U.S. patent application number 10/266892 was filed with the patent office on 2003-03-06 for microfabricated membranes and matrices.
This patent application is currently assigned to The General Hospital Corporation, a Massachusetts corporation. Invention is credited to Morgan, Jeffrey R., Pins, George D..
Application Number | 20030044395 10/266892 |
Document ID | / |
Family ID | 22386216 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030044395 |
Kind Code |
A1 |
Morgan, Jeffrey R. ; et
al. |
March 6, 2003 |
Microfabricated membranes and matrices
Abstract
The invention relates to microfabricated membranes and matrices
that have a highly controlled and complex three-dimensional
topography. The new microfabricated membranes and matrices can be
prepared of man-made as well as natural materials, such as
materials found in naturally occurring membranes, and thus can be
made in the form of tissue substitutes or analogs, such as basal
lamina, dermal, or skin analogs.
Inventors: |
Morgan, Jeffrey R.; (Sharon,
MA) ; Pins, George D.; (Randolph, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
The General Hospital Corporation, a
Massachusetts corporation
|
Family ID: |
22386216 |
Appl. No.: |
10/266892 |
Filed: |
October 8, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10266892 |
Oct 8, 2002 |
|
|
|
09500829 |
Feb 10, 2000 |
|
|
|
6479072 |
|
|
|
|
60119761 |
Feb 11, 1999 |
|
|
|
Current U.S.
Class: |
424/93.21 ;
424/423; 424/93.7; 435/371 |
Current CPC
Class: |
A61L 15/42 20130101;
A61L 15/325 20130101; A61L 15/26 20130101; A61L 15/32 20130101;
A61L 15/24 20130101 |
Class at
Publication: |
424/93.21 ;
435/371; 424/93.7; 424/423 |
International
Class: |
A61K 048/00; C12N
005/08 |
Goverment Interests
[0002] This invention was made, in part, with Government support
under grant number R29 AR42012-01A1 awarded by the National
Institutes of Health. The Government has certain rights in the
invention.
Claims
What is claimed is:
1. A dermal analog comprising a polymeric matrix and a basal lamina
analog fixed to a surface of the polymeric matrix, wherein the
basal lamina analog comprises a microfabricated membrane comprising
a sheet of conforming material that comprises a defined,
three-dimensional topography, wherein the membrane is 1 to 50
microns thick, and the three-dimensional topography mimics a
three-dimensional topography of a natural basal lamina.
2. A dermal analog of claim 1, wherein the polymeric matrix
comprises collagen.
3. A dermal analog of claim 1, wherein the polymeric matrix
comprises type I collagen and a glycosaminoglycan (GAG).
4. A dermal analog of claim 1, wherein the polymeric matrix
comprises hyaluronic acid.
5. A tissue substitute comprising a dermal analog of claim 1, and
mammalian cells grown on and in the dermal analog.
6. A tissue substitute of claim 5, wherein the mammalian cells are
epithelial cells.
7. A tissue substitute of claim 5, wherein the epithelial cells are
keratinocytes, and the tissue substitute is a skin substitute.
8. A tissue substitute of claim 5, wherein the mammalian cells are
human cells.
9. A tissue substitute of claim 5, wherein the mammalian cells are
engineered to include a nucleic acid construct that encodes a
heterologous polypeptide.
10. A tissue substitute of claim 5, wherein the mammalian cells are
engineered to include a nucleic acid construct that encodes a
therapeutic protein, a growth factor, a wound healing factor, or a
hormone.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of priority from U.S.
Provisional Patent Application No. 60/119,761, filed on Feb. 11,
1999, and U.S. patent application Ser. No. 09/500,829, filed on
Feb. 10, 2000, which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The invention relates to the preparation of synthetic
membranes, such as biocompatible membranes, and matrices.
BACKGROUND
[0004] Synthetic membranes have many uses, such as in the emerging
field of tissue engineering. In general, engineered tissue analogs,
often composed of cultured cells, biomaterials, or composites
combining cells and biomaterials, have achieved some clinical
success, for example, as substitutes for skin and cartilage.
[0005] Significant effort has been devoted to producing
biocompatible scaffolds with defined pore sizes that help ensure
proper cell-cell contacts, cell-matrix interactions, and to
preserve cellular function. For example, collagen sponges and
foams, cylindrical poly(1-lactic acid) (PLA) devices, and
polyglycolic acid (PGA) fibers processed into porous, nonwoven mesh
matrices, have been made to provide substitute skin, nerves, and
cartilage. Although sponges, foams, and matrices are useful for the
fabrication of relatively large, three-dimensional tissue analogs,
their use is limited for the creation of thin membranes such as the
basal lamina, a membranous layer of connective tissue found in many
organs and tissues.
[0006] The basal lamina or basement membrane is a thin membranous
layer of connective tissue that underlies all epithelial cell
sheets and tubes. For example, a basal lamina separates the
endothelial cell layer of blood vessels from the underlying tissue
and a basal lamina separates the epidermis from the underlying
dermal tissue. A basal lamina also surrounds individual muscle
cells, fat cells, and Schwann cells of nerve fibers. The basal
lamina separates these cells and cell sheets from the underlying or
surrounding connective tissue. In other locations, such as in the
kidney glomerulus and lung alveolus, a basal lamina lies between
two cell sheets and functions as a highly selective filter. Basal
laminae serve more than structural and filtering roles. They are
also able to determine cell polarity, influence cell metabolism,
organize the proteins in adjacent plasma membranes, induce cell
differentiation and serve as specific highways for cell migration.
The basal lamina can also serve as a selective barrier to the
movement of cells. The lamina beneath an epithelium, for example,
usually prevents fibroblasts in the underlying connective tissue
from contacting the epithelial cells, but does not prevent the
movement of immune cells in and out of the epidermis, nor does it
prevent the innervation of the epidermis. During wound healing as
well as during normal development, the basal lamina acts as a guide
and template that helps control cell migration and
differentiation.
SUMMARY
[0007] The invention is based on the discovery that a membrane can
be manufactured to have a highly controlled and complex
three-dimensional topography by using microfabrication techniques.
Similarly, a matrix can be manufactured to have a highly controlled
and complex three-dimensional surface topography. The new
microfabricated membranes and matrices can be prepared of man-made
as well as natural materials, such as materials found in naturally
occurring membranes, and thus can be made in the form of tissue
substitutes or analogs, such as basal lamina analogs.
