U.S. patent application number 12/555151 was filed with the patent office on 2009-12-31 for in-vitro mechanical loading of musculoskeletal tissues.
This patent application is currently assigned to Medldea, LLC. Invention is credited to Martin S. Bancroft, Michael A. Masini.
Application Number | 20090325295 12/555151 |
Document ID | / |
Family ID | 26897530 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090325295 |
Kind Code |
A1 |
Masini; Michael A. ; et
al. |
December 31, 2009 |
IN-VITRO MECHANICAL LOADING OF MUSCULOSKELETAL TISSUES
Abstract
Musculoskeletal tissues produced in vitro are optimized in
response to an externally applied mechanical load. The load applied
may vary from tissue to tissue, depending upon the response
desired, and may include intermittent axial, torsional, and bending
loads to produce cortical structures. Compression alone is
preferably applied to produce cancellous bone. A method according
to the invention for culturing bone in vitro comprises: providing a
culture vessel providing a scaffold material, supporting the
scaffold material within the tissue culture vessel so as to be
exposed to a tissue culture medium, and exerting a force on the
scaffold material during growth of a bone construct (a cultured
bone growth) around the scaffold material. Applicable apparatus
preferably includes a culture vessel, holders for holding a
scaffold within the tissue culture vessel, means for introducing a
tissue culture medium to the tissue culture vessel, and an actuator
adapted to apply a force to developing bone during the in vitro
culture of the tissue, whether bone, cartilage, ligament, or
composites thereof.
Inventors: |
Masini; Michael A.; (Ann
Arbor, MI) ; Bancroft; Martin S.; (Rochester,
NY) |
Correspondence
Address: |
GIFFORD, KRASS, SPRINKLE,ANDERSON & CITKOWSKI, P.C
PO BOX 7021
TROY
MI
48007-7021
US
|
Assignee: |
Medldea, LLC
|
Family ID: |
26897530 |
Appl. No.: |
12/555151 |
Filed: |
September 8, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09850659 |
May 7, 2001 |
7585323 |
|
|
12555151 |
|
|
|
|
60202282 |
May 5, 2000 |
|
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Current U.S.
Class: |
435/377 |
Current CPC
Class: |
C12N 5/0655 20130101;
C12N 5/0654 20130101; C12M 25/14 20130101; C12M 35/04 20130101;
C12M 21/08 20130101 |
Class at
Publication: |
435/377 |
International
Class: |
C12N 5/06 20060101
C12N005/06 |
Claims
1. A method of synthesizing musculoskeletal tissue, the method
comprising the steps of: providing a cell culture vessel containing
a cell culture medium; introducing at least one musculoskeletal
precursor to the medium; applying a force to the musculoskeletal
precursor in vitro through an actuator that mimics forces
experienced by a bone in a body, whereby the precursor develops
into a musculoskeletal tissue matrix having desired properties
through the application of the force.
2. The method of claim 1, wherein the culture medium includes
osteoblasts.
3. The method of claim 1, wherein the culture medium includes
chondroblasts.
4. The method of claim 1, wherein the culture medium includes
fibroblasts.
5. The method of claim 1, wherein the culture medium includes stem
cells.
6. The method of claim 1, wherein the matrix develops into bone
tissue.
7. The method of claim 1, wherein the matrix develops into
cartilage.
8. The method of claim 1, wherein the matrix develops into
ligament.
9. The method of claim 1, wherein the matrix develops into
tendon.
10. The method of claim 1, wherein the matrix develops into a
bone-tendon-bone composite.
11. The method of claim 1, wherein the matrix develops into a
cartilage-bone composite.
12. The method of claim 1, wherein the tissue matrix includes stem
cells.
13. The method of claim 1, wherein the actuator that mimics forces
produced in a natural bone joint.
14. The method of claim 1, wherein the actuator that mimics forces
produced in a knee joint.
15. The method of claim 1, wherein the actuator that mimics forces
produced in a hip joint.
16. The method of claim 1, wherein the actuator that mimics forces
produced in a shoulder joint.
17. The method of claim 1, wherein the actuator that mimics forces
produced in an elbow joint.
18. The method of claim 1, wherein the actuator that mimics forces
produced in a spine.
19. The method of claim 1, wherein the actuator applies a
compressive force.
20. The method of claim 1, wherein the actuator applies
tension.
