U.S. patent application number 10/480897 was filed with the patent office on 2004-10-28 for biomaterial substrates.
Invention is credited to Cousins, Brian G, Doherty, Patrick Joseph, Fink, John, Garvey, Michael Joseph, Williams, Rachel Lucinda.
Application Number | 20040214326 10/480897 |
Document ID | / |
Family ID | 9916492 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040214326 |
Kind Code |
A1 |
Cousins, Brian G ; et
al. |
October 28, 2004 |
Biomaterial substrates
Abstract
The invention concerns the use of a biomaterial substrate to
orient cell or tissue growth. The substrate comprises a base
portion and a surface layer covering at least part of the base
portion. The surface layer provides the substrate with
topographical features having at least one nanoscale dimension of
from about 1 to about 200 nm. The topographical features having the
capacity to orient cell or tissue growth thereon and/or
therebetween.
Inventors: |
Cousins, Brian G;
(Litherland, GB) ; Garvey, Michael Joseph;
(Heswall, GB) ; Fink, John; (Liverpool, GB)
; Williams, Rachel Lucinda; (Little Neston, GB) ;
Doherty, Patrick Joseph; (Crosby, GB) |
Correspondence
Address: |
WADDEY & PATTERSON
414 UNION STREET, SUITE 2020
BANK OF AMERICA PLAZA
NASHVILLE
TN
37219
|
Family ID: |
9916492 |
Appl. No.: |
10/480897 |
Filed: |
June 4, 2004 |
PCT Filed: |
June 11, 2002 |
PCT NO: |
PCT/GB02/02634 |
Current U.S.
Class: |
435/395 ;
435/325 |
Current CPC
Class: |
A61L 27/34 20130101;
A61L 27/34 20130101; A61L 15/42 20130101; C12N 2533/30 20130101;
A61L 2400/18 20130101; C08L 27/18 20130101; C08L 27/18 20130101;
A61L 15/24 20130101; A61L 27/50 20130101; C12N 2533/12 20130101;
A61L 15/24 20130101; C12N 5/0068 20130101; C12N 2535/10
20130101 |
Class at
Publication: |
435/395 ;
435/325 |
International
Class: |
C12N 005/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 2001 |
GB |
0114399.9 |
Claims
1. A substrate for use as a biomaterial to orient cell or tissue
growth, the substrate comprising: a base portion; and a
water-insoluble or sparingly water soluble polymeric surface layer
covering at least part of the base portion, the surface layer
providing the substrate with topographical features having at least
one nanoscale dimension of from about 1 to about 200 nm, said
topographical features having the capacity to orient cell or tissue
growth thereon and/or therebetween.
2. The substrate according to claim 1 wherein the topographical
features are provided in the form of at least one regular and/or
ordered array.
3. The substrate according to claim 2 wherein the array comprises a
series of longitudinal and/or lateral ridges.
4. The substrate according to claim 3 wherein the ridges have
substantially the same or similar dimensions and/or physical
characteristics.
5. The substrate according to claim 2 for use to orient cell or
tissue growth in a manner at least partially determined by the
configuration of the array.
6. The substrate according to claim 1 for use to orient mammalian
cell growth.
7. The substrate according to claim 1 wherein the polymeric
material is selected from, polyolefins and halogenated
polyolefins.
8. The substrate according to claim 1 wherein said topographical
features have at least one dimension of less than about 50 nm.
9. The substrate according to claim 1 wherein the surface layer
comprises topographical features separated from other nearest
neighbour similar topographical features by distances of up to
about 1000 nm.
10. The substrate according to claim 1 wherein the surface layer
comprises topographical features separated from other nearest
neighbour similar topographical features by distances of up to
about 500 nm.
11. The substrate according to claim 1 wherein said base portion
and said surface layer are of different materials.
12. The substrate according to claim 1 wherein the surface layer is
able to adhere directly to the base portion.
13. The substrate according to claim 1 wherein the base portion is
formed from a material selected from polymers, glasses, ceramics,
carbons, metals and composites.
14. The substrate according to claim 1 wherein the substrate is
used to orient cells in vitro.
15. The substrate according to claim 1 wherein the substrate is
used to orient cells in vivo.