[0008] Given the important role of the basal lamina membrane in
many different tissues and organs, the ability to produce basal
lamina analogs with a controlled and complex three-dimensional
topography has numerous applications in tissue engineering and the
manufacture of artificial organs. Because of their carefully
controlled and defined topographies and high surface areas,
synthetic, microfabricated membranes can be used, for example, in
air and water filters, cell culturing devices, and blood dialysis
devices.
[0009] In general, the invention features microfabricated membranes
including a sheet of conforming material that comprises a defined,
three-dimensional topography, e.g., invaginations and/or
projections. The membranes can be made of conforming materials such
as gelatins, collagens, polyurethanes, polylactic acids, TEFLON,
polystyrenes, epoxy resins, methacrylates, polycarbonates,
silicones, non-collagenous proteins, or polysaccharides. The
membranes can also be made of copolymers, such as blends of
polylactic acid and polyglycolic acid, or natural materials such as
proteoglycans and glycosaminoglycans, as well as blends of natural
and synthetic materials. The membranes can be, e.g., from 1 to 500,
or 1 to 5, 10, 15, 20, 35, or 50 microns thick. The topographic
features can have a height or depth of, e.g., 1.0 to 1000 microns,
or 10 or 20 to 100 or 200 microns. The topographic features can
have a width of, e.g., 1.0 to 500 microns, or 5, 10, or 20 to 100,
200, or 300 microns. The membranes can have a controlled porosity
or permeability.
[0010] The invention also features new basal lamina analogs that
include a microfabricated membrane, wherein the membrane is 1 to 50
microns thick, and the three-dimensional topography is defined to
mimic the three-dimensional topography of a natural basal
lamina.
[0011] In another aspect, the invention also features dermal or
tissue analogs that include a polymeric matrix and a basal lamina
analog fixed, e.g., laminated, to a surface of the polymeric, e.g.,
protein such as collagen, matrix. The polymeric matrix can include
type 1 or type IV collagen and a glycosaminoglycan (GAG). The
polymeric matrix can also be non-proteinaceous, and include, e.g.,
hyaluronic acid. The polymeric matrix can include any of the
conforming materials mentioned herein that can be used to form the
membranes.
[0012] The invention also features tissue substitutes including a
dermal or tissue analog, and mammalian, e.g., human, canine,
feline, bovine, equine, porcine, or ovine cells, e.g., epithelial
cells, grown on and/or in the dermal analog. When the epithelial
cells are keratinocytes, the tissue substitute is a skin
substitute. In some embodiments, the mammalian cells are engineered
to include a nucleic acid construct that encodes a heterologous
polypeptide, or a therapeutic protein, a growth factor, a wound
healing factor, or a hormone.
[0013] The invention also features a method of preparing a
microfabricated membrane comprising a defined, three-dimensional
topography, by preparing a master plate comprising a defined,
three-dimensional pattern; transferring the pattern or a negative
of the pattern to a membrane material; and allowing the membrane
material to solidify, e.g., polymerize, harden, or gel, to form the
microfabricated membrane, wherein the membrane has a defined,
three-dimensional topography that is substantially the same as the
three-dimensional pattern of the master plate or a negative of the
master plate pattern.
[0014] The pattern can be transferred from the master plate to the
membrane material by applying the material directly to the master
plate, to produce a microfabricated membrane that has a defined,
three-dimensional topography that is substantially the same as a
negative of the three-dimensional pattern of the master plate.
Alternatively, the pattern can be transferred from the master plate
to the membrane material by coating the master plate with a liquid
or semi-solid conforming material, e.g., polydimethyl-siloxane
silicone elastomer (PDMS); allowing the conforming material to
solidify, and removing the conforming material from the master
plate to form a negative replicate that comprises a negative of the
master plate pattern; applying a membrane material to the negative
replicate; and allowing the membrane material to solidify to form
the microfabricated membrane and removing the membrane from the
negative replicate, wherein the membrane has a defined,
three-dimensional topography that is substantially the same as the
three-dimensional pattern of the master plate. The invention also
includes microfabricated membranes prepared by the new methods.
[0015] In yet another embodiment, the invention covers a method of
preparing a microfabricated tissue analog comprising a defined,
three-dimensional surface topography, by preparing a master plate
comprising a defined, three-dimensional pattern; transferring the
pattern or a negative of the pattern to a matrix material; and
allowing the matrix material to solidify to form the
microfabricated tissue analog, wherein the analog has a defined,
three-dimensional surface topography that is substantially the same
as the three-dimensional pattern of the master plate or a negative
of the master plate pattern. The invention also includes tissue
analogs made by this method.
[0016] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0017] The invention provides several advantages. For example, the
invention provides virtually unlimited design possibilities for the
precise control of the three-dimensional topography of thin,
synthetic membranes, such as biologically active membranes.
Moreover, the new microfabricated, synthetic membranes can be
prepared in the form of novel skin substitutes, which have
applications in the treatment of burns, plastic surgery, ulcers,
and gene therapy.
[0018] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF DRAWINGS
[0019] FIGS. 1a and 1b are schematic diagrams (top and side view,
respectively) of a master pattern consisting of a series of
parallel channels with varied widths and depths.
[0020] FIG. 2 is a diagram of a master chip or plate made by laser
machining the channels specified in the master pattern in FIGS. 1a
and 1b into the surface of a polyimide chip. The scale bar
represents 5 mm.
[0021] FIG. 3 is a diagram of a negative replicate formed by curing
polydimethylsiloxane (PDMS) on the surface of the master chip. The
scale bar represents 5 mm.
[0022] FIG. 4 is a diagram of a microfabricated membrane produced
by air-drying a small volume of gelatin or collagen-GAG
coprecipitated on the surface of the negative replicate.
[0023] FIG. 5 is a computer-generated, two-dimensional surface
profile of a channel in the master plate, which can be used to
measure the width and depth of each channel.
[0024] FIG. 6 is a schematic representation of a section cut
perpendicular to the surface of a negative replicate, which shows
the width (W) and depth (D) of the protruding ridges that match the
channels cut into the master plate. The scale bar represents 250
microns.
[0025] FIGS. 7a to 7e are a composite series of surface plots of
the master plate and photographs of the negative replicates
comparing the dimensions of each protruding ridge of the negative
replicate with the surface profile of the corresponding channel in
the master chip. The scale bars represent 250 microns.
[0026] FIG. 8 is a top view of a rehydrated dermal analog produced
by laminating a microfabricated membrane to the surface of a
collagen-GAG sponge. The scale bar in the lower right corner
represents 5.0 mm.
