrocyte cells includes incubating the tissue-engineered ligament
after forming the slender structure.
32. The method of claim 24 wherein forming a slender structure
includes rolling the scaffold sheet to form a rolled tubular
structure.
33. The method of claim 24 wherein forming a slender structure
includes folding the scaffold sheet to form an accordion
structure.
34. The method of claim 24 wherein forming a slender structure
includes cutting a series of strips from the scaffold sheet and
stacking the series of strips on top of one another to form the
slender structure.
35. The method of claim 25 wherein attaching each end of the
ligament to implantation sites within a patient involves using an
interference screw for fixation.
36. The method of claim 24 further comprising molding threads into
each end of the tissue-engineered ligament for implantation.
37. The method of claim 24 further comprising providing
interlocking surfaces on the scaffold sheet for locking the
scaffold sheet into the tissue-engineered ligament when the method
step of forming a slender structure is accomplished.
38. The method of claim 24 further comprising applying cyclic
tensile loading of the seeded scaffold prior to implantation to
increase the strength of the scaffold.
39. The method of claim 24 further comprising applying cyclic
tensile loading of the slender structure prior to implantation to
strengthen the tissue-engineered scaffold.
40. The method of claim 24 further comprising forming spaces within
the slender structure to allow the fibrocyte forming cells to grow
therein.
41. The method of claim 24 wherein increasing the number of
fibrocyte forming cells further comprises implanting the seeded
scaffold in a penultimate section of a patient's body prior to
implantation in a functional location.
42. The method of claim 24 wherein increasing the number of
fibrocyte forming cells further comprises implanting the slender
structure in a penultimate location of a patient's body prior to
implantation in a functional location.
43. The method of claim 24 further comprising coating the scaffold
sheet with a material to promote adhesion of the fibrocyte forming
cells.
44. The method of claim 24 further comprising genetically altering
fibrocyte forming cells to increase production of fibrocyte
cells.
45. The apparatus of claim 1 wherein the apparatus further
comprises: means for attaching each end of the tissue-engineered
ligament to implantation sites within a patient.
46. (Canceled)
47. The method of claim 24 further comprising forming microchannels
in the scaffold sheet.
48. A tissue engineering ligament formed by forming a scaffold
sheet, seeding the scaffold sheet with cells, increasing the number
of cells, and forming a slender structure suitable for use as a
ligament.
Description
REFERENCE TO EARLIER APPLICATION
[0001] This application claims the benefit of pending U.S.
Provisional Patent Application Serial No. 60/165,331, filed Nov.
12, 1999 by James Cho Hong Goh and Kwan-Ho Chan. The aforementioned
document is hereby incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention generally relates to apparatus and methods
for the manufacture of replacement tissue using tissue engineering
methods. More particularly, this invention relates to apparatus and
methods for the manufacture of tissue-engineered ligaments suitable
for the treatment of ligament deficiencies in patients.
BACKGROUND OF THE INVENTION
[0003] Men and women who are athletically active experience the
majority of ligament tears, particularly tearing of the anterior
cruciate ligament of the knee. The anterior cruciate ligament is
commonly torn by forces applied to the knee during twisting,
cutting, deceleration or tackling. A torn anterior cruciate
ligament will generally not heal. An anterior cruciate deficient
knee is often unstable during pivoting activity. Repeated
instability episodes of the knee may lead to further damage of the
articular surface and cause tearing in the menisci. It is therefore
desirable to stabilize the knee by reconstructing a torn anterior
cruciate ligament. Attempts in the past to directly repair the torn
anterior cruciate ligaments have been relatively ineffective.
Prosthetic ligament replacements made of carbon fibers and Gore-Tex
materials do not last a long period of time. Repeated loading of a
prosthetic ligament in a young active patient leads to failure of
the ligament. The release of debris from a failed ligament results
in chronic inflammation of the joint, and osteolysis of bone, in
and around the area of ligament attachments.
[0004] The current standard practice is to reconstruct a torn
anterior cruciate ligament by substituting the torn ligament with a
patient's own tissue. The middle third of the patellar tendon or
the hamstring tendons are commonly used as substitution ligaments.
