U.S. patent application number 11/419918 was filed with the patent office on 2006-10-12 for layered aligned polymer structures and methods of making same.
This patent application is currently assigned to Cambridge Polymer Group, Inc.. Invention is credited to Gavin J. C. BRAITHWAITE, Jeffrey W. Ruberti.
Application Number | 20060228401 11/419918 |
Document ID | / |
Family ID | 46123743 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060228401 |
Kind Code |
A1 |
BRAITHWAITE; Gavin J. C. ;
et al. |
October 12, 2006 |
LAYERED ALIGNED POLYMER STRUCTURES AND METHODS OF MAKING SAME
Abstract
This invention includes a method of producing a thin, oriented
layer of polymer material. The material is preferably produced by
the method of introducing a shearing flow to a free surface in a
predominantly monomeric solution of the self-assembling polymer
sub-units, and inducing polymerization or growth of the monomer
while in this shearing flow. The system for forming the oriented
layer of material provides relative movement between a delivery
system and the substrate on or over which the material is
deposited. The rate of flow of the material from the delivery
system and the relative velocity between the deposition surface and
the material as it is developed to the surface are controlled to
properly orient the material and desired thickness. These rates can
be adjusted to vary the properties of the film in a controlled
manner. Preferred embodiments include either angular or linear
relative movement between the delivery system and the
substrate.
Inventors: |
BRAITHWAITE; Gavin J. C.;
(Cambridge, MA) ; Ruberti; Jeffrey W.; (Lexington,
MA) |
Correspondence
Address: |
PROSKAUER ROSE LLP
1001 PENNSYLVANIA AVE, N.W.,
SUITE 400 SOUTH
WASHINGTON
DC
20004
US
|
Assignee: |
Cambridge Polymer Group,
Inc.
Boston
MA
|
Family ID: |
46123743 |
Appl. No.: |
11/419918 |
Filed: |
May 23, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10306825 |
Nov 27, 2002 |
7048963 |
|
|
11419918 |
May 23, 2006 |
|
|
|
60337286 |
Nov 30, 2001 |
|
|
|
Current U.S.
Class: |
424/443 ;
530/356 |
Current CPC
Class: |
B29K 2089/00 20130101;
A61L 27/50 20130101; B29C 41/52 20130101; C08L 89/06 20130101; B29C
41/22 20130101; A61L 27/24 20130101; B29C 2037/90 20130101; A61L
2400/18 20130101; B29C 67/0003 20130101; D01D 5/18 20130101; B29C
41/045 20130101; B29C 48/022 20190201; B29C 41/36 20130101; B29C
67/24 20130101; D01F 4/00 20130101; B29C 48/08 20190201; B29K
2995/005 20130101; A61L 27/38 20130101; D01D 5/38 20130101 |
Class at
Publication: |
424/443 ;
530/356 |
International
Class: |
A61K 9/70 20060101
A61K009/70; A61K 38/39 20060101 A61K038/39 |
Claims
1. A method of producing a thin film of oriented polymer
structures, comprising the steps of: controlling the flow of a
polymer solution into a device having a substrate, the device
generating a shear flow to induce alignment of polymer structures;
controlling a plurality of parameters during polymerization; and
generating a layer of oriented polymer.
2. The method of claim 1, wherein the polymer is a biopolymer such
as collagen.
3. The method of claim 2 wherein the method further comprises the
steps of: mixing a solution of collagen with phosphate buffered
saline solution; adjusting the pH of the solution to 7.4.+-.0.2;
applying the solution at a controlled rate onto a substrate which
generates a shearing flow; causing preferential orientation of the
gelling collagen fibrils; and generating successive layers, each
layer representing a portion of the component.
4. The method of claim 3, wherein the layers have a uniform,
controllable thickness ranging from sub-micron to 100 microns.
5. The method of claim 2 wherein the collagen is either type I or
type V collagen.
6. The method of claim 1, wherein the principle orientation of the
aligned fibrils in a single layer alternates in each successive
layer.
7. The method of claim 3, wherein the angle between the principle
orientation of each layer is approximately in the range of 0 to 180
degrees.
8. The method of claim 1, wherein the solution properties,
including temperature, concentration and surfactant composition are
controlled.
9. The method of claim 1, wherein the shear flow is generated by
spinning the substrate at a controlled rate in a range of
approximately 50 to 50,000 Hz.
10. The method of claim 1, wherein the shear flow is generated by
drawing the substrate out of the collagen solution.
11. The method of claim 1 where the atmosphere is controlled to a
specified temperature and relative humidity.
12. The method of claim 1, wherein the solution conditions are
modulated to control the polymerization kinetics and
morphology.
13. The method of claim 1, wherein the use of shear flow aligns
polymerizing polymer chains in a layer such that polymers are
predominantly aligned parallel to each other.
14. The method of claim 1, further comprising angular rotation of
the substrate providing shear flow and confinement to orient the
polymerized polymers.
15. The method of claim 14, wherein an input flow rate, solution
viscosity and substrate rotational velocity combine to produce a
shear rate between 1 s.sup.-1 and 500,000 s.sup.-1.
16. The method of claim 14, wherein an input flow rate, solution
viscosity and substrate rotational velocity combine to produce a
shear rate preferably between the range 10 s.sup.-1 and 10,000
s.sup.-1.
17. The method of claim 1, wherein a second aligned polymer layer
is produced on top of a first polymer layer by repeating the
method.
18. The method of claim 17, wherein a rotating surface is moved to
change a deposition direction on the substrate.
19. The method of claim 17, wherein the second layer comprises a
different material than the first layer.
20. The method of claim 17, wherein the second layer is a promoter
of at least one of cell adhesion and proliferation.
21. The method of claim 17, wherein an additional layer comprising
collagen type IV and cell adhesion proteins such as, laminin,
fibronectin and/or any integrin is receptor is deposited between
aligned polymer layers.
22. The method of claim 15, wherein a construct of a plurality
aligned layers is used as a replacement or repair of the human
stroma.
23. The method of claim 17, wherein the alignment of the polymers
in a plane of second and subsequent layers is predominantly
parallel with the alignment of the polymers in a plane of the layer
in the first layers.
24. The method of claim 17, wherein the alignment of the polymers
in a plane of a layer in a second and subsequent layers is
predominantly orthogonal with the alignment of the polymers in the
plane of a layer in the first layers.
25. The method of claim 17, wherein the alignment of the polymers
in a plane of a layer in the second and subsequent layers does not
have a defined angular relationship to the alignment of the
polymers in a plane of a layer in the first layers.
26. The method of claim 1, wherein the end-associating biopolymer
monomer is included in an aqueous solution.
27. The method of claim 26, wherein the biopolymer monomer is
collagen.
28. The method of claim 26, wherein the biopolymer monomer is
extracted or recombinant collagen.
29. The method of claim 26, wherein the collagen is Type I as the
polymerizing medium.
30. The method of claim 26, wherein the collagen is Type I and Type
V to assist in creation of heterotypic fibrils.
31. The method of claim 1, wherein the polymer solution is injected
at a constant rate.
32. The method of claim 1, wherein the polymer solution is injected
with a flow rate between 0.05-1000 ml/min.
33. The method of claim 1, wherein the material is preferably
injected with a flow rate between of 0.1-100.0 ml/min.
34. The method of claim 1 further comprising a post-processing step
including spinning off any effluent material from the
substrate.
35. The method of claim 1, further comprising the substrate and a
substrate holder being modified to minimize waste of polymerization
solution.
36. The method of claim 1, wherein the solution is preferably
composed of 8:1:1 ratio of collagen type I (3 mg/ml) to
10.times.PBS to 0.1 M NaOH with pH adjusted to 7.4.
37. The method of claim 1, wherein the viscosity of the solution is
between 1 mPas and 100 Pas.
38. The method of claim 1, where the viscosity solution is
preferably between 5 mPas and 1 Pas.
39. The method of claim 1 wherein the substrate comprises one of a
flat surface or curved surface.
40. The method of claim 39, wherein the flat surface is optically
smooth.
41. The method of claim 39, wherein preferably the flat surface has
a surface roughness of approximately less than 10 microns.
42. The method of claim 39, wherein the substrate is a borosilicate
glass disk.