[0027] FIGS. 9a and 9b are schematics of scanning electron
micrographs of a dermal analog at two different magnifications
(19.times. and 490.times., respectively). The scale bar represents
1.0 mm in FIG. 9a, and 50 microns in FIG. 9b.
[0028] FIGS. 10a and 10b are schematics of the cross-section of a
dermal analog including a microfabricated membrane (having sets of
channels designed to range from 40-200 microns in width and 40
microns in depth (FIG. 10a) and 200 microns (FIG. 10b) in depth)
and a porous collagen-GAG sponge. The scale bar represents 500
microns in FIGS. 10a and 10b.
[0029] FIGS. 11a to 11c are a series of schematics of micrographs
of a skin substitute including a layer of keratinocytes, a
microfabricated membrane, and a porous collagen-GAG sponge. FIGS.
11a and 11b show a skin substitute with sets of channels designed
to be 40 microns or 200 microns in depth, respectively. FIG. 11c
shows differentiated and stratified keratinocytes in individual
channels of a microfabricated membrane. Scale bars represent 500
microns in FIGS. 11a and 11b and 100 microns in FIG. 11c.
[0030] FIG. 12 is a schematic diagram showing the parameters used
measure invaginations in epithelial layers formed on
microfabricated membranes.
[0031] FIGS. 13a and 13b are two graphs showing epithelial
thickness and epidermal invagination depth, respectively, versus
channel depth in microfabricated membranes.
DETAILED DESCRIPTION
[0032] The invention includes methods to produce microfabricated,
synthetic membranes and matrices with controlled and complex
three-dimensional topographies at the micron scale with sub-micron
resolution; the microfabricated membranes, such as basal lamina
analogs, and matrices produced by the new methods; and improved
compositions, such as tissue analogs (e.g., dermal analogs and skin
substitutes), that are made with the microfabricated membranes,
e.g., basal lamina analogs, and matrices.
[0033] General Methodology
[0034] The new methods include three basic steps: (1) creating a
master pattern, e.g., a three-dimensional pattern, (2)
microfabricating a master plate or chip that corresponds to the
master pattern (either as a duplicate or a negative of the master
pattern), and (3) transferring the three-dimensional pattern of the
master plate to a membrane, dense gel, or other matrix or
substrate. The goal is to create a microfabricated membrane or
matrix that has the same, or essentially the same,
three-dimensional pattern as the master pattern. Thus, the third
step can be performed by making the master plate a duplicate of the
master pattern, then preparing a negative replicate from the master
plate, and using the negative replicate to create the membrane.
Alternatively, the master plate can be made as a negative of the
master pattern, and used directly to cast the membrane.
[0035] The master plate or the negative replicate can also be used
to imprint a defined, three-dimensional topography into a polymeric
matrix, used as a dermal analog, for example, without a separate
membrane. In this embodiment, a polymeric matrix is applied to the
master plate or negative replicate in a liquid or semi-solid state,
and conforms to the pattern. It is solidified and then removed to
provide the patterned matrix.
[0036] Microfabricating the Master Plate
[0037] The production of a microfabricated membrane begins with the
design of a master pattern that includes the desired
three-dimensional surface topography for the membrane. The master
pattern can be drawn out, e.g., by hand or using a CAD-CAM device,
or programmed into a computer, or both. The range of sizes of
surface features over which the methods can be used to control the
topography of the new membranes ranges from the submicron scale to
microns, or tens or hundreds of microns (and even millimeters). The
range was tested by use of a test pattern.
[0038] FIGS. 1a and 1b show a master pattern 10 having channels 12
of different depths and widths. The test pattern was prepared to
have a series of 25 parallel channels 12 (10 mm long) in groups of
five with each channel in a group having the same depth, and each
group having a different depth ranging from 40 microns to 200
microns. Within each group, the five channels had widths ranging
from 40 microns to 200 microns.
[0039] The master pattern is then used to generate a master plate
or chip, by programming a microfabrication device to create the
features of the master pattern (or a negative of the master
pattern) in a three-dimensional, solid material. The master plate
can be machined from various metals and metal oxides such as
alumina, ceramics, diamond, polymers like polyimide, quartz, glass,
and silicon.
[0040] For example, as shown in FIG. 2, a master plate 20 was
produced by laser machining the specified channels 12 of the master
pattern 10 into a polyimide chip 21 (1.0 mm thick, Goodfellow
Corp., Berwyn, Pa.) using an excimer laser (Potomac Photonics,
Inc., Lanham, Md.). Briefly, rectangular images of an excimer laser
beam were line focused (10 mm.times.40 .mu.m) onto the surface of a
polyimide chip 21, and then the laser energy was pulsed at 10 mJ,
at an energy of 2 to 3 J/cm.sup.2, and at a pulse rate of 100 Hz to
ablate its surface. The image of the laser beam was repositioned
and the process was repeated until all channels 22 with the
specified geometries were machined.
[0041] Numerous other microfabrication technologies and materials
can be used to produce a master pattern with micron size and
smaller dimensions. Examples include microetching and
micromachining of metallic, metal oxide (e.g., alumina), ceramic,
glass, or polymeric surfaces (e.g., polyimide), as well as the
widely used photo-, x-ray-or UV-lithography methods used to make
micropatterns on the surface of silicon, or other semiconductors
used in the manufacture of computer chips. For a review of various
microfabrication techniques including photolithography, oxidation,
layer deposition, and etching, see Chapter One of Maly, "Atlas of
IC Technologies: An Introduction to VLSI Processes," The
Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif.,
1987.
[0042] Microstructures can also be fabricated using photosensitive
polyimide electroplating molds. For example, see, Frazier et al.,
J. Microelectromechanical Systems, 2:87-94 (I.E.E.E., 1993).
Standard reactive ion etching and the black silicon method can also
be used to fabricate microstructures in silicon having high aspect
ratios and smooth surface textures. See, e.g., Jansen et al., "The
Black Silicon Method IV," J. Microelectromechanical Systems, 88-93
(I.E.E.E., 1995).
[0043] In addition to channels, other types of structures,
patterns, and biological designs can be machined into the master
plate.
[0044] For example, to create a synthetic membrane to serve as a
basal lamina analog, the peaks and valleys of the three-dimensional
surface topography of the membrane should mimic the
three-dimensional surface of a naturally occurring basal lamina,
e.g., the lines of the hands or fingerprints for basal lamina that
are used to prepare skin substitutes for the hand or fingers. For
use in water filtration devices, the synthetic membranes can have a
straight channel design such as the test pattern described above
(but with all channels being of equal depth and width), or can have
spiral or serpentine channels to provide a significantly long path
length relative to the total area of the membrane through which the
water can flow.