Alternatively, the allograft patellar tendon, hamstring tendon or
Achilles tendon from a donor can be used for reconstructing the
ligament. However, donor materials are associated with a risk of
infectious disease transmission such as AIDS. Using a patient's own
tissue is also associated with morbidity at the donor site. For
example, stress fracture of the patellar, quadriceps muscle
weakness and a long rehabilitation period may result from the use
of a patient's own tissue. Furthermore, harvesting and preparation
of autogeneous tissue prolongs surgery time.
[0005] Previous attempts to use an artificial stent to replace a
damaged anterior cruciate ligament have not been successful. One
such example is the LAD Prosthetic Ligament, which was used as a
scaffold for tissue ingrowth. The LAD Prosthetic Ligament is not
bioabsorbable. Therefore, whatever initial fibrous tissue that
forms on the LAD Prosthetic Ligament is not subject to
accommodating increasing loads and there is no stimulus for the
fibrous tissue to proliferate to support increasing loads.
Furthermore, the LAD Prosthetic Ligament is not an optimal
structure for tissue ingrowth.
[0006] Recent progress in tissue engineering has made it possible
to harvest cells from a patient's own body or a donor. The
harvested cells are then grown into the desired tissues on
three-dimensional scaffolds, or hydrogel carriers, made of
biodegradable polymers. These tissues include, but are not limited
to, heart muscles, fat, cartilage, and skin. The tissue grown
outside of the body, together with the scaffold containing the
tissue, is then transplanted into the patient to correct an
existing defect. After transplantation, the cells may further
replicate, reorganize and mature, depending on the environment of
the host bed into which the cells were transplanted.
[0007] Two good sources of cells that are suitable for tissue
engineering are embryonic stem cells and mesenchymal stem cells.
These stem cells, when exposed to particular bioactive factors,
also known as growth factors, can be directed to differentiate into
different types of cell lines in a predictable way. For example,
mesenchymal stem cells can be directed to differentiate into
different types of tissue such as, but not limited to, skin,
tendon, ligament and bone under suitable conditions. These
conditions include exposing cells to certain growth factors. It is
known that mesenchymal cells are directed to differentiate into
fibroblast when exposed to interleukin. Furthermore, fibroblast is
only able to differentiate into fibrocytes that are the mature
cells of ligament tissue.
[0008] Mesenchymal cells are present in very small numbers in bone
marrow, periosteum, skin and muscle. A small piece of the tissue
containing a small number of mesenchymal cells is preferably
harvested from the patient's own body. For example, a piece of
periosteal tissue harvested from the patient or donor is
morsellised into small pieces. Using tissue culture techniques well
known to those skilled in the art, the mesenchymal cells are
isolated and the number of cells expanded. The mesenchymal cells
are then seeded onto scaffolds. These scaffolds are preferably made
of biodegradable materials to make the desired tissues.
[0009] There are two major challenges in growing tissue-engineered
ligaments outside the body. First, most cells cultured in vitro
tend to grow in a monolayer. Even if it is possible to culture
tissue to a few millimeters thick, deeper layers of the cells may
not have sufficient supplies of nutrients. Secondly, it is
difficult to adequately and uniformly seed the scaffold with cells
to initiate cell expansion.
[0010] It is, therefore, an object of the present invention to
provide tissue-engineered ligaments for reconstruction of
previously torn ligaments.
[0011] It is another object of the present invention to provide
tissue-engineered ligaments to reduce the time it takes to complete
the surgery and to eliminate donor site morbidity in the
patient.
[0012] It is still another object of the present invention to
provide tissue-engineered ligaments grown from a small amount of
tissue obtained from the patient.
[0013] It is another object of the present invention to provide a
scaffold for uniform and adequate seeding of cells to initiate cell
expansion for making tissue-engineered ligaments.
[0014] It is also another object of the present invention to
provide a scaffold for a tissue-engineered ligament with adequate
channels for nutrients to reach the cells.
[0015] It is also another object of the present invention to
provide a method to enhance the growth and alignment of the
fibrocytes and the extra cellular matrix during incubation of the
tissue-engineered ligament.
[0016] Another object of the present invention is to provide
tissue-engineered ligaments that will permanently anchor to a
patient's bone.