43. The method of claim 1, wherein a surface of the substrate is
treated to control adhesion of the polymer and wetting of the
solution.
44. The method of claim 1, wherein a surface of the substrate is
ultrasonicated in 10% micro90 (Brand) cleaner for a time
duration.
45. The method of claim 1, wherein a surface of the substrate is
plasma cleaned.
46. The method of claim 1, wherein a surface of tie substrate is
homogeneous.
47. The method of claim 1, wherein the substrate has a surface
treatment that is heterogeneous.
48. The method of claim 1, wherein the substrate has a surface
treatment that is patterned.
49. The method of claim 1, wherein a substrate is patterned to
constrain the flow.
50. The method of claim 1, wherein a surface of the substrate and
atmospheric conditions are modulated to control self-assembly.
51. The method of claim 50, wherein the atmospheric conditions
include a temperature range of 30.degree. C.-45.degree. C. and
humidity range of 80-100%.
52. The method of claim 51, wherein the preferred range is
35.degree. C.-42.degree. C. with 90-100%.
53. The method of claim 1, wherein a substrate of the rotation
velocity is used to control layer thickness and final polymerized
material morphology.
54. The method of claim 53, wherein a layer thickness is between
100 nm and 1 mm.
55. The method of claim 53, wherein layer thickness is preferably
between 0.5 .mu.m and 100 .mu.m.
56. The method of claim 53, wherein the substrate rotational
velocity is varied.
57. The method of claim 53, wherein the velocity is initially
between 10 to 5,000 rpm.
58. The method of claim 53, wherein the velocity is preferably
initially between 60 to 1,000 rpm.
59. The method of claim 53, wherein the velocity during
polymerization is constant.
60. The method of claim 53, wherein the velocity during
polymerization is varied.
61. The method of claim 53, wherein the velocity is in the range
100 to 50,000 rpm.
62. The method of claim 53, wherein the velocity is preferably in
the range 500 to 10,000 rpm.
63. The method of claim 53, wherein the average velocity is in the
range 100 to 50,000 rpm.
64. The method of claim 63, wherein the average velocity is
preferably in the range 500 to 10,000 rpm.
65. The method of claim 1, wherein additives are injected with the
polymer solution to control the polymerization process and final
morphology of the layer.
66. The method of claim 65, wherein the additives are
proteoglycans.
67. The method of claim 65, wherein the additives are at least one
of chondroitin sulfate, dermatan sulfate and keratan sulfate
proteoglycans.
68. The method of claim 65, wherein the proteoglycans are one of at
least or a combination of decorin, lumican, biglycan, keatocatn or
syndican.
69. The method of claim 65, wherein the percent (by weight) of
added proteoglycans is between 0.25 and 50.0.
70. The method of claim 65, wherein the percent by weight of added
proteoglycans is between 0.5 and 10.
71. The method of claim 1, wherein a network of channels is used to
guide the growth of the polymerizing polymers.
72. The method of claim 71, wherein the growing polymer is attached
to a fixed point and extruded from a channel as it polymerizes.
73. The method of claim 71, wherein the growing polymer is attached
to a moving plate pulled through a channel where conditions
conductive to polymerization are maintained.
74. The method of claim 71, wherein conditions outside the channels
are not conductive to polymerization.
75. The method of claim 71, where in said channel is part of an
array of identical channels.
76. The method of claim 71 wherein said channel is treated to
prevent adhesion of polymerizing material.
77. The method of claim 71, wherein said channels are manufactured
using any standard microfabrication process.
78. The method of claim 71, wherein said channels are obtained from
a self-assembled three dimensional network.
79. A system to align polymerizing polymer chains in a layer such
that polymers are predominantly aligned parallel to each other,
comprising: an apparatus to generate shear flow; a plurality of
sensors to monitor a plurality of parameters; and a processor to
modulate a plurality of control parameters.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/337,286 filed on Nov. 30, 2001, the entire
contents of which are incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] Accurate control of the orientation of polymeric structure
in thin layers is desired to maximize their mechanical, chemical,
and optical properties. While orientation can be preformed through
mechanical means, it is often more desirable to orient the
structure during the polymerization process, particularly if the
process involves polymerizing the system into a specific final
shape, where further mechanical manipulation is unfeasible.
[0003] Biomedical componets often require oriented structures.
Tendons, for example, contain highly oriented collagen fibrils, and
the spinal intervertebral disc is composed mainly of oriented
crystalline collagen fibrils and amorphous hydrophilic
proteoglycan. The prevalence of oriented collagen in the human body
makes formation of highly oriented layers of the polymer a desired
goal in modeling of tissue structure. Collagen is a biopolymer and
protein, and found in the structure of tendons, skin, bones and
blood vessels. Randomly oriented collagen materials are weak, and
degrade quickly when exposed to mechanical stress. Soluble collagen
is derived from animal tissue, and can be obtained in a monomeric
form. The collagen monomer will polymerize, termed
"fibrillogenesis," to form a gel-like structure. This process is
achieved when the collagen is in a solution of specific pH,
temperature, and ionic strength. The polymerization occurs as a
self-assembly process, producing native-type collagen fibers. The
fibers grow into a porous gel matrix.
[0004] The collagen molecule is a rod-like structure in the
unaggregated state, composed of three peptide chains interwined to
form a triple helix. Improvements over randomly oriented gels in
mechanical and optical properties can be realized if the aggregated
molecules can be coerced into an oriented, or aligend, state. Cast
films of collagen can form a randomly oriented gel structure, which
lacks desired mechanical and optical properties. It is desirable to
give a structured order for optical transparency in corneal
replacements.
[0005] Collagen fiber orientation techniques have included
mechanical deformation of already gelled matrices and laminar flow
of a getting matrix. These approaches yield gelled layers hundreds
of microns thick.
SUMMARY OF THE INVENTION
[0006] This invention includes a method of producing a thin,
oriented layer of polymer material. The material is preferably
produced by the method of introducing a shearing flow to a free
surface in a predominantly monomeric solution of the
self-assembling polymer sub-units, and inducing polymerization or
growth of the monomer while in this shearing flow. The system for
forming the oriented layer of material provides relative movement
between a delivery system and the substrate on or over which the
material is deposited. The rate of flow of the material from the
delivery system and the relative velocity between the deposition
surface and the material as it is delivered to the surface are
controlled to properly orient the material at the desired
thickness. These rates can be adjusted to vary the properties of
the film in a controlled manner. Preferred embodiments include
either angular of linear relative movement between the delivery
system and the substrate.
[0007] It should be noted that deposition off the polymer solution
can occur on a substrate having curved or spherical surfaces to
result in stress-free interlayer boundaries. This embodiment is
enabling for corneal constructs.
[0008] In a preferred embodiment, the step of controlling the
temperature, pH, solvent chemistry, and relative humidity during
the polymerization process is preformed on a local level.
[0009] The preferred embodiment of the present invention further
comprises a layered construct composed of layers of sub-micron to
10 .mu.m thick oriented polymeric films or fibers, with the
principle direction of orientation alternating in each subsequent
layer. A preferred embodiment of the invention uses this method to
form synthetic biocompatible or biopolymeric materials such as
implantable tissue material. These materials cab be implanted as
soft tissue replacement or for bone or joint replacement or
repair.
[0010] In another preferred embodiment of the present invention,
the method further includes inducing fibrillogenesis of the
collagen while in this shearing flow. The method further includes
controlling the collagen monomer concentration, temperature,
solution properties and relative humidity of the fibillogensis
process, producing collagen material having an oriented fibrillar
structure in a sheet with a uniform, controllable thickness. The
thickness can range from approximately 500 nm to 100 .mu.m.
[0011] In accordance with another preferred embodiment, the method
for producing a multi-layer construct can be used to form an
artificial corneal construct. The collagen layers can be seeded
with endothelial and epithelial cells, which generate a negative
pressure field in the construct, and compress the construct to a
thickness necessary for optical transparency.