16. The substrate according to claim 15 wherein the substrate is
used in a medical implant.
17. The substrate according to claim 15 wherein the substrate is
used as a wound dressing.
Description
[0001] The present invention relates to novel biomaterial
substrates, to methods of making them and to uses therefor. In
particular the invention concerns such materials, methods and uses
for the orientation of cell or tissue growth.
[0002] The properties of cells are influenced by their external
environment, both chemical and physical. Understanding interactions
which take place between a cell and its substrate is important in
connection with such fields as medical implants and prostheses,
tissue engineering and pharmaceutical development. One substrate
characteristic which has been shown to influence cellular
properties is topography and synthetic structured surfaces have
been used to investigate this influence. A review of such
investigations may be found in Biomaterials (1999), 200,
573-588.
[0003] The modification of surface topography for the control of
cellular response is an important area of research in medical
engineering that targets several potential end uses, particularly
relating to the biocompatibility of materials used in medical
devices. In this area, it is required to control the interfacial
reactions that mitigate the appropriate response for a specific
application. It is known that the interfacial reactions are
influenced by the surface properties of the biomaterial substrate
in terms of the surface chemistry, energy and topography. Of the
latter, current research is focused on etching techniques to create
the desired topography. Experimental Cell Research (1996), 223,
426-435, discusses the production of microfabricated grooves and
steps by means of dry etching a silica substrata with a reactive
ion etching unit. U.S. Pat No. 4,832,759 also discusses the
generation of a plurality of surface discontinuities by means of
ion beam etching. The majority of prior art studies have used photo
lithographic techniques to engineer surface features with
controlled morphology for the study of cell behaviour thereon.
Other techniques include glancing angle deposition, laser ablation,
laser deposition, replica molding of x-ray lithography masters,
imprint lithography, micro contact printing and etching and ink-jet
printing. For example, Canadian Pat No. 2,323,719 discusses the
production of structural elevations by the LIGA lithographic
process which incorporates x-ray lithography, electrodeposition and
moulding. Other substrates having nanoscale topography have also
been described (Wear (1997), 41, 383-398 and Nature (1991), 352,
414-417).
[0004] Cell-substrate interactions in the natural environment are
influenced by the surface topography of the substrate, the
topographical features of which are represented at the nanoscale
level. Some of the above-mentioned techniques for engineering
synthetic surface features are capable of generating topographical
features at the nanoscale level but none has so far offered a quick
and convenient means to study, manipulate or modify cellular
properties at this level.
[0005] It is an object of the present invention to provide a
substrate which can be used as a biomaterial for orienting cell or
tissue growth.
[0006] According to the present invention there is provided the use
of a substrate as a biomaterial to orient cell or tissue growth,
the substrate comprising:
[0007] a base portion; and
[0008] a surface layer covering at least part of the base portion,
the surface layer providing the substrate with topographical
features having at least one nanoscale dimension of from about 1 to
about 200 nm, said topographical features having the capacity to
orient cell or tissue growth thereon and/or therebetween.
[0009] The invention provides a novel use of a substrate which may
find application in a wide variety of circumstances. For example,
the invention may be used to provide medical implants which orient
cell growth thereon. Prior art substrates having nanoscale
topography have been reported as biomaterials but there has been no
satisfactory commercial development in the field of tissue or cell
growth orientation. There are a wide variety of medical end uses
for technology which can orient cell growth. Other examples include
wound dressings, nerve regeneration materials and dental implants.
"Orient" includes the exercising of directional influence over cell
or tissue growth, for example influencing a cell culture to grow in
a desired direction.
[0010] In the context of this document, "nanoscale" is used to
refer to topographical features having at least one dimension which
is measurable at the nanometre level, for example a feature which
measures from about 1 to about 200 nm, preferably from about 1 to
about 150 nm, even more preferably from about 1 to about 100 nm, in
at least one dimension.
[0011] The topographical features may form a random array on the
surface layer of the substrate. Such an array may comprise, for
example, an agglomeration of peaks and troughs, preferably having
substantially the same or similar dimensions and physical
characteristics.