21. The method of claim 1, wherein the actuator applies an
oscillating force.
22. The method of claim 1, wherein the actuator applies a torsional
or twisting force.
23. The method of claim 1, wherein intermittent axial, torsional
and bending loads are applied to produce a tubular bone.
24. The method of claim 1, wherein compression alone is applied to
produce cancellous bone.
25. The method of claim 1, further including the steps of:
supporting a scaffold material with the culture vessel; and
applying the force to the scaffold material through the culture
medium so as to synthesize the tissue on the scaffold material.
26. The method of claim 1, further including the step of measuring
a physical property of the tissue during the synthesis thereof.
27. The method of claim 26, wherein the magnitude of the force is
correlated with the measured property.
28. The method of claim 26, wherein the physical property is an
elastic modulus or a Young's modulus.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/850,659, filed May 7, 2001, which claims
priority from U.S. provisional patent application Ser. No.
60/202,282 filed May 5, 2000, the entire content of both of which
is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the in-vitro growth of
musculoskeletal tissues, in particular bone, cartilage, and
ligaments.
BACKGROUND OF THE INVENTION
[0003] Musculoskeletal tissues are composed of a composite of
cellular and matrix components. In vivo, the cells are generally
believed to be derived from undifferentiated cell lines that
respond to different stimuli, both chemical and mechanical, and
then ultimately differentiate and produce a particular matrix
providing a tissue with a given structure and function.
Furthermore, musculoskeletal tissues in living organisms have the
ability to adapt to mechanical and physiologic changes throughout
life.
[0004] An example is bone. The material properties of bone are
governed by the density (and microdensity) of the material. The
geometry of the bone determines its strength. The tubular structure
of long bones provides them with a greater moment of inertia than
would be true if bones were solid rods. Consequently, bones are
stronger withstanding bending or torsional stresses than they would
be if they were solid rods. As one grows older, the outer diameter
increases as does the inner periosteal diameter. Theoretically,
these changes allow one to maximize bone strength as bone mass
decreases with age.
[0005] Articular cartilage is similarly composed of cellular and
matrix components. The cells are uniquely isolated by the matrix
and highly responsive to their environment. The matrix is composed
primarily of collagen, proteoglycan, and water. The
three-dimensional lattice and hydrostatic forces give cartilage its
unique ability to withstand compressive forces.
[0006] In addition to what has been observed in-vivo, in-vitro
studies have shown chondrocytes respond to mechanical loads (P. M.
Freeman et al., J. Orth. Res., 12(3), 311-319 (1994)). This study
found a decrease in the cell volume of chondrocytes in response to
compressive loads. Other studies have shown an increase in
proteoglycan synthesis and deposition in response to intermittent
physiologic compression (G. P. J. van Kampen et al., Arthritis
Rheum. 28 419-424 (1985)). Bone changes in response to load have
been documented for many years and the appositional deposition of
bone in an effort to increase the structural strength of loads
areas is generally referred to as "Wolff's Law." Similarly, tendon
and ligament healing has been shown to be affected by the forces
applied to these tissues at various periods in the healing
process.
[0007] The last few years have seen a rapid increase in the number
of biomaterials available to augment and enhance the body's ability
to repair and replace damaged musculoskeletal tissues. A recent
article in the New England Journal of Medicine discusses autologous
cartilage transplantation as a treatment of deep cartilage defects
in the knee (M. Brittberg et al., New England J. Medicine, 331(14)
889-895 (1994). This method is currently available in the United
States and undergoing investigation. The patient's cartilage is
essentially "cloned" and reinserted in a cartilaginous defect after
being grown in vitro to the appropriate volume. It is injected as a
liquid paste and secured by an autologous periosteal patch. The
authors had "encouraging" results in femoral condyle defects
although the results were poor in the highly mechanically loaded
patella.
[0008] Bone morphogenic protein, growth hormone, coral bone
substitutes, bone paste, etc. are commercially available products
used to enhance repair of fractures, nonunions, or osseous defects.
These materials are gaining widespread acceptance within the
medical community for their applicability in complex cases. Most of
these products lack mechanical strength and structural properties
approximate to the tissues they will support and rely on the
healing of the host before adequate function can be restored
[0009] In U.S. Pat. No. 6,121,042, Peterson et al. disclose an
apparatus for applying an axial load to a cultured tendon or
ligament construct. However, the growth of bone is not disclosed.