[0012] A preferred embodiment includes a method of producing a thin
film of oriented polymer structures, including the steps of
controlling the flow of a polymer solution into a device having a
substrate, the device generating a shear flow to induce alignment
of polymer structures, controlling a plurality of parameters
including polymerization; and generating a layer of oriented
polymer. The method further includes the polymer being a biopolymer
such as collagen. The method further comprises the steps of mixing
a solution of collagen with phosphite buffered saline solution,
adjusting the pH of the solution to 7.4.+-.0.2, applying the
solution at a controlled rate onto a substrate which generates a
shearing flow, causing preferential orientation of the gelling
collagen fibrils; and generating successive layers, each layer
representing a portion of the component. The method layers have a
uniform, controllable thickness ranging from sub-micron to 100
microns. The collagen is either type I or type V collagen. The
principle orientation of the aligned fibrils in a single layer
alternates in each successive layer. The angle between the
principle orientation of each layer is approximately in the range
of 0 to 180 degrees. The solution properties, including
temperature, concentration and surfactant composition are
controlled. The shear flow is generated by spinning the substrate
at a controlled rate in a range of approximately 50 to 50,000
Hz.
[0013] In a preferred embodiment the shear flow is generated by
drawing the substrate out of the collagen solution. Further, a
preferred embodiment includes a system to align polymerizing
polymer chains in a layer such that polymers are predominantly
aligned parallel to each other, having an apparatus to generate
shear flow, a plurality of sensors to monitor a plurality of
parameters; and a processor to modulate a plurality of control
parameters.
[0014] The foregoing and other features and advantages of the
system and method for producing a multilayer construct of
sub-micron to 10 micron thick layers of oriented collagen fibers
will he apparent from the following more particular description of
preferred embodiments of the system and method as illustrated in
the accompanying drawings in which like reference characters refer
to the same parts throughout the different views. The drawings are
not necessarily to scale, emphasis instead being placed upon
illustrating the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 illustrates a preferred embodiment of the apparatus
used to generate a single layer of oriented self-assembled polymer
molecules in accordance with the present invention.
[0016] FIG. 2 illustrates a flow chart for producing aligned
collagen fibers in accordance with a preferred embodiment of the
present invention.
[0017] FIGS. 3A and 3B illustrate electron micrographs of collagen
fibers prepared by a spin-coating method where FIG. 3A particularly
illustrates random alignment of fibers and FIG. 3B illustrates
fibers aligned in flow field in accordance with preferred
embodiments of the present invention.
[0018] FIGS. 4A and 4B illustrate a preferred embodiment of the
apparatus used to generate a matrix of orthogonally-aligned layers
in accordance with the present invention.
[0019] FIGS. 5A-5C illustrate another preferred embodiment of all
apparatus used to generate a matrix of orthogonally-aligned layers
in accordance with the present invention.
[0020] FIGS. 6A-6C illustrate schematically a folding method to
generate a multilayer structure with orthogonally-aligned layers in
accordance with a preferred embodiment of the present
invention.
[0021] FIG. 7 schematically illustrates collagen fibers in the
lamellae of the stroma in accordance with a preferred embodiment of
the present invention.
[0022] FIG. 8A illustrates an apparatus having an offset holder
wherein a monomer solution is deposited onto the substrate at the
center of the offset disk holder in accordance with a preferred
embodiment of the present invention.
[0023] FIG. 8B illustrates a flow chart of a preferred embodiment
for producing aligned collagen fibers in accordance with a
preferred embodiment of the present invention.
[0024] FIG. 9 is a scanning electron microscope (SEM) image
demonstrating the deposition of a plurality of thin aligned layers
onto a glass substrate in accordance with a preferred embodiment of
the present invention.
[0025] FIG. 10 is a scanning electron microscope (SEM) image of a
single layer of predominately aligned collagen fibrils in
accordance with a preferred embodiment of the present
invention.
[0026] FIG. 11 is a scanning electron microscope (SEM) image of
aligned collagen fibrils generated by the spin-coating methodology
in accordance with a preferred embodiment of the present
invention.
[0027] FIG. 12 illustrates a scanning electron microscope (SEM)
image demonstrating layering of collagen in pseudolamellae in
accordance with a preferred embodiment of the present
invention.
[0028] FIG. 13 is a scanning electron microscope (SEM) image
illustrating the interaction of two individual layers of aligned
collagen in accordance with a preferred embodiment of the present
invention.
[0029] FIGS. 14A-14C schematically illustrate a nanofabrication
system and a flow-focussing method to manufacture layered, aligned
polymer structures in accordance with a preferred embodiment of the
present invention.
[0030] FIG. 15 schematically illustrates another preferred system
to manufacture layered, aligned polymer structures in accordance
with a preferred embodiment of the present invention.
[0031] FIGS. 16A and 16B illustrate a block diagram and a schematic
diagram, respectively, of a preferred embodiment system to
manufacture layered, aligned polymer structures in accordance with
a preferred embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The systems and methods of the present invention relate to
processes and resulting material, suitable for the guided
production of the self-assembly of aligned polymers in layers, and
structures having multiple, non-aligned layers. More particularly
the systems and methods of the present invention relate to the
production of self-assembled biocompatible non-aligned thin
lamellae.
[0033] This invention will be better understood with reference to
the following definitions:
[0034] "Self-assembly": a process by which a system spontaneously
combines a number of smaller structural elements to form a large
molecular or supramolecular complex. Self-assembly is the process
by which many biological complexes form, such as DNA and collagen.
This reaction sometimes requires that a threshold temperature,
solution condition, or pH is reached before the spontaneous
reaction occurs.
[0035] "Gel": a material that is crosslinked through bonds of
sufficient strength that it cannot be dissolved in ambient
conditions. The bonds can be covalent ionic, physical, or
other.
[0036] "Collagen": a self-assembling biopolymer produced by
reacting monomeric collagen in a neutral pH, slightly ionic
solution. Collagen is a structural building block in the body, and
is found in the cornea, skin, bones, and blood vessels.
[0037] "Polymerization": the process by which monomer-sized
molecules are assembled to form supramolecular structure. This
process can occur through the formation of permanent chemical
bonds, ionic bonds, or associative bonds. Fibrillogenesis is a form
of polymerization in collagen.
[0038] "Polymer": any supramolecular structure comprised of
repeating subunits. These structures can be naturally occurring,
such as proteins, or man-made, such as polyolefins.
[0039] A preferred embodiment of the invention comprises a method
to produce a highly oriented, thin film of self-assembled polymer.
The method comprises the process of subjecting a solution of
monomeric building blocks of the self-assembling polymer to a
shearing flow. While under the influence of this shearing flow, the
polymer self-assembles, forming oriented polymeric structure in a
thin film from approximately 500 nm to 100 .mu.m thick. The
preferred embodiments of the present invention provide for several
processes of generating this shearing flow, examples of which are
described herein below. The systems and methods of the present
invention also provide for a process of forming constructs with
multiple layers of aligned polymer structures, each layer having a
different principle direction of orientation. The preferred
embodiments provide for a method of forming constructs in both
biopolymers and synthetic polymers.
EXAMPLE 1
[0040] As one example of a preferred embodiment of this present
invention, a metal disk is used as a deposition substrate. The disk
is mounted on a spin-coating-type apparatus. A chamber is built
around the apparatus to control the temperature and the relative
humidity. The disk can be spun at a specified rate. This embodiment
is illustrated in FIG. 1.
[0041] A solution of Vitrogen collagen is chilled to 4-6.degree. C.
Eight ml of the collagen solution is mixed with 1 ml of 10.times.
phosphate-buffered saline solution (0.2 M Na.sub.2HPO.sub.4, 1.3M
NaCl, pH=7.4) and 1 ml of 0.1M NaOH. The pH is adjusted to
7.4.+-.0.2 by adding 0.1M HCl. The solution is warmed to the test
temperature, then steadily dripped onto the rotating substrate 16.
The collagen gels form a uniform sub-micron thick sheet. This
process is described with respect to the flow chart illustrated in
FIG. 2. Nematic stacks are prepared by cutting out sections of the
radially-aligned collagen fiber sheets, and stacking them
orthogonally by hand. Electron micrographs of randomly oriented
collagen fibers and oriented collagen fibers in a single layer
prepared by this method are shown in FIGS. 3A and 3B,
respectively.