[0012] Alternatively, the topographical features may form an
ordered array on the surface layer of the substrate. An ordered
array may comprise, for example, a series of longitudinal and/or
lateral ridges. A combination of random and ordered arrays may be
used also. Whatever form of array adopted by the topographical
features, the substrate of the invention preferably comprises
nanoscale topographical features separated from other nearest
neighbour similar nanoscale topographical features by distances of
up to about 1000 nm. For example, when the array comprises
individual peaks and troughs, each peak in the array may be
separated from its nearest neighbour peaks by distances of up to
about 500 nm, preferably no more than about 200 nm. Where the array
comprises a series of longitudinal ridges, each ridge in the array
may be separated from its nearest neighbour by distances of up to
about 1000 nm, preferably no more than about 500 nm.
[0013] Preferably the base portion and the surface layer are of
different materials. There may be further layer(s) between the base
portion and the surface layer. In other words, the surface layer
may adhere directly or indirectly to the base portion.
[0014] The topographical features are preferably provided by means
of controlled deposition onto the base portion of a surface layer
capable of adhering to the substrate. Such adherence may be
chemical or physical.
[0015] Thus, in one of its aspects this invention relates to
methods for tailoring the topography of surfaces using the
controlled deposition of thin films of nanoscale material onto an
underlying substrate so as to modify the cellular response to the
treated surface.
[0016] Thus, in one of its aspects the invention provides a
biomaterial substrate, modified with a surface of deposited
nanoscale material in order to control directionally the cellular
response that occurs as a result of cell contact or
interaction.
[0017] In one preferred embodiment of the invention there is
provided a biomaterial substrate for the study, manipulation or
modification of at least one cellular or tissue behaviour or
response, the biomaterial substrate comprising a base portion being
provided on a surface thereof, by means of controlled deposition
onto the base portion, with a substance capable of adhering to the
base portion, with topographical features having at least one
nanoscale dimension and a cell or tissue growth, growth inhibition
or growth control region thereon and/or therebetween, the
topographical features being deposited in a densely packed array
with a separation between nearest neighbour similar topographical
features of not more than 1000 nm.
[0018] In another preferred embodiment of the invention there is
provided a method of manufacturing a biomaterial substrate for the
study, manipulation or modification of at least one cellular or
tissue behaviour or response comprising a base portion, depositing
onto the base portion a substance capable of adhering thereto in
order to provide the biomaterial substrate with topographical
features having at least one nanoscale dimension the deposit being
densely packed with separation between the topographical features
of not more than 1000 nm, and providing the biomaterial substrate
with a cell or tissue growth, growth inhibition or growth control
region on and/or between the topographical features.
[0019] The base portion may comprise a single material or may
comprise two or more layers of different materials. For example,
the base portion may comprise a base material, such as polystyrene,
covered with a layer of a polymeric or surface active agent.
[0020] The base portion may be selected from any suitable material,
this depending on whether it is intended to promote, inhibit or
control cell or tissue growth on the base portion material itself
or only on a substance adhered thereto. Preferred base portion
materials according to the invention include polymers, glasses,
ceramics, carbon, metals and composites.
[0021] The surface layer is preferably formed from a polymeric
material. Polyolefins and halogenated polyolefins are particularly
suitable, for example poly(tetrafluoroethylene),
poly(trifluoroethylene), poly(chlorotrifluoroethylene),
poly(vinylidenefluoride), poly(2,2,3,3-tetrafluorooxetane),
poly(fluoroalcylacrylate), poly(hexafluoro-propylene),
poly(perfluoropropylene oxide), poly
(2,2-bis(trifluoromethyl)-4,5-difluoro-1,3-dioxole), poly(vinyl
perfluoroalkyl ether), poly(fluoro-alkyl methacrylate), or other
polymers or perfluoroalkoxy compounds, poly(ethylene),
poly)propylene), poly(isobutene), poly(isoprene),
poly(4-methyl-1-pentene), poly(vinyl alkanoates) and poly(vinyl
methyl ethers) as homo- or copolymers. One particularly preferred
material is PTFE. Preferably the surface layer material is
insoluble or sparingly soluble in water. The surface layer material
is preferably able to adhere to the base portion without any
additional binding material. However, additional binding agents may
be used if desired and the substrate may in that case have a
multi-layer structure comprising one or more layers of a binding
agent between the base portion and the surface layer.