Further the application of torsional forces is not disclosed.
Peterson further fails to disclose the application of forces scaled
by (such as proportional to) a relevant elastic modulus of the
cultured structure.
SUMMARY OF THE INVENTION
[0010] Broadly, this invention optimizes musculoskeletal tissues
produced in-vitro by utilizing their unique ability to respond to
mechanical load. The load applied may vary from tissue to tissue,
depending upon the response desired. For example, intermittent
axial, torsional, and bending loads can be applied to bone cells
and matrix when the desired response is to produce tubular bone.
Compression alone is preferably applied to produce cancellous
bone.
[0011] An improved method for culturing bone in vitro comprises:
providing a culture vessel providing a scaffold material,
supporting the scaffold material within the tissue culture vessel
so as to be exposed to a tissue culture medium, and exerting a
force on the scaffold material during growth of a bone construct (a
cultured bone growth) around the scaffold material.
[0012] An improved apparatus for culturing bone in vitro comprises:
a culture vessel, holders for holding a scaffold within the tissue
culture vessel, means for introducing a tissue culture medium to
the tissue culture vessel, and an actuator adapted to apply a force
to developing bone during the in vitro culture of the tissue,
whether bone, cartilage, ligament, or composites thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a system for in vitro growth of bone according
to the present invention;
[0014] FIG. 2 shows a system embodiment;
[0015] FIG. 3 is a flow chart illustrating an improved method of in
vitro bone growth;
[0016] FIG. 4 is a flow chart illustrating a method of applying a
force correlated with a measured physical property of the growing
bone matrix;
[0017] FIG. 5 is a flow chart illustrating a further method of
applying a force correlated with a measured physical property of
the growing bone matrix;
[0018] FIG. 6 shows another system for in vitro growth of bone
according to the present invention, using a motorized actuator to
apply a force;
[0019] FIG. 7 shows another system for in vitro growth of bone
according to the present invention, using magnetic repulsion to
apply a force;
[0020] FIG. 8 shows another system for in vitro growth of bone
according to the present invention, in which the force is applied
through a flexible component of the culture vessel enclosure;
[0021] FIG. 9 is a drawing of a joint simulator disposed within a
culture vessel according to the invention;
[0022] FIG. 10A shows the used of force applied directly to a
culture vessel;
[0023] FIG. 10B also shows the used of force applied directly to a
culture vessel; and
[0024] FIG. 11 shows an alternative "piston-like" culture vessel
according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0025] FIG. 1 shows a system applicable to the in-vitro culturing
of bone or tissue according to the present invention. Culture
vessel 10 has a culture medium inlet conduit 12, having an inlet
control 14, and a culture medium outlet conduit 16, having an
outlet control 18. A scaffold material 20 is supported in culture
medium 22 by first holder 24 and second holder 26.
[0026] The culture medium is enclosed by culture vessel 10 and
inner lid 28. Inner lid 28 is supported, relative to the walls of
vessel 10, by one or more pins such as 30. A spacer 32 passes
through a hole in the inner lid 28, maintaining a seal, and
connects to actuator 36 and force sensor 34 to outer lid 38, held
in place relative to vessel 10 by one or more pins 40. Electrical
connections 42 and 44 lead to electrical contacts 46 and 48,
allowing power to be provided to actuator, and signal to be
obtained from the force sensor.
Scaffold Material
[0027] The scaffold material can be any porous, fibrous, meshed,
woven, or other material suitable for growth of bone material. In a
preferred embodiment, the scaffold is formed from collagen fibers.
In alternative embodiments, the scaffold material may be a porous
glass, sol-gel, aerogel, xerogel, ormosil, polymer gel, porous
ceramic, proteins, biomaterial, hydroxyapatite framework, nylon,
other biocompatible polymer, or other biocompatible material,
including pieces of bone, cartilage, ligament, or other appropriate
tissue. The scaffold may provide intrinsic structural integrity, or
may be a flexible fiber network.
[0028] Polymer gels may be grown in solvent, then dried for use as
a scaffold material. Gels may be cross-linked by UV, other
radiation exposure, or other chemical means before use as a
scaffold material. The scaffold material may be a piece of
autogenous tissue (i.e., obtained From a patient), to ensure
biocompatibility any reduce the likelihood of a host/graft
rejection.