[0045] For size exclusion filtration, the microfabricated membranes
can be made with pores with highly accurately size control. To make
such membranes, the master plate is prepared with numerous circular
plateaus or spikes, e.g., that remain when the remainder of the
plate surface is etched away. See, e.g., Henry et al., J.
Pharmaceutical Sciences, 87:922-925 (1998), and Jansen et al., "The
Black Silicon Method IV," J. Microelectromechanical Systems, 88-93
(I.E.E.E., 1995). The plateaus or spikes are designed to have a
height greater than the desired thickness of the membrane. The
membrane is cast directly from the master plate, and the conforming
material used to prepare the membrane is applied to the master
plate so that the tops of the plateaus remain exposed. When the
membrane material solidifies, the membrane will have pores with
precisely controlled pore sizes and locations. Alternatively, the
master plate can have precise circular wells or cones cut into its
surface, a negative replicate is created that has round plateaus or
spikes, and the microfabricated membrane is cast form the negative
replicate.
[0046] To test the fidelity with which laser machining had
reproduced the specifications of the master pattern, the dimensions
of the channels in the master plate (chip) were analyzed with a
Zygo New View 200 Scanning White Light Interference (SWLI)
Microscope (Zygo Corp., Middlefield, Conn.) fitted with a 20.times.
Mirau objective lens. The microscope produced three-dimensional
images of each channel and two-dimensional surface profiles in
planes perpendicular to the surface of the master chip. The
shoulder-to-shoulder widths and maximum depths of each channel were
measured with the caliper tools in the microscope software. FIG. 5
is a computer-generated plot of the surface profile of a channel in
the master plate.
[0047] Analyses of this data indicated that the
shoulder-to-shoulder widths of the channels in the master chip
ranged from 114-310 microns, deviating from the 40-200 microns
chanmel widths specified in the master pattern. Nevertheless, the
varied channel widths in the master plate were useful to carry out
further tests, and the deviation merely indicated that the laser
etching had not be performed with a high degree of accuracy.
[0048] The maximum depths of the channels also deviated from the
original master pattern. The depths of the narrowest channels
deviated from the specified 40 or 80 microns by less than 14%, but
the depths of the wider channels deviated from the specified 120,
160, or 200 microns by 28 to 133%. The three-dimensional images
obtained by SWLI microscopy also showed that, unlike the flat
bottoms of our original design, the bottom surfaces of the wider
channels (specified 120, 160 or 200 microns) tapered to points or
had serrated and irregular surfaces. In addition, the widest
channels (specified 200 microns) had notched shoulders in the side
walls. In spite of these irregularities, these master plates were
useful to prepare negative replicates. In fact, the irregularities
were replicated in the final microfabricated membranes, providing
evidence that even details at the micron and submicron level can be
recreated using the new methods of preparing membranes.
[0049] Laser etching can provide higher degrees of accuracy, and
other microfabrication techniques discussed herein can also be used
to provide a greater degree of accuracy.
[0050] The degree of accuracy can be varied according to the
manufacturing technique and cost, and according to the intended use
of the microfabricated membranes made with the master plate. For
example, if a membrane is to be used in certain tissue substitutes,
for example, the master pattern should be recreated with a high
degree of accuracy, e.g., 90 percent or higher. For use in water
filtration or other mechanical devices, the degree of accuracy may
be lower, e.g., 75, 80, or 85 percent.
[0051] Preparing Negative Replicates
[0052] In the next step, which is optional, a negative replicate is
made using the master plate. To make a negative replicate, a
solution of liquid or semi-solid plastic or resin is applied to the
master plate and allowed to solidify. The solid plastic is then
removed to form the negative replicate, which has a negative
imprint of the master pattern as machined into the master plate.
Many materials can be used to prepare the negative replicate. The
main features of these materials are that they can conform to the
master plate, i.e., fill the three-dimensional topography, and that
they can thereafter solidify in some way. The materials must also
be separable from the master plate after they have solidified. This
can be achieved by judicious selection of the materials used for
the plate and the negative replicate, and can be assisted by the
use of release agents sprayed onto the plate.
[0053] Various materials can be used as the conforming material,
many of which are polymeric. Polyurethanes, polylactic acids,
TEFLON, polystyrenes, epoxy resins, methacrylates (e.g.,
polymethylmethacrylates)- , polycarbonates, silicones, collagens,
gelatins, glycosaminoglycans, proteoglycans, and polysaccharides
are all useful examples. Other materials are known, such as
copolymers, e.g., of polylactic acid and polyglycolic acid, or
blends of natural and synthetic materials, and can be used.
[0054] The negative replicates can be solidified using various
techniques, and depends on the specific conforming material used.
For example, the conforming material can be solidified by heating
or cooling, by changing the ambient pressure, by Uv or other
curing, by activation of a catalyst, e.g., by heat or light, or by
cross-linking, e.g., with fonnaldehyde, glutaraldehyde, UV, or
carboduimide. Other techniques are known to those of skill in this
field.
[0055] A negative replicate was made by pouring a
polydimethylsiloxane silicone elastomer (PDMS, Sylgard 184, Dow
Corning Corp., Midland, Mich.) over the master plate and
polymerizing the elastomer by incubating at 65.degree. C. for 2
hours. The PDMS overlay, which conformed to the fine
three-dimensional features (e.g., channels) of the master plate,
was carefully separated from the chip. As shown in FIG. 3, the
resulting negative replicate 30 had surfaces of protruding ridges
32 that were an exact negative replicate of the channels 22 cut
into the surface of the master plate 20 (FIG. 2).
[0056] To determine whether the PDMS faithfully reproduced the
channels in the master chip, the dimensions of the protruding
ridges on the surface of a negative replicate were analyzed with a
Nikon Diaphot 300 microscope coupled with MetaMorph Imaging
Software (West Chester, Pa.). Sections of a negative replicate were
collected by cutting samples in a plane perpendicular to the
microfabricated surface with a razor blade. Sections were placed on
glass slides, viewed with a low power objective lens and digitally
imaged with the microscope software. Shoulder-to-shoulder widths
and maximum depths of each channel were measured with the tools
provided in the software package.
[0057] The results are shown in FIGS. 6 and 7a to 7e. The
shoulder-to-shoulder widths and the maximum heights of the
protruding ridges replicated the channels in the master chip with
an accuracy greater than 91% (analyses not shown).
[0058] Using the new methods it is possible to produce many
negative replicates of a single master pattern, for example, to
prevent the master plate from being worn out too rapidly if used to
cast membranes directly without a negative replicate.