[0022] The biomaterial substrates, methods and uses of the
invention have advantages over conventional engineered surfaces
used in medical engineering and methods for making them. Cell
behaviour can be directionally manipulated and controlled by
selecting different topographies as cells respond differently to
different physical environments. The invention therefore provides a
valuable tool for use in medical engineering.
[0023] This technique can be envisaged to be useful in a number of
applications. One such application would be to use the substrate in
the production of bioactive glass coated substrates for nerve
regeneration. The technology may also prove useful in dental
implants, employed to produce a gingival fibroblast attachment to
the implant abutment that would secrete collagen and create
circumferential tissue fibres around the implant. This would create
a tight, fibrous collar, which would protect the soft-tissue
attachment and reduce the incidence of peri-implantitis. These are
a few applications where the technology may prove valuable, but
those skilled in the art would realise that there are many other
applications that may benefit from this technique.
[0024] The invention will now be more particularly described with
reference to the following Figures and Examples in which:
[0025] FIG. 1 shows an image of a biomaterial substrate in
accordance with the invention, the image being generated by means
of atomic force microscopy (AFM).
[0026] FIG. 2 shows a high magnification image generated by a light
microscope of cells on a biomaterial substrate in accordance with
the invention.
[0027] FIG. 3 shows a light microscope image of fibroblast cells on
an alternative biomaterial substrate in accordance with the
invention.
[0028] FIG. 4 shows an image generated by a light microscope of
bovine fibroblast cells on a biomaterial substrate in accordance
with the invention.
[0029] FIG. 5 shows a high magnification image generated by a light
microscope of bovine fibroblast cells in FIG. 4.
EXAMPLE 1
[0030] A standard glass microscope slide was washed once with
methanol followed by copious rinsing with deionised water
(Millipore-Q 18.2 M). The glass slide was then heated to
220.degree. C. on a hot plate for approximately 10 minutes to allow
for thermal equilibrium to take place. A thin strip of
polytetrafluoroethylene (PTFE) (3.times.2.times.0.5 cm) was then
applied to the glass slide once in one direction along the long
axis of the slide while still on the hot plate by means of a wiping
action. The glass slide was then removed from the hot plate and
allowed to cool. The PTFE-wiped glass slide was then gently wetted
with water to determine if the PTFE was deposited successfully as
shown by the appearance of "water-lines" on the glass slide due to
the hydrophobicity of the PTFE fibres. The surfaces of the PTFE
coated slides were then scanned using an atomic force microscopy
(AFM) as shown in FIG. 1. FIG. 1 shows the formation of
approximately 20 nm high PTFE fibres spaced apart from
approximately 100 nm. The next stage was to use the PTFE coated
glass slide in cell culture experiments.
[0031] A suspension of clone L 929 mouse fibroblast established
cell line was prepared from a culture maintained in Eagle's Minimum
Essential medium with a 5% foetal calf serum supplement. The
suspension was prepared at cell concentration of approximately
1.times.10.sup.5 cell/ml. This was performed by immersing each PTFE
coated slide in a cell culture medium containing established
fibroblast cells for approximately 24 hours in an incubator at
37.degree. C.
[0032] After this period the PTFE coated slides were removed from
the culture medium and examined with an inverted phase-contrast
light microscope. The results clearly show that the surface
influences the cell growth in the direction of the PTFE fibres, as
shown in FIG. 2. A control (results not shown) of untreated glass
slide showed no orientation or elongation of the cells, indicating
that the effects observed with the PTFE/glass matrix resulted from
the directionality of the PTFE fibres.