Culture Medium
[0029] The culture medium contains a mixture so as to induce growth
of bone material on the scaffold. Preferably, this is an aqueous
solution containing osteoblasts. Other culture medium components
may include: protein sources, carbohydrates, fats, and minerals for
deposition including calcium and phosphate. Any appropriate
enzymes, co-enzymes, hormones or growth modifiers may be included
as well. Stem cells can advantageously be derived from fat or other
tissue obtained from the patient receiving the implant, and used in
the culture medium for any of the embodiments of the present
invention.
Actuator
[0030] Actuator 36 is preferably a stacked piezoelectric actuator,
so as to provide stress (compression force) and/or strain
(extensive force) to the scaffold and growing matrix through a
displacement of the upper end of the scaffold. Other actuators may
be used, such as solenoids or electric motors. The matrix itself
may be bone, cartilage, or ligament tissue produced by osteoblasts,
chondroblasts, and fibroblasts, respectively. Pluripotential stem
cells can differentiate into the various cell lines, depending upon
the environment created by the culture medium and force construct
utilized.
[0031] The signal from force sensor 34 can be used to control the
force applied to the scaffold, and thereby to the bone matrix on
the scaffold. The compression modules can be determined for the
displacement of the actuator and the force signal provide by the
force sensor. Initially, the force signal may be very small or
zero, particularly if a fibrous scaffold is used, and compressive
force is applied. As a bone matrix develops, the force signal will
increase as the bone provides resistance to compression. The
displacement applied by the actuator can be correlated with the
force signal. For example, the displacement applied by the actuator
can be controlled to be correlated with the force signal obtained
per unit displacement.
System Embodiment
[0032] FIG. 2 shows a system according to the present invention. A
computer 50 receives a force signal from force sensor 52. The
computer 50 also has a software application program adapted to send
a signal to the actuator control, so as to control the
actuator.
Compression Modulus
[0033] The program determines the compression modulus of the bone
growth from the ratio of the force measured to actuator
displacement. The actuator is then controlled so as to apply a
force correlated (for example, proportional to) the compression
modulus of the bone.
[0034] An effective compression modulus M can be defined by
M = .DELTA. X F ##EQU00001##
where F is the applied force, and .DELTA.X is the actuator
displacement or other equivalent measure of bone compression.
[0035] Equivalently, the modulus can be determined by applying a
force to a cultured structure, and monitoring the consequent
displacement. A force provider and displacement sensor can be used
in place of an actuator (providing displacement) and a force
sensor.
Improved Method of In Vitro Culture
[0036] FIG. 3 shows a flow chart illustrating an improved method of
preparing a cultured structure. The cultured structure may be bone,
cartilage, ligament, skin, tendons, organ components such as heart
valves, blood vessel components, other connective tissue, other
cellular tissue, teeth, or other biological materials.
[0037] Box 60 corresponds to the provision of a culture vessel,
such as described in relation to FIG. 1.
[0038] Other culture vessels known by skill of those in the art may
be advantageously adapted for use with the present method, for
example, such as described in U.S. Pat. Nos. 5,153,136 to
Vanderburgh; and 4,839,280; 6,037,141; 6,048,723 to Banes;
6,171,812 to Smith and 6,121,042 to Peterson. The entire contents
of any patent or publication referred to in this specification are
incorporated herein by reference.
[0039] Box 62 corresponds to the support of a scaffold material
within the culture vessel. Suitable scaffold materials have been
discussed above. Box 64 corresponds to the exposure of the matrix
material to a culture medium so as to grow the cultured matrix
structure on and around the scaffold. The culture vessel will be
fully or partially filled with culture medium. In a preferred
embodiment, an aqueous solution containing osteoblasts is used.
(The scaffold material may also be exposed to a gel containing
osteoblasts, or other culture medium components discussed above).
The culture medium can be periodically or continuously replaced by
fresh culture medium. The culture medium with the vessel may also
be stirred or agitated.
[0040] Box 66 corresponds to the monitoring of the cultured
structure. This monitoring will be discussed in detail below. Box
68 corresponds to the application of a force to the cultured matrix
structure, so as to improve the properties of the structure. In the
case of bone culture, a compressive force is preferably applied to
the culture so as to improve the development and mechanical
properties of the cultured matrix structure. The apparatus and
systems described above are preferably used. Other embodiments and
methods are described below.