EXAMPLE 2
[0042] As a second example of a preferred embodiment of the present
invention, sample disks 104 with T-slots cut into their surfaces
are mounted on a disk 102 rotating at a specified rate as shown in
FIGS. 4A and 4B. In accordance with a preferred embodiment of the
present invention the first layer of aligned associating fibrils is
prepared by flowing the polymeric solution into channel of sample
disks, which are on the rotating bottom disk. The centrifugal
motion generates a shearing flow, which produces a thin layer and
adds in aligning the growing associating polymers. The sample disks
are then rotated 90 degrees, or to another specified angle, and the
second layer is applied, which aligns with the specified angle
relative to the first. A solution of Vitrogen collagen is chilled
to 4-6.degree. C. Eight ml of the collagen solution is mixed with 1
ml of 10.times. phosphate-buffered saline solution (0.2 M
Na.sub.2HPO.sub.4, 1.3M NaCl, pH=7.4) and 1 ml of 0.1M NaOH. The pH
is adjusted to 7.4.+-.0.2 by adding 0.1M HCl. The solution is
warmed to the test temperature, then steadily dripped onto the
center of the rotating disk, so that the solution travels down the
channels extending to the outer radius of the rotating disk. After
the first layer has gelled into an oriented collagen film, the
sample disks are rotated 90 degrees, and the second layer is
applied, so that the orientation direction of the second layer is
90 degrees with respect to the first layer. This process is
repeated many times to generate a matrix of orthogonally-oriented
collagen fibril layers.
EXAMPLE 3
[0043] Another preferred embodiment of the present invention
includes the monomer solution being placed in a
temperature-controlled bowl as shown in FIGS. 5A-5C. In FIG. 5A,
the polymer solution is subjected to a shearing flow by rotating
the ball in one direction, or by oscillating in the same rotation
plane. The polymer associates during this process. In FIG. 5B, the
ball is rotated or oscillated in the orthogonal plane, to generate
a layer of associated polymers orthogonal to the first layer. In
FIG. 5C a three-dimensional (3D) rendering of the method is
illustrated. A ball 122 with a diameter a few microns smaller than
the bowl 128 diameter is placed in the bowl. A shaft 126 attached
to the ball rotates the ball first in one direction, generating a
shearing flow, during which time the monomer polymerizes. After a
designated gelation period, fresh monomer solution may be
introduced to the bowl, and the ball is rotated in the orthogonal
direction, creating a layer orthogonal aligned to the first
layer.
EXAMPLE 4
[0044] As another preferred embodiment of the material described in
this invention, epithelial cells and endothelial cell layers are
established in the structure generated in either of the first three
examples with collagen to develop a corneal construct. This
construct produces a cornea that is similar physiologically,
ultrastructurally and biochemically to normal mammalian corneas.
The construct is suitable as a physiological or biomechanical
model, and may find utility as a material for transplant into human
subjects.
EXAMPLE 5
[0045] As another embodiment of the material described in this
invention, highly oriented collagen layers prepared as described
herein can be bundled to form the annulus fibrosus fund in the
spinal intervertebral disk. This disk acts primarily as a
weight-bearing and flexible joint. The load bearing capability and
flexibility in selected directions is achieved by the combination
of the annulus fibrosus and nucleus pulposus. Annulus fibrosus is a
layered structure that is rigid in the radial direction bit
deformable in the axial direction and by torque. This structure has
alternating layers of oriented collagen fibrils, similar to that
described in the cornea. Each layer has its collagen fibrils wound
at an angle, and subsequent layers have an alternate orientation.
Such a structure helps to achieved a maximum resistance to radial
stress, while allowing a deformation in torque and bending.
EXAMPLE 6
[0046] As another preferred embodiment of this invention, single
layers of aligned polymer structure are generated with the
techniques and methods discussed herein. The layers are folded as
shown in FIGS. 6A-6C, to generate successively crossed layers of
aligned polymer fibrils or other superstructure.
[0047] The extracellular matrix of the mature intact cornea
comprises an extremely varied yet highly structured array of
collagen, proteoglycans, glycoproteins and soluble
macromolecules.
[0048] Current attempts to generate a biomimetic cornea construct
have yielded corneas that behave similarly to in vivo corneas with
respect to the function of the cellular layers. However, these
corneal constructs have severe limitations with regard to the
structure of the stromal extracellular matrix (ECM), which was
constructed to ensure biocompatibility, nominal transmittance and
the ability to promote adherence of the superficial cell layers and
little more. It appears that previous investigators focused on the
cellular layers but lost sight of the importance of the stroma
itself. The three major functions of the cornea: protection,
refraction and transmission, are performed primarily by the stromal
ECM, the structure of which is optimally designed to accomplish
these objectives. Thus, the cellular layers serve only to maintain
and defend the stroma, which provides the principle functions of
the cornea:
[0049] Strength. The major structural collagen of the stroma (type
I collagen) is arranged in 300 to 500 lamellae of parallel,
non-crosslinked fibrils. The lamellae are stacked in the anterior
posterior (AP) direction and the fibrils of adjacent lamellae are
nearly perpendicular to each other. Free ends of the fibrils have
not been discerned in the cornea, which suggests that they run
uninterrupted from limbus-to-limbus. This "plywood" arrangement of
the lamellae give the cornea remarkable strength tangential to the
surface. Randomly crosslinked collagen networks of similar
thickness (used in current constructs) cannot provide similar yield
strength.
[0050] Refraction. The ability of the fibrils to slide relative to
each other with ease allows the natural cornea to distribute the
load imposed by the intraocular pressure (IOP) uniformly. This
enables the anterior surface of the eye to form a nearly perfectly
spherical shape for refraction. Stroma as comprising randomly
cross-linked collagen are likely to form imperfect surfaces for
refraction upon inflation to normal IOP. This is due to the complex
stress fields that would be produced when they are loaded.
[0051] Transmission of light. The uniform diameter of natural
corneal fibrils and their short-range ordering allows light to pass
through virtually unimpeded. Randomly crosslinked collagen
networks, though nominally transparent, cannot produce the same
quality optical properties. The process described in the
embodiments of this invention can produce orthogonally stacked
arrays of aligned type I/type V collagen fibrils. The general
biomechanical and optical properties of such a construct should be
similar to native corneal stroma.
[0052] The difficulties in forming a fully complete, functional,
corneal stroma, constructed de nova, by artificial means have led
some to indicate that it may not be a viable given the technology
available today. However, it is not likely to be necessary to
complete the construction of the stroma purely by mechano/chemical
manipulation of biochemical components in the laboratory. A
partially completed primary corneal stroma comprising orthogonal
layers of aligned type I/type V heterotypic collagen fibers can
suffice as a suitable scaffolding or starting point, as it does
during embryogenesis. In support of this approach, it is known that
following full-thickness trephination wounds in rabbit corneas, the
healing response is capable of transforming the initially opaque
fibrous plug to tissue that is similar to normal corneal stroma.
The initially opaque scars comprise large interfibrillar spaces,
unusually large chondroitin sulfate (CS) proteoglycans, hyaluronic
acid and no detectable keratan sulfate proteoglycan. After one year
of healing and remodeling, normal interfibrillar spacing, size and
distribution of proteoglycans is restored. This mechanism works
even if tissue of similar biochemistry but not structure is
implanted into the cornea. It has been found that the corneal
healing response has the capacity to partially resolve collagen
fibril structure even when scleral tissue is used to repair corneal
wounds (Winkelman 1951 Am J Ophthalmol; Kurz 1953 Cs Oftal; Maurice
an Singh Cornea 1996) incorporated herein by reference in its
entirety. The present invention takes advantage of the natural
healing response and remodeling ability of the corneal stroma. A
stromal scaffolding remodeled by the corneal wound healing
response, can be implanted. However, implanting a biomimetic stroma
that is not sufficiently strong, transparent, or smooth at the
outset is not viable. The artificial, stroma at the outset, must
preserve the optical qualities of the cornea during the remodeling
period. Current corneal constructs, which employ randomly
cross-linked type I collagen fibrils, cannot meet this
requirement.
[0053] The closer the tissue or scaffolding to be remodeled mimics
native stroma, the less time it takes to fully resolve the
structure and fully incorporate the graft. Further, if the initial
scaffolding is capable of performing the three major functions of
the stroma (protection, refraction and transmission), remodeling in
vivo may process while vision remains clinically acceptable.