[0017] Yet another object of the present invention is to provide
tissue-engineered ligaments that will mature and resist
physiological load across the joint.
[0018] Still another object of the present invention is to provide
a method of making tissue-engineered ligaments.
SUMMARY OF THE INVENTION
[0019] The present invention comprises an apparatus and method for
the reconstruction of a previously torn ligament using a
tissue-engineered ligament. The tissue-engineered ligament includes
a scaffold of biocompatible material having at least one layer and
forming a sheet. The scaffold is placed in a cultured medium for
seeding with fibrocyte forming cells. The seeded scaffold is then
placed in an incubator to increase the number of cells. The seeded
scaffold is then formed into a slender structure suitable for
implantation. The method of making a tissue-engineered ligament
includes forming a scaffold of biocompatible material having at
least one layer forming a sheet. Next, the scaffold sheet is seeded
with fibrocyte forming cells. The method further includes
increasing the number of cells on the seeded scaffold and forming a
slender structure suitable for implantation from the scaffold.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] Referring first to FIGS. 1-12, the starting material for a
tissue-engineered ligament 10 is a sheet 15 of biocompatible
material which is preferably bioabsorbable. The sheet 15 may also
be a porous structure. The pores of the porous structure may be
partially or fully interconnected across the thickness of the
sheet. The pore structure essentially forms perforations when fully
and directly interconnected across the thickness of the sheet. A
sheet of woven fabric made of a biocompatible and preferably
biodegradable material is an example of such a construct.
Additionally, the sheet may contain slits 20 which extend
completely through sheet 15. A method of forming a
tissue-engineered ligament includes placing the biocompatible sheet
15 in a cultured medium and seeding the sheet with fibrocyte
forming cells 25 (FIG. 2). Next, the seeded sheet is incubated to
increase the number of fibrocyte forming cells. After a sufficient
cell increase, sheet 15 is reformed into a slender structure 35,
such as a tissue-engineered ligament suitable for implantation into
a patient. In this respect it should be appreciated that the
presence of slits 20 in sheet 15 permit nutrients and the like to
pass readily into the interior of slender structure 35. In
addition, slits 20 permit cells 25 to grow through sheet 15.
Furthermore, the presence of slits 20 provides greater flexibility
to slender structure 35, whereby it may function more like a
natural ligament. If there is sufficient porosity in biocompatible
sheet 15, additional perforations or slits may not be necessary for
diffusion of nutrients to cells 25 when sheet 15 is formed into
slender structure 35.
[0021] The first embodiment of the invention shown in FIGS. 1-4
provides rectangular sheet 15 of biocompatible material with
multiple longitudinal slits 20 precut into the material. Slits 20
start and end at a prescribed distance from the edge of rectangular
sheet 15, thereby leaving uncut margins 30 connecting the adjacent
strips as shown in FIG. 2. The whole construct is immersed in
culture medium solution (not shown) and then seeded with fibrocyte
forming cells such as mesenchymal stem cells or fibroblast cells.
When a sufficient quantity of fibrous tissue has formed, sheet 15
is rolled into a slender structure 35, preferably with the strips
oriented longitudinally and the uncut edges 30 forming the ends of
the slender structure. Slender structure 35 is then implanted into
a patient to reconstruct a missing ligament. Slender structure 35
may be further incubated prior to implantation to allow for further
growth of fibrous tissue. Alternatively, slender structure 35 can
be implanted in a penultimate location in a patient's body such as
the peritoneal cavity, muscle bed or any other soft tissue bed.
This implantation allows further growth of fibrous tissue prior to
implantation in a functional location, such as the knee joint for
reconstruction of the anterior cruciate ligament.
[0022] Other methods of forming slender structure 35 from sheet 15
are also possible. Examples include, but are not limited to,
folding sheet 15 back and forth in an accordion style fold 40 with
a width 45 corresponding to the desired width of tissue-engineered
ligament 10 as shown in FIGS. 5-8, or cutting sheet 15 into strips
50 corresponding to a desired width 55 of tissue-engineered
ligament 10 and stacking one strip 50 on top of another, thereby
forming stack 60 as shown in FIGS. 9-12.