[0041] Box 70 corresponds to the removal of the culture from the
culture vessel. Other processes can be applied to the culture after
removal from the vessel, and before implantation. These may include
cleaning, shaping, further mechanical processing (such as loading),
sterilization and other characterization tests such as ultrasound
density measurements and immunological tests. An unsatisfactorily
cultured matrix structure may be discarded, returned to the culture
vessel, or otherwise further processed before implantation. Box 72
corresponds to implantation of the cultured structure into a
patient.
Monitoring of Cultured Structure
[0042] During early stages of bone culture, it may be unhelpful to
apply forces to the cultured bone, as the culture may be overly
brittle at this stage. The culture process may be divided into two
or more periods; the first period with no forces applied, and the
later stage(s) characterized by the application of forces.
[0043] The bone culture can be visually inspected by a technician
during growth to monitor expected development and to initiate
corrective measures if development is less than expected. The walls
of the culture vessel may be in full or in part transparent, or
windows provided, to allow visual monitoring. Ultrasound, x-rays,
and other radiation/tomography may be used to monitor the density
of bone during in-vitro culture. The x-rays, MRI and/or CAT scans
may be analyzed automatically, through pattern recognition, for
example.
[0044] Electric fields generated from bone compression may also be
monitored. An oscillating compression can be applied to the bone
sample and a resulting electric field detected and monitored so as
to characterize the properties of the bone. This may be achieved
even in a conducting medium if the signal frequency is rapid,
compared with ion diffusion time in the conducting medium. The
detected electric field is related to the piezoelectric properties
of bone, which are correlated with the mechanical strength of the
bone. Hence, a force can be applied to the bone culture scaled
according to the detected piezoelectric signal.
[0045] The force applied to the bone can also be scaled according
to an estimate elastic modulus (Young's modulus, compression
modulus, and similar) based on ultrasonic, x-ray, or other
radiation absorption measurements.
[0046] Acoustic waves can also be used to characterize the cultured
bone sample, e.g. from the noise generated by a mechanical impulse.
The resonant frequencies of a cultured bone sample can be used to
determine mechanical strength.
[0047] Total applied forces may be limited by known properties of
native biological material.
Force Application
[0048] In preferred embodiments, a compressive force is applied to
a cultured bone sample, and the magnitude of the applied force is
correlated with the mechanical and/or structural properties.
[0049] It can also be beneficial to apply tension to a cultured
structure, such as a tendon, ligament, or cartilage. In this case,
the Young's modulus of the structure can be determined, and a
tensioning force applied having a magnitude correlated with (e.g.
proportional to) the Young's modulus.
[0050] It can further be beneficial to apply an oscillating force
to the cultured structure, having compressive and tensioning
components. Conventionally, a symmetrical oscillating force can be
applied, however by determining elastic moduli for tension and
compression separately, a non-symmetrical oscillating force can be
applied, having, a tensioning component scaled by the tension
modulus; and a compression component scaled by the compression
modulus.
[0051] It can further be beneficial to apply a torsional (twisting)
force to the cultured structure during its growth. One end of a
cultured bone sample can be fixed, relative to the culture vessel,
and a rotating actuator such as a micro-stepper motor used to apply
a twist to the other end of the bone sample. The torsional modulus
can be determined, and the angular displacement scaled by the
torsional modulus.
[0052] In all cases of force application, the appropriate modulus
can be determined as the force is applied and increased. If the
behavior of the measured modulus indicates a yield point, fracture,
or other structural breakdown, the force then can be limited to
lower values.
[0053] A microphone can be used to detect signals characteristic of
structural breakdown during the application of force(s), and the
signals used to limit the maximum applied force. A maximum force
can be defined by known properties of fully cultured tissue.
Force Control
[0054] FIG. 4 shows a flow chart illustrating a method of
controlling a force applied to a cultured structure (bone,
ligament, cartilage, and the like) during growth. For convenience,
this method concerns application of a compressive force, but this
is intended to be non-limiting, as tension, torsional, bending and
combination forces can also be applied according to this
method.
[0055] Box 80 corresponds to the application of a displacement to
the cultured structure. For example, using a piezoelectric
actuator, the displacement is correlated with the voltage applied
to the actuator. Using a stepper motor, the displacement is
correlated with the number of step pulses applied. Actuators can
also comprise an integrated displacement sensor.