[0054] Thus, from investigations into the developmental biology of
the chicken, it has been learned that corneal embryogenesis is an
intricately orchestrated and complex phenomenon. Initially, from
superficial epithelial cells derived from an offshoot of the
developing brain, an orthogonally organized primary corneal stroma
of type I collagen is secreted layer-by-layer. The extracellular
matrix (ECM) of the mature intact cornea comprises an extremely
varied yet highly structured array of collagens, proteoglycans,
glycoproteins and soluble macromolecules.
[0055] The three major functions of the cornea: protection,
refraction and transmission, are performed primarily by the stromal
ECM, the structure of which is optimal to accomplish these
objectives. Thus, the cellular layers serve only to maintain and
defend the stroma. In addition the major structural collagen of the
stroma (type I collagen) is arranged in approximately 300 lamellae
of parallel, non-crosslinked fibrils. The lamella are stacked in
the anterior posterior (AP) direction and the fibrils of adjacent
lamella are nearly perpendicular to each other. Free ends of the
fibrils have not been discerned in the cornea, which suggests that
they run uninterrupted from limbus-to-limbus. This "plywood"
arrangement of the lamellae gives the cornea remarkable strength in
tension tangential to the surface. Randomly crosslinked collagen
networks of similar thickness (used in content constructs) cannot
provide similar yield strength.
[0056] The ability of the fibrils to slide relative to each other
with ease, allows the natural cornea to distribute the load imposed
by the intraocular pressure (IOP). This enables the anterior
surface of the eye to form a nearly perfectly spherical shape for
refraction. Stromas comprise randomly cross-linked collagen are
likely to form imperfect surfaces for refraction upon inflation to
normal IOP. When these random networks are loaded, complex stress
fields are formed that result in inhomogeneous refraction and thus
compromised optical qualities.
[0057] The uniform diameter of natural corneal fibrils and their
short-range ordering allows light to pass through the cornea
virtually unimpeded. Randomly crosslinked collagen networks, though
nominally transparent, cannot produce the same quality optical
properties because of the varying spacing in the random
network.
[0058] With regard to biomimetic corneal constructs, recent
attempts have been made to develop full corneal constructs de novo.
These efforts have yielded corneas that behave similarly to in vivo
corneas with respect to the function of the cellular layers.
However, both of these cornea constructs have severe limitations
with regard to the structure of the stromal ECM, which was
constructed to ensure biocompatibility, adequate transmittance and
the ability to promote adherence of the superficial cell layers and
little more.
[0059] Completely artificial corneal replacements, such as the
K-pro, have not met with widespread clinical success. Such devices
are not yet qualified for use in clinical situations where a
transplant would be considered even marginally effective because of
the high potential for devastating complications and the need for
continual high quality follow-up. Current incarnations of corneal
constructs generated from biomimetic materials and live cell layers
do not include physiologically, ultrastructurally or biochemically
realistic stromas. In addition there have been a number of efforts
towards the production of an artificial cornea which have not met
with complete success.
[0060] With regard to effective scaffolding and wound healing
response, a fully complete, functional, corneal stroma, constructed
de novo, by artificial means may not be a viable approach given the
technology available today. However, it is not likely to be
necessary to complete the construction of the stroma purely by
mechano/chemical manipulation of biochemical components in the
laboratory. A partially completed primary corneal stroma comprising
orthogonal layers of aligned type I/type V heterotypic collagen
fibers suffice as a suitable scaffolding or starting point, as it
does during embryogenesis. Such a partial solution would be a
marked improvement over current systems, aligning easy cell
infiltration and critically, immediate functionality both optically
and mechanically. In support of this approach, it is known that
following full-thickness trephination wounds in rabbit corneas, the
healing response is capable of transforming the initial opaque
fibrous plug/scar to tissue that is similar to normal corneal
stroma. The initially opaque scars comprise large interfibrillar
spaces, unusually large chondroitin sulfate (CS) proteoglycans,
hyaluronic acid and no detectable keratan sulfate proteoglycan.
After one year of healing and remodeling, normal interfibrillar
spacing, size, and distribution of proteoglycans is restored. This
mechanism works even if tissue of similar biochemistry, but not
structure is implanted into the cornea. It has been found that the
corneal healing response has a capacity to partially resolve
collagen fibril structure even when scleral tissue is used to
repair corneal wounds. This suggests that it might be possible to
take advantage of the natural healing response and remodeling
ability of the corneal stroma. Stromal scaffolding, which can be
remodeled by the cornea wound healing response can be implanted.
However, implanting a biomimetic stroma that is not sufficiently
strong, transparent, or smooth is not acceptable. The artificial
stroma, at the outset, has to preserve the optical qualities of the
cornea during the remodeling period. Current corneal constructs,
which employ randomly cross-linked type I collagen fibrils, cannot
meet this requirement.
[0061] FIG. 7 illustrates collagen fibers in the lamellae of the
stroma in accordance with a preferred embodiment of the present
invention. The behavior of associate rods (collagen) in solution is
exploited. Collagen fibrils are composed of triple-helix collagen
macromolecules that are approximately 300 nm long and 1.5 nm in
diameter. These segments are produced inside cells and are excreted
as procollagen whereupon the ends are removed to form a
macromolecule that naturally self-assembles. Although there is some
debate over the exact mechanism of this self-assembly process, it
is understood that the assembly of collagen macromolecules into
fibrils is all entropy driven process. The most favorable structure
is therefore a cylinder 184 which minimizes the surface area. This
fibril assembly is very similar to other protein polymerizations.
Each of these cylindrical bundles is composed of segments of
associated molecules in the familiar 67 nm repeating structure.
There is still some debate over the exact molecular process of
association but it appears that the cylindrical bundles of segments
end-associate whereby the terminating C-telopeptide 192 associates
with the N-telopeptide on the adjacent chain. The telopeptides also
influence the diameter of the fibril and the segment packing, but
there are also probably influences from glycoseaminoglycans (GAGs),
proteoglycans, solution temperature and concentration. All of these
components can be controllable in a manufacturing process. Further,
there is some evidence that the association is driven by a further
hydrophobic effect between the telopeptides. These fibrils then
associate to form fibers.
[0062] Thus with no other influence, a solution composed of
collagen macromolecules (i.e. the basic triple helix unit) forms an
isotropic random gel. However, with the correct driving force,
these gels can be forced to grow in an aligned manner. Magnetism,
drainage flows and confining effects have both been used
successfully, but only with weak influence on the morphlology of
the resulting gels.
[0063] Orientation of a rigid-rod in a flow field is determined by
the balance of hydrodynamic forces (aligning) and rotary Brownian
motion (randomizing) which is described by the Peclet number
Pe=II.sub.2D/D.sub.r, where D.sub.r is the Brownian diffusion
coefficient and II.sub.2D is the convective diffusion coefficient.
Simple estimates suggest that the flow field can orient the
collagen monomer because of their size (Pe<1) but that in fact
for the monomers the flow is very close to the transition ranges.
However, as the monomers associate and the collagen fibrils grow,
the aspect ratio increases and consequently the Peclet number
increases rapidly as it is related to the cube of the length of the
rod. Pe = II 2 .times. D 1 / 2 D r = .gamma. . D r Equation .times.
.times. 1 = 8 .times. .pi..eta. x .times. a 3 .times. .gamma. . 3
.times. kT .function. ( ln .function. ( 2 .times. r p ) - 1 / 2 )
.times. .times. for .times. .times. thin .times. .times. prolate
.times. .times. spheroids .times. .times. ( r p >> 1 )
.times. .times. in .times. .times. shear .times. .times. flow ,
Equation .times. .times. 2 ##EQU1## wherein r.sub.P= a/b is the
aspect ratio of spheroid (a is long dimension). At 300 K for rigid
rods of length 300 nm and diameter 30 nm in a solvent of 1 mPas and
at a shear rate of 600 s.sup.-1 the Peclet number is 13.
[0064] In addition, the proximity of walls also act to orient the
monomers in the plane of the wall. This influence is especially
strong in the confined films which are only of an order of
magnitude larger than the longest axis of the collagen monomers.