[0023] The fibrocyte forming cells can be harvested from a human
donor. Preferably, fibrocyte forming cells are harvested from the
patient's own body. Fibrocyte forming cells, which include
mesenchymal stem cells or fibroblast cells, can be derived from a
number of sources such as skin, bone marrow and periosteum.
[0024] The biocompatible material of sheet 15, which is preferably
also a bioabsorbable material, can be, but is not limited to, one
or more of the following: polyglycolic acid, polylactic acid, a
mixture of PGA/PLA, chitin and collagen. This material may also be
porous. Sheet 15 may comprise a uniform structure, a woven
structure, a composite structure (e.g., a sheet with incorporated
filaments, including aligned filaments such as for reinforcement),
etc. Flat sheets 15 of autograft or allograft tissue may also be
used. One example is fascia lata. The fascia lata tissue can be
preprocessed to reduce reaction. Several methods, including
freeze-drying, exist for preprocessing fascia lata.
[0025] The biocompatible material of sheet 15 may also be coated
with collagen and other factors to promote adhesion of the
fibrocyte forming cells 25. The expansion of the number of cells
can be promoted by the addition of growth factors to the
biocompatible material of sheet 15 or the culture medium.
[0026] To induce mesenchymal cells to differentiate into
fibroblast, fibroblast growth factors such as interleukin may be
added to the biocompatible material or the culture medium.
[0027] The strength of the cultured fibrous tissue is made stronger
by orientating the growth of the fibrocytes and deposition of the
collagen fibers in the longitudinal direction of the slender
structure. The fibrocytes are induced to orientate longitudinally
by incorporating longitudinal microchannels 201 (FIG. 2A), with a
width and depth of the order 1 to 200 microns, on the surface of
the biocompatible material of sheet 15. The longitudinal
microchannels 201 encourage the fibrocytes to cluster along the
microchannels and, additionally, urges the fibrocyte cells to
orient themselves parallel to the axis of the microchannels. The
application of cyclic loading to the biocompatible materials during
incubation further enhances orientation of the cells and collagen
fibers. Cyclic loading can also increase the growth of the fibrous
tissue. The cyclic loading can be applied to the biocompatible
material in the rolled or unrolled configuration. It is to be noted
that when the slender structure is loaded in longitudinal tension,
the strips 105 will be taut and fluid will be forced out through
the perforations or slits. When the tension is released or
lessened, the slender structure tends to assume a slightly larger
diameter, thus permitting fluid to flow into the slender structure
through the slits or perforations. Such cyclic loading will create
an environment of circulating fluid providing fresh nutrient fluid
to the cells within the slender structure. Additionally, the cells
suspended in the fluid for seeding are carried by the circulating
fluid into the interior of the slender structure 35 thereby
increasing the chance of cells attaching in the interior of the
slender structure 35.
[0028] In an alternative embodiment of this invention (not shown),
fibrocyte forming cells 25 on the biocompatible material of sheet
15 are cultured and, when a sufficient expansion of cells 25 is
achieved, slits 20 are then made in the sheets as described above.
Some of cells 25 will be damaged during the creation of slits 20.
This damage is compensated for by the more efficient cell expansion
on an uninterrupted (i.e., slit-less) flat surface during the
incubation period.
[0029] In order to increase the rate at which the number of cells
25 increase, the mesenchymal stem cells or fibroblast can be
genetically altered to increase local production of desired growth
factors. This can be done by viral transfection or by incorporating
plasmid genes into the matrix of the biocompatible material.
[0030] Now looking at FIG. 13, the ligament 10 is ready for
transplantation into a patient after incubating cells 25 attached
to scaffold sheet 15. Incubation usually occurs for a period of
time ranging from one to twelve weeks to allow substantial growth
and expansion of cells 25. In general, as shown in FIG. 13, two
bone tunnels 65 are prepared during surgery on opposite sides of
the joint using techniques well known in the art. The ligament 10
is then fixed in the bone tunnels 65 with an interference screw 70
at both ends. This is similar to the fixation of hamstring
ligaments with an interference screw in standard arthroscopic
anterior cruciate reconstruction. Alternatively, if desired, other
surgical techniques well known in the art may be used to secure the
tissue-engineered ligament 10 within the knee joint. Without
harvesting autologous tissue, such as the patellar tendon or
hamstring tendons, the complexity and time required for completing
this surgery is greatly reduced. Also any morbidity associated with
the harvesting of autologous tendon is eliminated.