[0056] Box 82 corresponds to the measurement of the force on the
structure corresponding to the applied displacement. A signal from
a force sensor is obtained.
[0057] Box 84 corresponds to the determination of the modulus of
the structure. An electronic device, such as a computer, can be
used to control the actuator, receive a signal from the force
sensor, and determine a modulus using a software program or
algorithm
[0058] Box 86 corresponds to the application of a displacement to
the structure correlated with the modulus. A stronger structure has
a stronger force applied to it, up to a predetermined maximum
force.
[0059] FIG. 5 illustrates a method similar to that shown in FIG. 4,
except that a known force is applied to the structure, and a
corresponding displacement is measured.
[0060] Box 90 corresponds to the application of a known force, Box
92 corresponds to a determination of corresponding compression,
extension, twist, bend or other deformation, and Box 94 corresponds
to the application of a force with a magnitude correlated with the
determined modulus.
[0061] In another embodiment, a compressive force is applied to the
end of a cultured bone and the consequent bending determined,
allowing an elastic modulus to be determined.
[0062] Displacement sensors can use any conventional technique,
such as micrometers, laser reflection, capacitance, and other
effects.
[0063] Force sensors also can use any conventional technique, such
as piezoelectric effects, and other stress and/or strain
sensors.
Other Embodiments
[0064] Other means of applying forces to a cultured construct
include using a pumped fluid (such as compressed air), a flexible
or elastic structure or sheet, a thermal expansion element, other
piezoelectric or electrical induced expansion elements, gravity,
magnetic fields (such as attraction or repulsion between magnetic
elements), shock waves, acoustic waves, impacts, motors, expansion
chambers, or other elements or devices which expand, contract, or
deform in a controllable manner.
[0065] FIG. 6 shows a device in cross-section having frame 100,
supporting a culture vessel comprising cylindrical housing 102 and
housing end 104. Scaffold 108, of some biocompatible material, is
supported by first and second supports 106 and 110. The frame 100
supports a motor-driven micrometer 114, having shaft 112
controlling position of housing end 104, and hence the force on a
cultured bone grown on the scaffold 108. The motor driven
micrometer comprises a force sensor. Micrometer position and force
readings are electrically accessible through contacts 116. The
housing 102 can be rotated about the long axis periodically to
average the effects of gravity. Growth culture medium inlet and
outlet connectors are provided on the housing 102 or housing end
106 (not shown). The shear flows generated by housing rotation can
further be used to improve the properties of grown culture matrix
constructs.
[0066] FIG. 7 shows another system having a culture vessel 120
further comprising an electromagnet 122 embedded in the vessel, the
electromagnet providing a variable repulsive force to suitable
aligned magnet 124, the magnet propagating the force to scaffold
128 (or culture or matrix) through scaffold attachment 126.
Scaffold holder 130 is connected to force sensor 132, which
measures the force applied to the scaffold 128. This system is
advantageous over other systems (such as disclose in U.S. Pat. No.
6,191,042) in that the electromagnet is contained within the
culture vessel, and in that a repulsive magnetic force is
applied.
[0067] FIG. 8 shows a further culture vessel having housing 150,
containing flexible membrane 152. culture medium is contained by
the housing below the flexible membrane. Scaffold 148 is supported
by first and second supports 142 and 144, and support 144 is
connected to the membrane by spacer element 146. An electrically
controlled actuator 154, having electrical contacts 156, applies a
compressive force to the scaffold through shaft 152 and through the
membranes.
[0068] The supports or holders for the scaffold material can be any
convenient design, such as clips, adhesives, cement, hook-and-loop
structures (hooks and/or loops can be provided by the matrix), and
the like.
[0069] Other mechanisms can be used to apply compressive or
extensive force to the scaffold and growing matrix construct
through the membrane, such as gas pressure (or vacuum), weights
placed on the membrane (which can be increased with time),
expansion chambers, expanding materials such as hydrating gels,
springs, torsion wires, elastic structures, compressed resilient
materials, and the like. In other embodiments, such mechanisms can
be used in place of the actuator shown in the system of FIG. 1.
Acoustic waves and mechanical shock waves can also be applied to
provide tissue compression.