Concentrated solutions of rigid non-interacting rods are known to
behave in a liquid crystalline fashion, forming nematic structures
where there is long range order in the direction of the long axis
of the rod. In theory, the direction of this ordering is arbitrary
but is usually generated by perturbations, such as the confining
effect of a wall, or solvent flow. Although nematic structures
built from non-interacting rods require a critical concentration to
transition from an isotropic to a nematic phase. In the case of
interacting rods, the situation is more complex, but the
interactions can clearly influence the ability of the molecules to
align. Once fibrils have begun to grow the flowing monomers passing
across the substrate in the shear field naturally end attach since
there is a pronounced anisotropy between side addition and end
addition for interacting rods. In addition magnetic (or electric)
fields are known to strongly align nematic liquid crystals and have
been proven to influence the alignment of growing collagen
gels.
[0065] With regard to the alignment of polymers, it is well known
that flow aligns polymers. Both shear and extensional flows align
polymers in different manners but in general if the flow is strong
enough there is some form of alignment in the direction of the
flow. Spin-coating, and other similar flows, such as film drainage
and steady shear, have been observed to produce roughly oriented
polymers from the melt, for rigid-rod molecules and for liquid
crystals. Alignment in these systems is often however only local
and is usually for systems where the macromolecules themselves are
being aligned. The alignment of growing fibers or polymers is less
well recognized a process.
[0066] It has also been observed that shear induces alignment in
polymers during the polymerization process of rod-like molecules.
However simple shear does not provide sufficient force to counter
the random fluctuations of the chains, aid their natural propensity
move under Brownian motion. The preferred embodiments of the
present invention use spin-coating methods to provide a flow regime
suitable for initiation and growth of aligned collagen fibrils. The
solution conditions can be modulated while the polymerization
process is proceeding, and the naturally radial nature of the flow
assists in maintaining the fiber separation and promotes growth
from a central core area. In addition the spin-coating process
provides a natural length scale that can be easily adjusted to
control the layer thickness and rate of polymerization. Also, since
it is known that the proximity of a surface influences the position
of an adjacent molecule, the thin confinement layer promotes
alignment in the most critical direction, parallel to the layers
longest axis. Spin-coating has been used to produce thin
homogeneous films for a number of years. In melts and liquid
crystals this deposition process is known to align polymers. This
method is extremely well understood for evaporating and
non-evaporating solvents since it is used heavily commercially in
silicon microfabrication processes and in the optics industry.
[0067] With regard to the alignment of collagen, examples of
artificial alignment of collagen are relatively common in the
literature, but rarely result in the highly aligned configuration
that is required for a truly biomimetic system. Previously noted
parameters such as gravity, shear, diffusion and other external
forces can influence the morphology of the fibers, built in all
cases the alignment is weak. Other observations include alignment
of collagen-like self-assemblies using shear while cooling from a
hot solution. All of these cases require relatively concentrated
solutions which limit the control over the fiber morphology. In
addition collagen has been aligned using the natural propensity of
collagen monomers to self-assemble in concentrated solutions.
However, this method allows no control of the fiber sizes and
shapes. To provide the highly aligned morphology two things are
critically required, a thin confinement layer, and a unidirectional
flow. Spin-coating provides both requirements readily. Although
spin-coating has been observed to generate alignment in thin films,
these are usually from melts and in polymerized systems. It should
be noted that alignment as traditionally detected for many of these
systems using Circular Dichroism, does not indicate alignment on
the levels required by a corneal analog.
[0068] FIG. 8A illustrates an apparatus having an offset holder
wherein a monomer solution is deposited onto the substrate at the
center of the offset dish holder in accordance with a preferred
embodiment of the present invention. FIG. 8B illustrates a
preferred methodology to create single and multiple layers of
aligned polymer films in accordance with a preferred embodiment of
the present invention. In this system and method a substrate is
placed off-center on a substrate holder. The holder 206 is rotated
at a prescribed velocity or sequence of velocities to create a thin
shearing flow. The monomer solution 202 is added to the deposition
substrate 210 (either by steady flow or by unsteady dripping) and
is carried radially away from the injection point by the
centripetal acceleration. Thus a single layer (film) of aligned
collagen fibrils is created. To generate multiple layers with
different alignment directions, the deposition substrate 210 is
rotated at any prescribed angle and another layer may be
deposited.
[0069] FIG. 8B is a flow chart describing method 216 for generating
aligned collagen via spin-coating in accordance with a referred
embodiment of the present invention. The method begins with step
218 of obtaining commercially available type I collagen monomers.
They are kept refrigerated at 4-6.degree. C. Extracted or
recombinant human and/or animal collagen type I monomers may be
used. Collagen type I monomer concentrations are in the range of
0.01 mg/ml to 100 mg/ml with a preferred range of 0.5 to 10 mg/ml.
A preferred embodiment utilizes Vitrogen (brand) bovine collagen
(3.0 mg/ml). In an alternate embodiment, if heterotypic fibrils are
being made for diameter control, type V collagen monomer is
obtained as well. Extracted or recombinant human and or animal
collagen type V monomers may be used.
[0070] Per step 220, collagen type I is prepared for
polymerization. In a preferred embodiment, Vitrogen is neutralized
in preparation for self-assembly adding 8:1:1 ratio of collagen
type 1:10.times.PBS: 0.1M NaOH. The pH is adjusted to 7.4. If type
V is also included, the solution is further processed to ensure
neutralization of type V as well. In preferred embodiments,
additives may be used to alter the viscosity of the collagen
solution to change the shear stress on the growing fibrils. Such
additives include, for example, but are not limited to, glycerol.
The viscosity of the final solution of monomer is in the range of
1.0 mPa-s to 100.0 Pa-s. The preferred range is from 5.0 mPa-s to
1.0 Pa-s. In a preferred embodiment, the viscosity of the collagen
monomer solution is approximately 10.0 mPa-s
[0071] The method 216 in accordance with the preferred embodiment
includes the step 222 of substrate selection. The substrate that
accepts the collagen coating may comprise of any material that can
be generated with a uniform optically flat surface to promote the
establishment of a uniform shear flow field during spin-coating.
Asperities on the surface of the material may be in a range of 0.0
to 10 microns, with a preferred range of 0.1 to 0.5 microns. A
preferred embodiment, for example, utilizes a 2 inch diameter
borosilicate glass disk.
[0072] The method 216 also includes the step 224 of preparation of
the substrate to be coated with collagen. Preparation may include
modulation of the surface of substrate to be uniformly hydrophilic,
uniformly hydrophobic, gradient hydrophilic, or to preferentially
bind collagen, for example, antibody inclusion. A preferred
embodiment for hydrophilic treatment for glass substrate utilizes,
for example, 1.5 hour ultrasonication of substrate in 10% micro90
(brand) cleaner. Glass is stored in deionized water until use.
[0073] Per step 222, the substrate to be coated with collagen is
positioned into the device designed to generate centripetal
accelerations. In a preferred embodiment, the substrate is placed
directly onto a vacuum chuck of a commercial spin-coater.
[0074] If multiple layers of collagen oriented at differing
relative angles are desired another substrate handling system is
required, for example, in a preferred embodiment a borosilicate
glass disk is placed in an offset disk holder of FIG. 8 or a system
as shown in FIG. 4 is utilized.
[0075] The method 216 includes the step 228 of modulating the
environment surrounding substrate to create conditions conducive to
initiate polymerization of collagen. The environmental conditions
include a temperature range of 30.degree. C. to 45.degree. C. with
a preferred range of 35.degree. C. to 42.degree. C. and 80-100%
humidity with a preferred range of 90-100% humidity. In a preferred
embodiment, a steam heat humidifier is attached to vent ports in
the housing of the substrate rotating device as shown in FIG. 1. In
alternate embodiments, the substrate or substrate holder may also
incorporate a heating device to locally control temperature of the
substrate surface. Such devices require rotating electrical
contacts.
[0076] A preferred embodiment of the present invention includes
pre-processing or pre-wetting the substrate. Prior to sustained
addition of the monomer to the substrate, a bolus of cold monomer
is added to the substrate to ensure that the substrate is fully
wet. In a preferred embodiment, an amount capable of covering the
entire substrate is injected and is used to be spread on the
substrate.