EXAMPLE 2
[0033] The effect of time on the cell growth in the direction of
the PTFE fibres was assessed over a 6 day period. Slides were
presented with an inoculum of L 929 murine fibroblasts cells in
culture medium [approx 0.2 ml cell conc. 10.sup.6 cell/ml] and
applied to the PTFE surfaces as outlined in Example 1. The dish was
then incubated for 30 minutes to allow the cells to attach to the
glass slide surface. After 30 mins the plate was flooded with
culture medium and re-incubated. The plates were then examined by
phase contrast microscope at 24 hour periods. After each time
period, the dishes were removed from the incubator and the slides
examined by phase contrast microscopy. The slides were stained with
methylene blue after the end of a 6 day period in order to assess
areas of viable cells. The results were as follows:
[0034] After one day, a high concentration of cells was observed at
the site of the inoculum. These cells were well spread and adhering
to the substrate. Where the fibres were observed a small number of
the cells were aligning.
[0035] After two days, the cells were clearly aligning. The cells
were becoming confluent.
[0036] After six days, The cells had spread only in the direction
of the applied fibres and the cells were viable.
[0037] These results suggested that cells could be grown in a
directional manner by using PTFE fibres as a substrate.
EXAMPLE 3
[0038] In order to assess what the effect of two PTFE fibres of
differing orientations had on cell growth, PTFE coated glass slides
were prepared as in Example 1, but the PTFE fibres were deposited
at right angles to each other. Cell culture experiments using L929
fibroblast cells were carried out as in Example 2.
[0039] A suspension of clone L 929 mouse fibroblast established
cell line was prepared from a culture maintained in Eagle's Minimum
Essential medium with a 5% foetal calf serum supplement. The
suspension was prepared at cell concentration of approximately
1.times.10.sup.5 cell/ml.
[0040] 1 ml of the cell suspension was directly applied to both
treated and untreated surface of a substrate prepared as described
in Example 1. The cells were left in contact with the substrate for
30 minutes to allow cells adhesion, then the substrate was flooded
with culture medium and maintained at 37.degree. C./5% CO.sub.2 for
48 hours. After this time the culture medium and non-adherent cells
are removed. The substrate was then treated with 100% methanol to
fix the cells and the substrate is stained with 0.04% methylene
blue for 10 minutes.
[0041] FIG. 3 show that the cells again aligned and elongated along
the direction of the fibres but at crossover points between sets of
fibres it was also observed that the cells oriented at right angles
to each other. This demonstrates that by controlling the way in
which the PTFE is deposited on the surface one can control the
directionality and orientation of cells that are grown on that
surface.
EXAMPLE 4
[0042] Further experiments were directed towards the growth of
bovine fibroblast cells on PTFE fibres in order to assess whether a
different type of fibroblast cells would react in the same manner
to the PTFE fibres as the L 929 mouse fibroblast cell line.
[0043] Primary bovine fibroblast cells were obtained from the Unit
of Opthalmology, University of Liverpool at second or third passage
and maintained in Dulbecco's Minimal essential Medium supplemented
with 10% foetal calf serum. A cell suspension was prepared at a
cell concentration of approximately 5.times.10.sup.4 cells/ml. 1 ml
of this cell suspension was directly applied to the surface of a
substrate prepared as described in Example 1. The cells were left
in contact with the substrate for 30 minutes to allow cells
adhesion, then the substrate was flooded with culture medium and
maintained at 37.degree. C./5% CO.sub.2 for 48 hours. After this
time the culture medium and non-adherent cells are removed. The
substrate was treated with 100% methanol to fix the cells and the
substrate is stained with 0.04% methylene blue for 10 minutes.
[0044] FIG. 4 shows an optical micrograph detailing alignment of
the bovine fibroblast cells with the PTFE fibres. The cell
morphology is very different to that seen on an untreated glass
base substrate or polystyrene base substrate.
[0045] FIG. 5 is a higher magnification of FIG. 4 showing bovine
fibroblast cells in contact with nano-fibres applied to a glass
substrate.
EXAMPLE 5
[0046] As other cell and tissue types may have an application
utilising PTFE fibres as a substrate for orientated cell growth,
murine neuroblast cells were assessed to investigate their growth
behaviour on the substrate.
[0047] The neuroblast cells were grown in accordance with the
protocol as outlined in Example 5 and also showed evidence of
alignment of cells relative to the PTFE fibres. The alignment with
neuroblast cells was not as clearly defined as the alignment shown
by fibroblasts, but was consistent and repeatable.
* * * * *