[0059] Preparing Microfabricated Membranes and Matrices
[0060] In the final step, the synthetic membrane is created by
applying a conforming material in a liquid or semi-solid state to
the negative replicate (or directly to the master plate if no
negative replicate is used), and then allowing the material to
solidify to its final consistency. The conforming materials used to
prepare the microfabricated membranes can be selected from the same
set of conforming materials used to make the negative replicates,
but will generally be applied thinly to provide a thin membrane
(e.g., 5, 10, 15, 20 microns thick, or more in certain embodiments)
rather than applied more thickly to provide a more solid structure
for the negative replicate. The conditions used to solidify the
conforming membrane materials depend on the material used, and are
the same as the conditions described herein for preparing the
negative replicate. In one manufacturing run, the negative
replicate and the membrane can be made of the same or different
materials.
[0061] Once solidified, the synthetic, microfabricated membrane can
be removed from the negative replicate. Some membranes are rigid,
while others can be flexible or rubbery in consistency, depending
on their intended utility. In all cases, the synthetic membranes
have a three-dimensional surface topography that is substantially a
negative of the surface topography of the negative replicate, and
thus a topography that is substantially identical to the topography
of the master plate, and to the master pattern. The degree of
similarity or identity between the membrane and the master plate
can vary, and can be more or less critical depending on the use of
the membrane. In some uses, for example, filtration, the precise
topography, e.g., of pores in the membrane, can be critical, and
thus the membrane should have a very high degree of similarity to
the master plate, and ideally to the master pattern, e.g., 95
percent or higher. In other uses, the degree of similarity is not
as important, e.g., for certain basal lamina analogs (as describe
herein), where the precise configuration is not as important as the
overall three-dimensional nature of the topography.
[0062] Basal Lamina Analogs
[0063] As with most tissues, the basal lamina in the skin is not a
simple flat plane of connective tissue, rather it conforms to a
series of ridges and invaginations known as rete ridges and
papillary projections. The depth and patterns of these ridges and
invaginations of epidermis and dermis make important contributions
to the function of the skin. For example, rete ridges are deep and
numerous in the palms of the hands and soles of the feet and serve
to significantly increase the surface area of contact between the
epidermis/dermis, thus providing strong resistance to the shear
forces experienced by the hands and the feet. The pattern of ridges
and projections between the epidermis/dermis are also a significant
part of the underlying features that contribute to the gross
outward appearance of skin in the form of fine lines, small pores,
and natural wrinkles. In addition, the adnexal structures of skin
(hair and sweat glands) are deep invaginations of the epidermis
into the dermis and a basal lamina conforms to these structures as
well.
[0064] In addition to effects on the biomechanical properties of
the skin, the pattern and depths of these ridges are thought to
have a role in the proliferation and differentiation of epidermal
keratinocytes. The fabrication of a basal lamina analog with
controlled dimensions would help elucidate the influence of
topography on cell function and has applications in tissue
engineering of skin substitutes as well as other basal lamina
containing tissues.
[0065] To form microfabricated basal lamina analogs, two types of
materials were used with the negative replicates: gelatin or a
white coprecipitate containing type I collagen (5 mg/ml) and
glycosaminoglycan (GAG, 0.18 mg/ml). A 1% (w/v) gelatin solution
was prepared by stirring 1.0 g of gelatin (Sigma Chemical, St.
Louis, Mo.) into 100 ml of a 0.05 M acetic acid solution that was
warmed to 65.degree. C. until the gelatin was completely dissolved.
The coprecipitate was prepared according to published protocols
(Chamberlain, L. J. and Yannas, L. V. (1998) Preparation of
collagen-GAG copolymers for tissue regeneration. In Tissue
Engineering Methods and Protocols (Morgan, J. R. and Yarmush, M.
L., eds) pp. 3-17, Humana Press, Totowa, N.J.). Briefly, 3.6 g of
lyophilized bovine collagen (Medicol F, Integra Medicus, West
Chester, Pa.) were dispersed in 600 ml of a 0.05 M acetic acid
solution by blending at 20,000 rpm for 90 minutes at 4.degree. C.
in a refrigerated homogenizer. The coprecipitate was formed by
adding 120 ml of a 0.11% w/v solution of shark cartilage
chondroitin 6-sulfate (Sigma Chemical) to the blending collagen
dispersion, and then blending the collagen-GAG copolymer for an
additional 90 minutes. The collagen-GAG dispersion was degassed
under vacuum to remove trapped air, and then stored at 4.degree.
C.
[0066] To create membranes, a small volume of either gelatin or
collagen-GAG dispersion (220-330 ml/cm.sup.2) was poured onto the
PDMS negative replicate where it conformed to the surface. The
dispersion was air dried at room temperature in a laminar flow hood
and the resulting dried collagen membrane was gently peeled from
the negative replicate. Finally, dried membranes were covalently
crosslinked by thermal dehydration at 105.degree. C. in a vacuum of
100 mTorr for 24 hours.
[0067] As can be seen in FIG. 4, the resulting basal lamina analog
40 contains a series of channels 42 substantially identical to the
channels 12 in the master pattern 10 and the master plate 20.
[0068] Many materials other than collagen type I and GAGs can also
be used to make basal lamina analogs. Examples include naturally
occurring substances such as gelatin and purified basement proteins
(laminin, collagen type IV, heparin sulfate proteoglycan, entactin,
fibronectin, and nidogen) as well as biocompatible synthetic
materials such as polylactic acid or copolymers of polylactic acid
and polyglycolic acid, as well as blends of natural and synthetic
materials. See, e.g., Boyce, U.S. Pat. No. 5,273,900 (column 7),
which describes the use of bovine collagen and mucopolysaccharides
such as GAGs like chondroitin-6-sulfate. See, also, Kleinman et
al., U.S. Pat. No. 4,829,000.
[0069] In addition, negative replicates can be used to produce
topographic patterns on microfabricated membranes made of other
conforming materials that do not require a drying step. One useful
example is gelled collagen.
[0070] Dermal and Other Tissue Analols
[0071] Dermal or other tissue analogs can be prepared by combining
a microfabricated basal lamina analog with an analog of tissue
normally underlying the natural basal lamina. The basal lamina
analog is attached to the surface, e.g., laminated as described in
Boyce, U.S. Pat. No. 5,273,900 (columns 7-9), of the tissue analog
to provide a composite synthetic tissue.
[0072] Composite dermal analogs were prepared by laminating a
microfabricated basal lamina analog to the surface of a collagen
sponge. The collagen sponge was produced by methods similar to
those previously described by Yannas et al., "Design of an
Artificial Skin, II. Control of Chemical Composition," J. Biomed.