[0077] The method 216 further includes step 230 of initiating a
flow of the monomer units. Prior to substrate rotation, a flow of
monomer is begun. For neutralized Vitrogen solution the start up
range is 0.05-1000 ml/min with a preferred range of 0.1-100.0
ml/min. In a preferred embodiment, the start up flow rate for
neutralized Vitrogen solution is 2.0 ml/min.
[0078] The method then includes the step 232 of initiating rotation
of the substrate. The rotation of substrate is initiated and may
proceed in a series of steps to aid in the uniform spreading of the
collagen monomer solution over the substrate surface. A range of
initial angular velocities during startup is 10 to 5000 rpm with a
preferred range of 60 to 1000 rpm. In a preferred embodiment the
initial angular velocity utilized is 250 rpm.
[0079] The method further includes the step 234 of controlling a
plurality of parameters during polymerization. For example, the
rotational velocity of the substrate during polymerization is set.
During the polymerization of the collagen monomer on the warm
substrate, the rotational velocity may remain steady or undergo
modulation of any kind. However, the average velocity (based on
averaging over each minute of operation) of the substrate has a
range of 100 to 50,000 rpm with a preferred range of 500 to 10,000
rpm. In a preferred embodiment, the average rotational velocity of
the substrate is 1600 rpm.
[0080] Further, during polymerization, the flow rate of monomer is
controlled. The monomer solution flow rate provides a monomer for
polymerization to the substrate surface while generating suitable
shear force under rotation to induce alignment of the growing
collagen fibrils. The flow rate of collagen solution over the
rotating substrate may remain steady or undergo modulation of any
kind. However, the average flow rate of collage solution (based on
averaging over each minute during operation) is in a range of
between 0.05 ml/min to 1000 ml/min with a preferred range of 0.1 to
100 ml/min. In a preferred embodiment, the average flow rate over
the substrate during polymerization is 0.25 to 2.0 ml/min.
[0081] Another parameter that is controlled during polymerization
is the optimum shear rate at the substrate interface. The flow rate
over the growing monomers tends to align them among the flow
direction and provides the monomer units to the free end. The
combination of monomer solution input flow rate, viscosity and
substrate rotational velocity combines to produce a rage of shear
rates from 1.0 to 500,000 Hz with a preferred range of 50 to 50,000
Hz. In a preferred embodiment, the shear rate at the substrate
surface is approximately 700 Hz.
[0082] The duration of polymerization is another parameter that can
be controlled. Rheological experiments have demonstrated that the
gelation of the collagen solution begins once optimum conditions
are achieved and take approximately six minutes following addition
to a substrate warmed to 37.degree. C. In the apparatus in
accordance with a preferred embodiment of the present invention,
the range of time of exposure of the monomer flow to the rotating
substrate lies between 1 minute and 2 hours with a preferred range
of 3 minutes to 1 hour. In a preferred embodiment, to generate a
single layer of polymerized collagen, flow is sustained over the
rotating substrate for 15 to 20 minutes.
[0083] In preferred embodiments, a layer including collagen type IV
and cell adhesion proteins such as, for example, but not limited
to, laminin, fibronectic and/or any integrin receptor is deposited
between aligned polymer layers.
[0084] The method 216 in accordance with the present invention
includes the step 236 of initiating a spin-down produce. Following
the addition of collagen to the rotating substrate, the rotating
substrate continues spinning to remove excess collagen monomer.
This spinning down procedure may include a rinse step where a
solution containing no collagen monomer is applied to the rotating
substrate to enhance the removal of unreacted monomer or to add a
layer of material to separate collagen layer, for example, to
promote cell attachment and proliferation. Such materials may
include proteoglycans, laminin, fibronectin and/or vinculin or
integrin moieties. During the spin-down procedure the rotational
velocity may remain steady or undergo modulation of any kind.
However, the average velocity (based on averaging over each minute
of operation) of the substrate is in a range of 100 to 50,000 rpm
with a preferred range of 500 to 10,000 rpm. In a preferred
embodiment, the average rotational velocity of the substrate is
1600 rpm.
[0085] The method 216 concludes with a post-processing step 238. To
ensure polymerization of the deposited layer, the substrate may be
post-processed. Post-processing may include an extended exposure to
the warm humid environment for a period of 0.1 to 60 minutes with a
preferred range of exposure of 3 to 10 minutes. In a preferred
embodiment, the exposure time during post-processing is 5 minutes.
The method 216 includes all of the essential steps to produce a
single layer of aligned collagen via spin-coating in accordance
with a preferred embodiment of the present invention.
[0086] FIG. 9 is a scanning electron microscope (SEM) image
demonstrating the deposition of a plurality of thin aligned layers
244 onto a glass substrate 242 in accordance with a preferred
embodiment of the present invention.
[0087] FIG. 10 is a scanning electron microscope (SEM) image of a
single layer of predominately aligned collagen fibrils in
accordance with a preferred embodiment of the present
invention.
[0088] FIG. 11 is a scanning electron microscope (SEM) image of
aligned collagen fibrils generated by the spin-coating methodology
in accordance with a preferred embodiment of the present
invention.
[0089] FIG. 12 illustrates a scanning electron microscope (SEM)
image demonstrating layering of collagen in pseudolaviellae in
accordance with a preferred embodiment of the present invention.
Multiple layers of aligned collagen polymer may be achieved by
repeating (indefinitely) the procedure for generating a single
layer. The method 216 may be repeated immediately following the
post-processing step or repeated following a dryout period or a
deionized water soak period. In a preferred embodiment, multiple
layers are achieved by repeating the single layer procedure
following a 24 hour soaking period in deionized water.
[0090] FIG. 13 is a SEM image 320 illustrating the intersection of
two individual layer of aligned collagen in accordance with a
preferred embodiment of the present invention. The arrows 322, 324
indicate the alignment directions for each layer. To generate
multiple layers of aligned collagen polymer where the relative
angle of alignment of the collagen is changed between depositions,
the method 216 for producing multiple layers is performed. However
the substrate is rotated through any angle relative to its previous
position on the substrate holder. Any range of angles is possible
from a range of 0 to 2.pi. (and any integer multiple thereof) with
a preferred range of 0 to .pi.. In a preferred embodiment, the
angle between aligned collagen layers is .pi./2.
[0091] With regard to microfluids methodologies, the fundamental
principle in the embodiments of the present invention is to use
flow regimes to control the growth of individual fibers of
collagen, or some other polymerizable material. The method 216
outlined the use of a common industrial process, namely
spin-coating, to provide the necessary constraints to the growing
fibers to allow control of alignment, length and diameter. However
in a more general embodiment it is possible to directly manipulate
the flow field, and thus the local environment, around a growing
filament. To this end the use of the emerging field of
microfluidics can be used.
[0092] The use of microfabricated fluid handling is described by
Giordano, N. and Cheng, J.-T. (2001) in the Journal of Physics:
Condensed Matter, 13, R271-R295 entitled "Microfluid mechanics:
progress and opportunities", the entire teachings of which is
hereby incorporated by reference. These methods have matured over
the last few years resulting in commercial products such as DNA
sorting systems. These systems can be used to handle fluids on
sub-micron scales using features and channels that can be applied
to sub-micron dimensions.
[0093] To control single growing filaments, the length scales
required must be closer to the characteristic of the filament,
approximately 50 nm. Confinement in tubes rapidly allows extension
of the filament and the narrow confines of such a channel rapidly
increase the shear rates around the filament. However these narrow
channel sizes are currently difficult to manufacture and use. A
solution is described by a preferred embodiment of the present
invention and uses a form of flow focussing illustrated in FIGS.
14A-14C which provides a schematic of the flow focussing concept.
At the length scales discussed herein flow is almost always
laminar. This means that in fact mixing is very difficult to
encourage in such flow regimes. In reality then the mixing process
is controlled by diffusion processes. Consequently if two jets
impinge on each other at an angle they do not mix directly but must
mix by diffusion across the interface. If one of these jets
contains the un-polymerized collagen (or other species) discussed
hereinbefore, and other jet contains the polymerizing agent then
there is a finite time before enough diffusion occurs to allow
mixing.