[0070] In addition to the above-referenced mechanical constructs, a
"joint simulator" such as that used for testing prosthetic joint
implants or performing experimentation (including cadaveric joints)
may be used with appropriate load cells applied to recreate the
appropriate forces to induce a desired matrix construct from cell
culture. An applicable fixture is illustrated in FIG. 9 with
respect to a knee simulator within a culture vessel, with the
understanding that other joints, including the hip, shoulder,
spine, elbow and those within the hands and feet may be
accommodated through appropriate modification. In FIG. 9, a distal
femur is shown at 902, interacting with a proximal tibia 908. An
actuator 906 is used with respect to the distal femur, in
conjunction with a force sensor 904, whereas actuator 910 is used
with sensor 909 relative to the tibia 908. Flow into the vessel
through conduit 912 is regulated at 914, with flow out through
conduit 916 being regulated by device 918. A scaffold vessel for
chondrocytes is depicted at 990, along with optional flexible
fusion 991. Forces are applied through the actuator(s) to simulate
the load of walling, compression, gliding and torsion may be
simultaneously simulated as well.
[0071] Through the use of a system such as that depicted in FIG. 9,
the culture vessel, with appropriate scaffold, positioned as
necessary to simulate the forces encountered in a natural joint. In
this way, multiple small tissue matrix constructs, or fewer larger
tissue matrix constructs, may be developed simultaneously. In
addition, composite tissue constructs may be created, such as
bone-tendon-bone or cartilage-bone, which would make implantation
into patients more predictable. Either a joint simulator of the
type shown in FIG. 9 or alternative, modified systems to use with
one or more scaffolds with one or more cultured vessels and
multiple cell lines or pluripotential stem cells exposed to
different media and forces may be used to illicit the production of
composite matrix components according to the invention.
[0072] Through the use of certain of the chambers referenced above,
it may be further possible to induce and modify matrix constructs
without the need for a pre-existing scaffold. As shown in FIGS. 10A
and 10B, a deformable culture vessel containing culture medium 210
and inlet/outlet ports 214 and 212, respectively, may be modified
by force 202 through actuator 200 to provide a compression of the
medium 210, or, with a force of a different direction such as 203
shown in FIG. 10A, a tension may be realized. As described
elsewhere herein, the force may be long-term, intermittent, or even
randomly effectuated, through oscillation, for example, using any
of the actuation devices herein disclosed.
[0073] FIG. 11 depicts a further option in the form of a "piston"
type of culture vessel including portions 302 and 304 movable and
rotatable with respect to one another by virtue of a flexible seal.
The culture medium 310 may be introduced through port 314 and
expelled through port 316, while compression, tension, or torsional
forces may be applied to either side of the system, through
oscillatory or other types of interaction.
[0074] In addition to, or instead of, mechanical forces, cultured
tissue can be exposed to other processes to improve properties. In
the case of bone growth, the cultured bone may be exposed to
electromagnetic radiation, electric fields, magnetic fields,
electrolytic effects, chemical exposure, biomolecule exposure (e.g.
exposure to enzymes, hormones, and the like), thermal processes
such as thermal cycling, chemical effects including photochemical
effects, ion implantation, other radiation exposure including
ultrasound exposure, and other effects so as to improve bone
quality. These processes can be performed on tissue cultures within
the culture vessel, or where appropriate, outside of the vessel,
such as prior to implantation in a patient.
[0075] The matrix structure (or scaffold) can be non-uniform in
cross section. For example, the density can be higher around the
edges. Using a fibrous scaffold to support bone growth, the fiber
density can be higher around the periphery of the matrix structure.
This approach is useful to grow bones having a natural structure
with a higher density around the periphery. The force applied to
the bone construct can also be non-uniform, for example using an
array of actuators. For example, using an array of programmable
actuators, such as piezoelectric actuators controlled by a computer
system, a higher force can be applied around the periphery of the
bone construct, so as to encourage the growth of stronger bone
around the periphery. The force can be lower in central regions.
The force applied to the bone growth can be correlated with the
density of the bone growth, for example as determined using
ultrasonic attenuation. The force applied can also be correlated
with the piezoelectric response of the bone construct. The culture
environment of the bone growth can also be varied, for example
through release of chemicals, hormones, bioactive agents and the
like, through additional fluid inlets, local heating, time-release
elements incorporated into the matrix structure, variable surface
processing of the scaffold material, non-uniform irradiation of the
growing structure (e.g. with electromagnetic radiation), and the
like. Scaffold materials can also be derived from organic sources,
such as animal bone processed to substantially remove organic
components.
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