Mater. Res., 14:107-131 (1980), and Boyce et al., "Structure of a
Collagen-GAG Dermal Skin Substitute Optimized for Cultured Human
Epidermal Keratinocytes," J. Biomed. Mater. Res., 22:939-957
(1988). See also, Boyce, U.S. Pat. No. 5,273,900 (column 7).
Briefly, 10 ml of a collagen-GAG dispersion were poured into an
aluminum pan with a surface area of 38.5 cm.sup.2 (Fisher
Scientific, Springfield, N.J.), and a microfabricated basal lamina
analog was gently floated on the surface of the dispersion. The GAG
was chondroitin-6-sulfate, but others can be used.
[0073] The dispersion was rapidly frozen at -80.degree. C., placed
on a shelf in a freeze dryer initially set at -45.degree. C., then
lyophilized overnight (Virtis Genesis, Virtis, Gardner, N.Y.) at a
vacuum of 100 mTorr. After lyophilization, the composites were
covalently crosslinked by thermal dehydration at 105.degree. C. in
a vacuum of 100 mTorr for 24 hours, rehydrated in a 0.05 M acetic
acid solution for 24 hours, crosslinked in a 0.25% glutaraldehyde
solution for 24 hours, and then washed exhaustively with sterilized
water, phosphate buffered saline and keratinocyte seeding medium
(described below).
[0074] A top view of the rehydrated dermal analog 50 is shown in
FIG. 8. FIGS. 9a and 9b show schematics of electron microscope
photos of cross sections of the dermal analog at different
magnifications. FIG. 9a shows numerous channels 42 of varying
depths, and FIG. 9b shows a cross-section of the basal lamina
analog 40 (microfabricated membrane) and one channel 42. The scale
bar represents 1.0 mm in FIG. 9a, and 50 microns in FIG. 9b.
[0075] To determine whether the microfabricated membranes 40
faithfully reproduced the surface of the negative replicates, the
dimensions of the channels were measured. Dermal analogs were
fixed, embedded in glycolmethacrylate and 5 microns sections were
cut perpendicular to the surface and were stained with hematoxylin
and eosin. Histological observations showed that the dermal analog
was composed of a 25 microns thick microfabricated membrane, with
25 channels of various widths and depths, that was supported by a
porous collagen-GAG sponge. The channels in the membrane exhibited
progressive increases in width and depth that were consistent with
the corresponding ridges in the negative replicate. FIG. 10a shows
very shallow channels 42 at one end of the analog, while FIG. 10b
shows more significant channels 42 at the other end. Collagen
sponge material 43 is also shown. Many of the small irregular
surface features that were present in the master chip, such as
serrated bottom surfaces, were also replicated in the
membranes.
[0076] Tissue analogs can also be prepared without a
microfabricated membrane on their surface, but still having a
controlled three-dimensional surface, by applying the analog
matrix, e.g., collagen gel, directly to the master plate or
negative replicate in the desired thickness. The matrix material is
allowed to solidify, and the resulting analog is removed and
maintains the three-dimensional pattern of the master plate or
pattern. Such matrices can be made of dense gels, such as collagen
gels or hydrogels. Polymeric 2-hydroxyethyl-methacrylate (pHEMA)
can be used to prepare such matrices and membranes.
[0077] Skin and Other Tissue Substitutes
[0078] The final step to make a skin or other tissue substitute is
to seed the surface and/or interior of a dermal or other tissue
analog with cultured mammalian cells, e.g., human cells, such as
epithelial cells, e.g., epidermal cells, keratinocytes, lung cells,
blood vessel cells, kidney cells, dermal cells, or other cells such
as fibroblasts, nerve cells, and hair follicle cells.
[0079] To prepare a man-made or synthetic human skin substitute,
normal human keratinocytes derived from neonatal foreskins were
cultured by the method of Rheinwald and Green, "Formation of a
Keratinizing Epithelium in Culture by a Cloned Cell Line Derived
from a Teratoma," Cell, 6:317-330 (1975). Keratinocytes were
co-cultivated with 3T3-J2 mouse fibroblasts, which had been
pretreated with 15 mg/ml mitomycin C (Boehringer Mannheim Co.,
Indianapolis, Ind.). Culture medium was changed every 3-4 days with
a 3:1 mixture of Dulbecco's modified Eagle's medium (DMEM) (high
glucose) (GIBCO-BRL, Gaithersburg, MD) and Ham's F-12 medium
(GIBCO-BRL) with 10% fetal bovine serum (FBS, JRH Bioscience,
Lenexa, Kans.). Supplements such as adenine, hydrocortisone,
cholera toxin, insulin, transferrin, triiodo-L-thyronine, and
penicillin-streptomycin, were added as described in Medalie et al.,
J. Invest. Dermatol., 107:121-127 (1996).
[0080] Cells were subcultured by first removing the feeder layer
cells with a brief EDTA wash, 5 mM in phosphate-buffered saline
(PBS), and then treating the keratinocytes with trypsin-EDTA.
[0081] Keratinocytes were seeded onto the dernal analogs using
methods similar to those previously described (Medalic et al.,
Transplantation, 64:454-465, 1997) with media changes as described
by Ponec et al., J. Invest. Dermatol., 109:348-355 (1997). Dermal
analogs were placed into 35-mm tissue culture dishes,
microfabricated membrane side up, and cells in keratinocyte seeding
medium (described below) were seeded onto the surface
(5.times.10.sup.5 cells/cm.sup.2). After approximately 2 hours, the
cell-seeded dermal analogs were submerged in keratinocyte seeding
medium for 24 hours. Keratinocyte seeding medium was a 3:1 mixture
of Dulbecco's modified Eagle's medium (high glucose) (GIBCO-BRL)
and Ham's F-12 medium (GIBCO-BRL) supplemented with 1% FBS (JRH
Bioscience), 10-10 M cholera toxin (Vibrio Cholerae, Type Inaba 569
B; Calbiochem, La Jolla, Calif.), 0.2 mg/ml hydrocortisone
(Calbiochem), 5 mg/ml insulin (Novo Nordisk, Princeton, N.J.), 50
mg/ml ascorbic acid (Sigma Chemical) and 100 IU/ml, and 100 mg/ml
penicillin-streptomycin (Boehringer Mannheim Co.).