[0031] After successful implantation of tissue-engineered ligament
10, further growth of the fibroblast and fibrocytes will further
anchor ligament 10 in the bone tunnels 65 by tissue ingrowth. Also
repeated cyclic loading will encourage hypertrophy of
tissue-engineering ligament 10. With time, the scaffold sheet 15
will be gradually absorbed and the entire load across the joint
will eventually be carried by tissue-engineered ligament 10.
Ligament 10 is a live tissue and will eventually mature into a more
stable structure capable of resisting normal transient increases in
physiologic load.
[0032] Now looking at FIGS. 14-16, fixation can be enhanced by
molding threads 75 into the ends 80 of slender structure 35 using a
molding device 85 before implantation. Threaded ends 75 of
tissue-engineered ligament 10 engage the thread of interference
screw 70, as seen in FIG. 17. Advancement of interference screw 70
urges threaded end 75 of tissue-engineered ligament 10 against the
wall of bone tunnel 65 and causes threads 75 of end 80 of
tissue-engineered ligament 10 to embed into bone tunnel 65. This
action further enhances fixation. Alternatively, a rigid body (not
shown) secured to the end of the tissue-engineered ligament may be
provided for an interference screw to press against to provide
fixation. The rigid body may contain threads corresponding to the
screw. Alternatively, the rigid body may not have any threads.
[0033] In a preferred embodiment, the starting material is a thin
rectangular sheet 15 of a biocompatible material including a
biodegradable polymeric material, a natural material, and/or an
allograft or autograft fascia lata material. Examples of
biodegradable polymeric materials include, but are not limited to,
PGA, PLA, or mixture of PLA/PGA, etc. Examples of natural materials
include, but are not limited to chitin, chitosan, etc.
[0034] Looking at FIG. 18, prescribed sections 85 at both ends of
sheet 15 may be thicker than the rest of sheet 15. In a preferred
embodiment, sections 85 have a specific male pattern 90 on one side
and matching female pattern 95 on the other. The matching male and
female patterns 90, 95 contact and inter-digitate when sheet 15 is
rolled up. These inter-digitations at both ends of the sheet
prevent sliding between adjacent layers of the rolled-up structure
to maintain a stable, slender structure 35. Also, the slightly
greater thickness at both ends helps to separate the layers to
provide room for cellular growth between the layers.
[0035] In this configuration, a middle thinner portion 100 of sheet
10 has many tiny strips 105. These strips 105 are made with
multiple longitudinal slits pre-cut into the material. The slits
start and end at a prescribed distance from the edge of the
rectangular material to leave uncut margins 30 connecting the
adjacent strips 105. These uncut margins 30 form the earlier
mentioned thicker sections preferably having interlocking patterns.
Each of the tiny strips 105 has longitudinal microchannels 201.
These microchannels each have a width and depth of the order of 1
to 200 microns. The sheet may be hydrophilised and coated with
collagen and growth factors such as TGF beta, IGF, etc.
[0036] Cell seeding may be performed using various methods
consistent with the present invention. Several such methods will
now be set forth by way of example but not limitation.
EXAMPLE I
[0037] One method involves clamping sheet 15 at both ends 80 of the
thicker, uncut sections and immersing the sheet in a culture medium
110. Sheet 15 is initially held in tension and mesenchymal stem
cells or fibroblasts are introduced into culture medium 110 and
onto strips 105. Cells 25 are allowed to attach to strips 105 and
proliferate. Nutrients and growth factors are added periodically as
desired. To increase the rate at which the number of cells 25
increase, the mesenchymal stem cells or fibroblast can be
genetically altered to increase local production of desired growth
factors. This can be done by viral transfection or by incorporating
plasmid genes into the matrix of the biocompatible material.
[0038] The mesenchymal stem cells are directed to differentiate
into fibroblasts and subsequently to fibrocytes. Differentiation
occurs under suitable conditions such as exposure to growth
factors. Matrix materials are then produced by the fibrocytes. A
cyclic tensile load introduced through sheet 15 stimulates the
fibrocytes to orientate and align in the direction of tension.