[0094] If the polymerizing jet has a higher volume flow rate and
both jets enter the same size channel, then the amount of the
channel used by each fluid "species" is proportional to the
incoming flow rate. Consequently this behavior allows the fluids to
control the active zone in the flow field, both through
constriction of the width as shown in FIG. 14B, related to the
relative flow rates (this is the flow focusing described
hereinbefore), and through diffusion related to fluid
concentrations.
[0095] The procedure outlined with respect to FIGS. 14A-14C allow a
confined area to be generated in a flow field that can be arranged
to have the correct solution conditions for polymerization. In
addition other fluids can be added to influence the filament
diameter and, if necessary, stop polymerization. If the growing
polymer is advanced such that the growing tip is always in this
critical region, the polymer can be extruded indefinitely. This
approach allows the nanofabrication of a single polymer.
[0096] FIG. 15 illustrates another preferred system to manufacture
layered aligned polymer structures in accordance with a preferred
embodiment of the present invention. This preferred embodiment
utilizes the recognition that if an array of channels such as
described in FIGS. 14A-14C is manufactured an array of controlled
polymers can be generated. In FIG. 15, initially the collagen is
polymerized against a fixed wall 384 to ensure a dangling collagen
chain is present in the nanoloom 386. As the collagen filament
grows this nanoloom 386 advances relative to the fixed wall, thus
extruding a single collagen filament as it advances. An array of
these filaments allow construction of a single layer of aligned
collagen. It may also be possible to produce woven materials in
this manner.
[0097] FIGS. 16A and 16B illustrate a block diagram 420 and a
schematic diagram 490, respectively, of a preferred embodiment
system to manufacture layered, aligned polymer structures in
accordance with the present invention. The apparatus 432 used to
generate layered, aligned polymer structures has a plurality of
input parameters 440 that can be modulated using a processor 480.
The input parameters, include, without limitation, volume flow rate
of monomer, temperature of monomer, monomer solution concentration,
concentrations of monomer solution additives, substrate
temperature, rotational velocity of substrate, inlet tube
temperature, relative humidity of environment, and temperature of
air. It should be noted that all parameters can vary with time. A
plurality of output parameters 460 are also monitored and modulated
by the processor 480. The output parameters include, without
limitation, layer thickness, orientation of polymerizing layer, and
radius of polymerized layer. The system includes a plurality of
sensing elements and monitoring elements that enables the processor
480 to process, control and monitor different parameters. The
processor through an input/output interface 442 interfaxes with a
pump to monitor and/or control the volume flow rate of the monomer,
the temperature of the monomer, the concentration of the monomer
solution and the concentration of any additives to the solution.
Further, the solution environment, for example, the temperature of
the solution is monitored using the interface 424. The substrate
environment, for example, the relative humidity and the temperature
can be monitored and modulated using an interface 426. The
substrate conditions are monitored such as, for example, substrate
temperature and the rotational velocity of the substrate. The
parameters associated with the layers of aligned polymers, for
example, layer thickness, orientation of polymerizing and radius of
polymerized layers are also monitored using an interface 428. The
processor 480 can have an integrated display device or provide data
to another display device and/or processor that is not co-located
with the apparatus 432. Post-processing of the polymer structure as
described with respect to the method illustrated in FIG. 8B can be
controlled by the processor 480. It should be noted that the device
432 can comprise a distribution network for the polymer solution
that includes a plurality of nozzles that can be rotated and
deposit the polymer/monomer solution onto a substrate that can be
stationary. The shear flow is generated by a relative motion and/or
velocity between the distribution system and the substrate.
[0098] The system 420 may also include a microprocessor and a
memory device that stores display data. The microprocessor may
include an operating system, as well as application and
communication software to implement the functions with respect to
controlling device 432 operation. The operating system for the
system of the present invention includes a processing system with
at least one high speed processing unit and a memory system. In
accordance with the practice of persons skilled in the art of
computer programming, the present invention has been described
wherein with reference to acts and symbolic representations of
operations or instructions that are performed by the processing
system. Such acts, operations and instructions are also referred to
sometimes as being computer executed or processing unit
executed.
[0099] It will be appreciated that the acts and symbolically
represented operations or instructions include the manipulation of
electrical signals by the processing unit. An electrical system
with data bits causes a resulting transformation or reduction of
the electrical signal representation, and the maintenance of data
bits at a memory location in the memory system to thereby
reconfigure or otherwise alter the processing unit's operation, as
well as other processing of signals. The memory locations where
data bits are maintained are physical locations that have
particular electrical, magnetic, optical, or organic properties
corresponding to the data bits.
[0100] The data bits nay also be maintained on a computer readable
medium including magnetic disks, optical disks, organic disks, and
any other volatile or non-volatile mass storage system readable by
the processing unit. The computer readable medium includes
cooperating or interconnected computer readable media, which exist
exclusively on the processing system or is distributed among
multiple interconnected processing systems that may be local or
remote to the processing system.
[0101] It should be understood that the programs, processes,
methods and systems described herein are not related or limited to
any particular type of computer or network system (hardware or
software), unless indicated otherwise. Various types of general
purpose or specialized computer systems may be used with or
performed operations in accordance with the teachings described
herein.
[0102] In view of the wide variety of embodiments to which the
principles of the present invention can be applied, it should be
understood that the illustrated embodiments are exemplary only, and
should not be taken as limiting the scope of the present invention.
For example, the steps of the flow diagrams may be taken in
sequences other than those described, and more or fewer elements
may be used in the block diagrams. While various elements of the
preferred embodiments have been described as being implemented in
software, other embodiments in hardware or firmware implementations
may alternatively be used, and vice-versa.
[0103] It will be apparent to those of ordinary skill in the art
that methods involved in layered aligned polymer structures and
methods of making same may be embodied in a computer program
product that includes a computer usable medium. For example, such a
computer usable medium can include a readable memory device, such
as, a hard drive device, a CD-ROM, a DVD-ROM, or a computer
diskette, having computer readable program code segments stored
thereon. The computer readable medium can also include a
communications or transmission medium, such as, a bus or a
communications link, either optical, wired, or wireless having
program code segments carried thereon as digital or analog data
signals.
[0104] The systems and methods of the present invention can be used
in a plurality of applications. For example, single layers of
aligned collagen can be manufactured which can be used as a test
bed for assessing effects of aligned collagen matrices on cellular
behavior in connective tissue fibroblasts, in epithelial and in
endothelia. Further they can generate scaffolding to promote
adhesion and proliferation of cell populations in corneal
epithelium and/or corneal endothelium.
[0105] Further, these embodiments of the present invention can be
used to generate multiple layers of aligned collagen which can be
used as a test bed for examination of the behavior of cells in
anisotropic extracellular matrix including cartilage fibroblasts,
corneal keratocytes, and tendon fibroblasts. They can generate
connective tissue scaffolding for repair and promotion of cellular
adhesion and proliferation that can be used in, for example, but
not limited to, artificial corneal replacement, corneal repair
material, transfer scaffolding for epithelial transplants, transfer
scaffolding for endothelial transplants, tendon replacement or
repair, ligament replacement or repair and annulus fibrosis
replacement or repair. In addition, biocompatible strengtheners for
natural and/or artificial materials for use in tissue repair or
replacement can be generated using the embodiments of the present
invention. These can be rolled up to perform annulus fibrosis
function while embedded in a poly(vinyl alcohol) matrix, or be used
as strengtheners for corneas made from artificial materials, for
example, poly HEMA and/or be a resorbable anchor for tissue
repair.
[0106] Further applications of the preferred embodiments include
generating multiple layers of other aligned biopolymers that can be
used as biocompatible scaffolding, for example, "braids" to
strengthen stents, or other implants. They can also be used as
guidance for nerves, for example, nerve cuffs. Other applications
having layers where alignment is not important that can benefit
from the layered polymer structures of the present invention
include support for species embedded in multiple layers such as
cells and drug release applications.
[0107] In addition, applications that include non-biopolymers may
benefit from the deposition of layered, aligned polymer structures,
for example, generation of optical storage media.
[0108] The claims should not be read as limited to the described
order or elements unless stated to that effect. Therefore, all
embodiments that come within the scope and spirit of the following
claims and equivalents thereto are claimed as the invention.
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