[0082] After 24 hours, the keratinocyte seeding medium was removed,
and the skin equivalents were submerged for an additional 48 hours
in a keratinocyte priming medium. Keratinocyte priming medium was
composed of keratinocyte seeding medium supplemented with 24 mM
bovine serum albumin (Sigma Chemical), 1.0 mM L-serine (Sigma
Chemical), 10 mM L-carnitine (Sigma Chemical), and a cocktail of
fatty acids including 25 mM oleic acid (Sigma Chemical), 15 mM
linoleic acid (Sigma Chemical), 7 mM arachidonic acid (Sigma
Chemical), and 25 mM palmitic acid (Sigma Chemical). See, Boyce and
Williams, J. Invest. Dermatol., 101:180-184 (1993).
[0083] After 48 hours in priming medium, skin equivalents were
placed on stainless steel screens, raised to the air-liquid
interface and cultured for 7 days with an air-liquid interface
medium composed of serum-free keratinocyte priming medium
supplemented with 1.0 ng/ml epidermal growth factor (Collaborative
Biomedical Products, Bedford, Mass.).
[0084] Other materials can be used to make dermal analogs and basal
lamina analogs can be used as part of the construction of tissues
other than skin.
[0085] Histological and Quantitative Morphometric Analyses
[0086] For histological analysis, skin substitutes were fixed in a
3% glutaraldehyde/4% paraformaldehyde solution, dehydrated with
increasing concentrations of ethanol, infiltrated first at
-80.degree. C. and then at 4.degree. C. with glycolmethacrylate
(JB-4, Polysciences, Inc., Warrington, Pa.), and finally embedded
in glycolmethacrylate. Sections of skin equivalents, 5 mm thick,
were collected by cutting samples in a plane perpendicular to the
surface of the microfabricated membrane. Sections were mounted on
glass slides with Tissue-Tack Adhesive (Polysciences, Inc.),
stained with Gill's hematoxylin and ethanolic eosin solutions, and
then viewed with a Nikon Eclipse 800 microscope.
[0087] For scanning electron microscopy, skin equivalents were
fixed in a 3% glutaraldehyde/4% paraformaldehyde solution, post
fixed with a 1% osmium tetroxide solution, en bloc stained with a
2% uranyl acetate solution, dehydrated with increasing
concentrations of ethanol, then critical point dried with liquid
carbon dioxide under pressure. Samples were sputter coated with a
thin layer of gold-palladium and viewed with an Amray 1000 scanning
electron microscope.
[0088] As shown in FIGS. 11a to 11c, histology showed that the
surface of the skin substitutes 60 contained a series of ridges
(channels) 42 of varying heights and widths and that these ridges
as well as the spaces between the ridges were covered with one or
more stratified epidermis layers 45. Depending on its depth as well
as its width, each ridge had an epidermis with different numbers of
stratified layers. Epidermal thickness of the flat inter-ridge
areas was about 37 microns, and the thickness increased as the
depth of each channel increased. Deeper ridges contained more cells
and had more stratified layers than shallow ridges as well as the
flat inter-ridge areas. In addition, the top surface of the
cornified layers conformed to the channels as well as the
inter-ridge pattern and created a macroscopic pattern. Infolds of
the epidermis occurred when the depth of the channels were greater
than about 25 microns, and these invaginations increased in size as
channel depth increased. Thus, some of the channels of the master
pattern were deep enough to create gross topological features
obvious to the naked eye.
[0089] Specifically, low magnification micrographs show a skin
substitute 60 with a basal lamina analog 40 having sets of channels
42 designed to be 40 microns (FIG. 11a) or 200 microns (FIG. 11b)
in depth. Dermal analog 43 is located beneath the basal lamina
analog, while keratinocytes form epidermal layers 45 above the
basal lamina analog 40. High magnification micrographs show
differentiated and stratified keratinocyte layer 45 in individual
channels 42 of a microfabricated membrane 40 (FIG. 11c). Scale bars
represent 500 microns in FIGS. 11a and 11b and 100 microns in FIG.
11c.
[0090] Morphometric analysis of the epidermal layer on the
microfabricated membrane was conducted and measured as shown in
FIG. 12. The schematic diagram of FIG. 12 shows the parameters used
to measure the channel width (CW), channel depth (CD), epidermal
thickness (ET), and epidermal invagination depth (EI) in each
channel of the membrane. The graph in FIG. 13a illustrates the
change in epidermal thickness versus channel depth, while the graph
in FIG. 13b shows the epidermal invagination depth versus channel
depth for each channel. For linear regression lines, r.sup.2=0.900
(FIG. 13a) and 0.961 (FIG. 13b). The geometric shapes (squares, +,
triangle) denote the different ranges of channel widths. Dashed
lines are the 95% confidence levels for the linear regression
lines. As shown in the graphs, the best fit for both of these
linear relationships was for those channels whose widths were
between 250 and 350 .mu.m.
[0091] Uses
[0092] Microfabricated basal lamina analogs will have numerous
applications in the emerging commercial field of tissue
engineering, which seeks to create substitutes for a wide variety
of human organs and tissues, many of which have basement membranes.
For tissue engineered products which are assembled ex vivo,
microfabricated basal laminae will allow for precise control of the
three-dimensional organization of different types of cells as well
as connective tissue layers. In the much the same way as naturally
occurring basal laminae, microfabricated basal lamina analogs can
be designed to control cell polarity, influence cell metabolism,
act as a highly selective filter, induce cell differentiation,
serve as specific highways for cell migration, and function as a
selective barrier to the movement of cells. Thus, microfabricated
basal lamina analogs will have an important role in the function as
well as fabrication of complex tissues and tissue substitutes.
[0093] Cell differentiation can be controlled by topography by
creating specific designs that mimic certain biological structures.
For example, the test membranes with channels described herein
demonstrate that the stratification of the epidermis is influenced
by the depth of the channel, the deeper the channel the more
stratified the cell layers. Thus, cell differentiation and/or
proliferation can be influenced by controlling the topographical
microenvironment of the cells.
[0094] As another example, a basal lamina microfabricated as a
narrow tube or circular pocket with a closed bottom can be used to
mimic the structure of a hair follicle. Such tubes or pockets can
be made by laser etching a series of pockets of the desired size
into the master plate, and then applying a conforming material that
conforms to the walls of, but does not fill, the pocket. The tubes
or circular pockets in the resulting microfabricated membrane are
seeded with dermal papilla cells in the bottom and the rest of the
tube is seeded with keratinocytes. This crowded tubular
configuration of keratinocytes is what occurs in a naturally
occurring hair follicle and may be the critical arrangement of
keratinocytes that programs them to differentiate into and start
growing as a hair. In this way, the arrangement of the
keratinocytes within this tubular analog of the basal lamina
influences cell metabolism, cell polarity, cell migration, and cell
differentiation.