Fibrous tissue forms on sheet 15 over a period of time and sheet 15
is then rolled up along strips 105. The ends 80 of sheet 15 are
crimped and then clamped in a cyclic loading machine as
schematically depicted in FIG. 19. Further cyclic tensile load is
applied to the structure immersed in culture medium 110 to promote
growth, organization and maturation of cells 25. Tissue-engineered
ligament 10 is then implanted into a patient when there is
sufficient maturation.
EXAMPLE II
[0039] Another method for cell seeding involves clamping sheet 15
at both ends 80 of the patterned sections 90, 95 and immersing
sheet 15 in culture medium solution 110. Sheet 15 is initially held
in tension and mesenchymal stem cells or fibroblasts are introduced
into solution 110 and onto strips 105. Cells 25 are allowed to
attach to strips 105 and proliferate. Nutrients and growth factors
are added periodically. To increase the rate at which the number of
cells 25 increase, the mesenchymal stem cells or fibroblast can be
genetically altered to increase local production of desired growth
factors. This can be done by viral transfection or by incorporating
plasmid genes into the matrix of the biocompatible material.
[0040] Once there is evidence of cell adhesion, sheet 15 is
rolled-up along the strips 105 to form a slender structure 35. Ends
80 are then crimped and then clamped. As the rolled-up slender
structure 35 continues to be immersed in culture medium 110, a
cyclic tensile load is applied to structure 35. The mesenchymal
stem cells now differentiate into fibroblasts and secretion of
matrix materials takes place. The fibroblasts form fibrocytes and
these fibrocytes become aligned in the direction of tension. Over a
period of time, the fibrocytes mature into fibrous tissue which
forms over rolled-up slender structure 35.
EXAMPLE III
[0041] A third method involves forming a slender structure 35 by
any of the methods previously described. Both ends 80 are crimped
and clamped, and immersed in culture medium solution 110. The
slender structure 35 is initially held in slight tension and
mesenchymal stem cells are introduced into the solution and onto
the slender structure 35. The cells attach to strips 105 and
proliferate. Nutrients and growth factors are added periodically as
desired. To increase the rate at which the number of cells 25
increase, the mesenchymal stem cells or fibroblast can be
genetically altered to increase local production of desired growth
factors. This can be done by viral transfection or by incorporating
plasmid genes into the matrix of the biocompatible material.
[0042] The mesenchymal stem cells are directed to differentiate
into fibroblasts and subsequently to fibrocytes under suitable
conditions, including exposure to growth factors. Matrix materials
are then produced by the fibrocytes. A cyclic tensile load is
introduced to slender structure 35. As the ends of the slender
structure 35 move towards and away from one another, the space
between strips 105 opens and closes, thereby permitting cells and
nutrients to flow between the strips, and thereby giving the cells
the opportunity to attach to the interior of the structure. This
loading also stimulates cells 25 further. The fibrocytes orientate
and align in the direction of tension. Over a period of time, the
fibrocytes mature into fibrous tissue which forms over slender
structure 35.
[0043] The in-vitro tissue-engineered ligament is ready for
implantation once ligamentous tissue has formed on slender
structure 35. Crimped ends 80 of in-vitro tissue-engineered
ligament 10 are then inserted and secured into bone tunnels 65.
Preferably, tissue-engineered ligament 10 is secured with
interference screws 70. Alternatively, other methods well known in
the art may be used.
[0044] Initially, the material of scaffold sheet 15 of
tissue-engineered ligament 10 supports loading across the joint.
For scaffold sheet 15 made of biodegradable material, as the
scaffold material of the implanted tissue-engineered ligament 10
degrades over time, the load is gradually transferred to the newly
formed tissue. Eventually, when scaffold sheet 15 is completely
absorbed, the entire load is transferred to the newly formed tissue
of ligament 10.
[0045] The preferred embodiments described above contain many
examples which are not limitations on the scope of the invention
but illustrations of alternate embodiments. Many other variations
are possible within the scope of the invention, as those skilled in
the art will recognize from the following claims.
* * * * *