[0095] Another possibility is to make analogs of the basal lamina
whose topography mimics the structures that occur during normal
embryogenesis, morphogenesis, and organogenesis. These mimics could
be seeded with cells, for example, cells grown from embryonic stem
cells that have the capacity to differentiate into numerous
different cell types, or they can be seeded with one or more
differentiated cell types that occur during development. In this
way, the basal analogs would position the cells into the correct
places, thus facilitating the establishment of correct spatial
gradients of growth factors and morphogens, that resume the
development process and allow these analogs to proceed with
morphogenesis or organogenesis.
[0096] One example of the use and advantages of microfabricated
basal lamina analogs in tissue engineering is the fabrication of
skin substitutes described herein. All of the current skin
substitutes have a flat, thus unnatural, interface between the
epidermal and dermal layers. By using a microfabricated basal
lamina analog, a skin substitute was created with a complex
interdigitating interface between the epidermal and dermal layers.
This interdigitating interface has several advantages.
[0097] First, the mechanical strength bonding the epidermal and
dermal layers together is significantly increased compared to a
flat interface. This mechanical strength can be measured using
known devices and techniques. For example, the mechanical strength
of the composite materials could be tested by subjecting the
materials to different shear stresses or by applying varying
amounts of a vacuum in a blistering device and determining what
force of vacuum is required to make these materials separate or
blister. Thus, skin substitutes with a microfabricated basal lamina
analog can be made which are more resistant to shear forces, a
problem in external wound beds which often leads to graft
failure.
[0098] Second, the interdigitating interface between epidermal and
dermal layers facilitates improved mass transport between layers.
Mass transport of nutrients, waste products, and growth factors to
and from the epidermal layer is critical to the successful
engraftment of any skin substitute. This process is significantly
enhanced in skin substitutes containing microfabricated basal
lamina analogs because of the greatly increased surface area, and
the varying depths to which the basal lamina "reaches into" the
underlying dermal layer.
[0099] Third, the outward appearance or cosmesis of a skin
substitute can be precisely controlled using a microfabricated
basal lamina analog. Present skin substitutes have an unnatural
appearance due in part to the flat interface between the epidermal
and dermal layers. By using a microfabricated basal lamina analog,
it will be possible to design more natural and cosmetically
appealing skin substitutes which have fine lines, wrinkles, and
pore structures characteristic of native skin. Such properties are
important to burn patients as well as other patients who receive
skin substitutes for the treatment of ulcers or undergoing
cosmetic/reconstructive surgery.
[0100] Fourth, microfabricated basal lamina analogs have
applications in the fabrication of adnexal structures of the skin
such as hair and sweat glands. Adnexal structures are characterized
by deep invaginations of the epidermis into the dermis and are a
complex interaction between cells of the epidermis and cells of the
dermis. At the interface of these two layers is a basement membrane
with a complex three-dimensional topography. Microfabricated basal
lamina analogs that control the organization and interactions of
epidermal and dermal cells are useful for the fabrication of these
structures.
[0101] In addition, microfabricated basal lamina analogs and other
microfabricated membranes can be used to introduce engineered cells
into a patient, e.g., to include cells that produce polypeptides
(e.g., heterologous polypeptides that the cells do not normally
produce, or autologous polypeptides that the cells do produce, but
not in such large amounts) such as growth factors (epidermal growth
factor, fibroblast growth factor, platelet-derived growth factor,
transforming growth factors, and the like), wound healing factors,
hormones, or other proteins. In other applications, the cells in
the new tissue analogs can be used for gene therapy to secrete
polypeptides that the patient does not normally produce because of
a defect, e.g., hormones and blood coagulation factors.
[0102] The skin and other tissues are an attractive target for
applications in gene therapy because, for example, the
keratinocytes of the epidermis can be easily cultured, genetically
modified, assembled as part of a skin graft, and easily
transplanted to a patient. Gene modified skin grafts can be used
for the systemic delivery of therapeutic proteins (e.g., insulin),
or for the local delivery of growth factors for wound healing
(e.g., PDGF). Regardless of the application, the use of a
microfabricated basal lamina analog would help to maximize the mass
transport of the therapeutic protein from the skin graft, as well
as to maximize the number of genetically modified keratinocytes per
surface area of the graft, thus reducing the size of the needed
skin graft.
[0103] The insertion of desired genes or other nucleic acid
constructs into cells seeded onto the new microfabricated membranes
or into the new tissue analogs or substitutes can be accomplished
using routine genetic and recombinant engineering techniques, e.g.,
as described in Ausubel et al., eds., 1989, Current Protocols in
Molecular Biology, Green Publishing Associates, Inc. and John Wiley
& Sons, Inc., New York.
[0104] The new tissue analogs and substitutes, such as skin
substitutes, can be tested in animal models to determine longevity
and "take" of the material, vascularization, and maintenance of the
defined three-dimensional topography of the microfabricated
membranes. For example, the tissue substitutes can be implanted
into athymic mice as described in Medalie et al., J. Invest.
Dermatol., 107:121-127, 122 (1996).
[0105] The new basal lamina can also be used to enhance mass
transport in tissues or tissue analogs or equivalents other than
skin. For example, the new membranes can be used to create
engineered small intestines using a microfabricated membrane that
mimics the topography of the intestinal mucosa.
[0106] The new microfabricated membranes can also be used in
non-biological settings, e.g., in medical devices, water and other
liquid filtration and/or purification systems, dialysis devices,
cell culturing systems, and artificial organs such as bioartificial
livers. In these devices, the three-dimensional topography of the
microfabricated membranes is selected to achieve a high surface
area, and to direct the flow of gases or liquids through tortuous
pathways to increase the pathlength, and thus the time that the
fluid remains in the device. The membranes can be located adjacent
walls in the device or additional membranes to form stacks of
membranes that allow flow between the membranes through the
channels in the membranes, but not through the membranes. In other
devices, such as size-exclusion filters, the membranes may be
located to force fluids to pass through one or more membranes.
[0107] In various embodiments, the membranes can be manufactured of
polymers that attract ions or charged proteins, e.g., for use in
water purification, deionization, or desalination. The membranes
can be made to have precise pores for filtration and dialysis. The
membranes can include charged moieties or chelating agents to
scavenge ions, heavy metals, or charged proteins or other
molecules. Membranes with pores and charged moieties can be
prepared to provide selectively permeable membranes.
OTHER EMBODIMENTS
[0108] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *