Note: Descriptions are shown in the official language in which they were submitted.
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Structured surfaces with properties which inhibit cell adhesion
and cell proliferation
Field of the Invention
The present invention relates to microstructured
surfaces of articles with low surface energy which reduce the
adhesion and proliferation of cells.
Background Art
Microstructured surfaces of articles are known and
are used in particular for self-cleaning surfaces.
An important feature of these surfaces is a low
wettability of the surface with water or aqueous systems.
Surfaces from which water readily runs off or is repelled have
to be either highly hydrophilic or hydrophobic. Hydrophilic
surfaces have low contact angles with water. This brings about
rapid dispersion of the water on the surface and finally rapid
run-off of the resultant film of water from the surface. The
use of hydrophobic materials for producing hydrophobic surfaces
is known. A further development of these surfaces consists in
structuring the surfaces in the millimeter to nanometer range.
H. Seito et al., in Surface Coating International V,
1997, pp. 168 et seq., for example, describe a hydrophobic,
microstructured surface of this type. In this case, particles
made from fluoropolymers are applied to metal surfaces,
whereupon the wettability of the resultant surfaces with
respect to water, and their tendency to icing, were found to be
markedly reduced. However, in this case there was no
demonstration of any properties, which inhibit adhesion or
proliferation with regard to cell cultures.
U.S. Patent No. 3,354,022 and WO 96/04123 describe
other processes for lowering the wettability of articles by
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changes to surface topology. Here, artificial elevations
(protrusions? and depressions with a height of from 5 to 100 ~m
and a separation of from 5 to 500 ~m are applied to hydrophobic
materials or to materials hydrophobicized after structuring.
Surfaces of this type give rapid drop formation, whereupon the
drops as they roll off pick-up particles of dirt and clean the
surface. There is no description of studies with completely
wetted surfaces, e.g., using a physiological nutrient solution.
Nor are there any data on the aspect ratio of the elevations.
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From a different sector of industry, the biological sector, it is known that
surface topographies in the range from about 20 nm to 50 ~m (nano- and
microtopography) can affect the physiological behavior of cells. This range
of sizes covers _supramolecular and cellular dimensions. Topographies
exceeding 50 ~.m (macrotopography) are used as rough contact structures
on hard bone implants, in the form of webs and fabrics, e.g. for vascular
prostheses, and porous matrices for cell cultures in tissue engineering
(R.P. Lanza, "Principles of Tissue Engineering", Academic Press, ISBN
1-57059-342-6, Chapter 11 ).
to
Various surface topographies are used for controlling the growth of cells.
In the case of structures with a regular periodicity, the topography is
described by giving the shape of an element and the periodicity. In the
case of random topographies, which are also used in cell culture,
statistical data are required for the distribution of the geometric parameters
(heights, gradients, correlation lengths, etc.). Eisenbart et al., in
Biomaterials (1996), 17, 1399-1403, Curtis et al., in Biomaterials (1997),
18, 1577-1583 and Wen et al., in Biomed. Mater. Eng. (1996), 6, 173-189
describe a number of types of topography which have been tested hitherto
2 o in cell culture techniques. Some of the results of this study are highly
contradictory. Despite much effort, there has been no success in deriving
general principles for the growth of cells from the surface topographies.
Although a very wide variety of geometric structures, periodicities and
dimensions have been studied, there has hitherto been no success in
finding surface structures whose properties inhibit cell adhesion and cell
proliferation.
Surfaces with properties which reduce cell adhesion and/or cell
proliferation would be useful in many applications.
It is highly undesirable that bacteria or cells should become established or
spread on the surfaces of pipelines, containers or packaging. Frequently,
slime layers form and permit sharp rises in microbial populations, and
these can lead to persistent impairment of the quality of water, drinks or
foods, and even to spoilage of the product and harm to the health of
consumers.
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Bacteria or cells must be kept away from all areas of
life in which hygiene is important. This affects textiles for
direct body contact, especially involving the genital area or
the care of the sick or elderly. Cells and bacteria must also
be kept away from the surfaces of furniture and of instruments
in wards, especially in areas for intensive care or neonatal
care, in hospitals, especially in areas intended for medical
intervention, and in isolation wards for critical cases of
infection, and also in toilets.
A current method of treating equipment, or the
surfaces of furniture or textiles, to resist bacteria, either
when this becomes necessary or else as a precautionary measure,
is to use chemicals or solutions or mixtures of these which as
disinfectants have fairly broad and general antimicrobial
action. Chemical agents of this type act nonspecifically and
are frequently themselves toxic or irritant or form degradation
products which are hazardous to health. In addition, people
frequently exhibit intolerance to these materials once they
have become sensitized.
Summary of the Invention
It would therefore be desirable to provide articles
having surfaces with properties, which inhibit cell
proliferation and/or cell adhesion.
Surprisingly, it has been found that articles having
structured surfaces having a particular aspect ratio, i.e. a
particular ratio of height to average widths, give substantial
and long-lasting avoidance of the establishment and spread of
cells.
Examples of cells for the purposes of the present
invention are fibroblasts, immortalized and primary cell lines,
and other eukaryotic non-microorganism cells, especially
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mammalian cells, but not bacteria. These cells differ markedly
from bacteria in their adhesion and proliferation behavior.
The adhesion of eukaryotic cells and bacteria to
different materials is a very complex phenomenon, the nature of
which plays a decisive part in biological function. Eukaryotic
cells use anchor proteins for adhesion to the surface, and
these permit directed movement (migration) and differentiation
of the cells. Since eukaryotic cells can only survive in
association, this function is important for giving a form to
the cells. In an aqueous solution, without binding to a
surface, eukaryotic cells are not able to divide. Bacteria use
simply structured proteins to adhere to the surface, but they
do not need these proteins for migration: rather, they are
exclusively for protection and for optimizing conditions for
survival. Division can take place even in aqueous solution.
The proliferation of bacteria on surfaces is therefore
controlled by mechanisms, which differ from those for the
proliferation of eukaryotic cells on surfaces.
The present invention therefore provides structured
(i.e., rough) surfaces of articles which are capable of
inhibiting proliferation of eukaryotic cells, wherein the
surfaces have elevations (or protrusions) with an average
height of from 50 nm to 10 ~m and an average separation (or
distance) of from 10 nm to 50 ~m and the surfaces are made of
materials which, as measured in their unstructured form, have a
surface energy of at least 20 mN/m, preferably from 20 to 80
mN/m, particularly preferably from 20 to 60 mN/m.
Brief Description of the Drawings
Fig. 1 is a graph showing an adhesion assay;
Fig. 2 is a graph showing an MTT assay;
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Fig. 3 is a graph showing a live/dead assay; and
Fig. 4 is a scanning electron micrograph with
overstructure and fine structure.
Description of Preferred Embodiments
5 Surfaces and elevations of this order of magnitude
are already known and are described, for example, in DE 19 80
3787.2 and DE 19 91 4007.3.
However, these surfaces are distinctly hydrophobic
and therefore strongly water-repellent. They have a very high
contact angle with respect to water and can be cleaned of
contamination by rinsing with water; or they promote the
running-off of water.
In the present case, these are undesirable
properties, since eukaryotic cells only live in aqueous
systems, and therefore complete wetting of the surfaces is
required.
To adhere to a surface or to proliferate on a
surface, bacteria and other microorganisms require water, which
is unavailable on hydrophobic surfaces, but very likely to be
available on the surfaces of the present invention. Surfaces
structured according to the invention prevent the establishment
and growth of eukaryotic cells, and thus inhibit cell
proliferation. However, the novel surfaces do permit localized
growth of bacteria and of other microorganisms on the
unstructured portions under appropriate conditions, such as
humidity and temperature. Since the fundamental effect is not
based on antimicrobial substances but on a physical effect, it
is impossible for the structured areas to impair the growth of
cells on the unstructured portions e.g. as a result of
migration and/or diffusion of active substances.
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The use of structured or rough surfaces in medicine
and biology has been established, for example, by E. Eisenbarth
et al., in "Influence of the surface structure of titanium
materials on the adhesion of fibroblasts", Biomaterials 1996,
Vol. 17, No. 14, pp. 1399-1403. It is shown here that the
number of cells, which have adhered, rises as roughness
increases, whatever material is used; this is contrary to the
effect of the present invention. In addition, R.G. Richard has
shown that differing roughnesses do not affect the adhesion of
fibroblasts ("The effects of surface roughness on fibroblast
adhesion in vitro, Injury" 1996, Vol. 27 Suppl. 3, pp.
C38-C43).
Technology has therefore not been moving towards the
use of microstructured surfaces for reducing cell proliferation
and/or cell adhesion.
The novel articles having structured surfaces may be
produced from a wide variety of materials, as long as these
have a surface energy of at least 20 mN/m in the unstructured
state. Use may be made, for example, of metals such as gold
and titanium; non-metallic elemental solid materials such as
silicon and carbon; ceramic materials such as quartz glass,
lithium niobate, silicon nitride, silicon carbide and
hydroxyapatite.
The materials for forming the articles may comprise
these substances, or be composed of these substances, either
entirely or only at the surface to be structured.
The materials may also contain organic polymer
materials, such as silicones, polydioxanes, fibronectin,
collagen, fibrin, polyurethanes, polymethyl methacrylate,
polyacrylic acid, polyvinyl chloride, polyethylene,
polypropylene, polycarbonate, polyester, polyimides or
polyamides, in each case in the form of a homo- or copolymer,
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or be composed of these substances either entirely or only at
the surface to be structured.
The materials are nontoxic and can also be used in
cell culture techniques.
The wettability of surfaces can be characterized by
measuring their surface energy. This variable is accessible,
for example, via measurement of the contact angle of various
liquids on a smooth, i.e. unstructured, material (D. K. Owens,
R.C. Wendt. J. Appl. Polym. Sci. 13, 1741 (1969)) and is given
in mN/m (millinewtons per meter). As determined by Owens et
al., smooth polytetrafluoroethylene surfaces have a surface
energy of 19.1 mN/m, and the contact angle (dynamic contact
angle) with water is 120°. Hydrophobic materials generally have
a contact angle (dynamic angle) with water of above 90°. For
example, polypropylene has a dynamic angle of about 105° with
respect to water, and a surface energy of 29-30 mN/m (depending
on the molecular structure).
Determination of the contact angle and the surface
energy is usefully carried out on smooth surfaces in order to
ensure better comparability. The material properties
"hydrophobicity", "liquid-repellent" and "wettable" are also
determined to some extent by the chemical composition of the
uppermost molecular layers of the surface. A higher or lower
contact angle and a lower or higher surface energy, for a
material may therefore also be achieved by coating processes.
The hydrophobic or hydrophilic properties of a
surface may therefore be defined via the surface energy, and
the contact angle of different liquids on the smooth, i.e.
unstructured, material is a measure of the surface energy,
which is given in mN/m.
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For certain application sectors, e.g. cell culture
technology, it is also possible to use other dimensions of the
elevations. It is therefore preferable for the average height
of the elevation to be from 50 nm to 4 Vim, with an average
separation of from 50 nm to 10 Vim. An alternative is for the
average height of the elevations to be from 50 nm to 10 Vim,
with an average separation of from 50 nm to 4 Vim. The
elevations particularly preferably have a height of from 50 nm
to 4 Vim, with an average separation of from 50 nm to 4 Vim,
especially a height of 0.5 to 5 ~m with an average separation
of from 0.2 to 5 Vim.
As mentioned above, the ratio of height to width of
the elevations, the aspect ratio, is of great importance. The
elevations may have aspect ratios of from 0.5 to 20, preferably
from 1 to 10, particularly preferably from 1 to 5. The
chemical composition of the uppermost monolayer of the material
is also significant. The use of the novel surfaces in cell
culture technology requires that the surfaces have specified
chemical properties. The surface must be cell-tolerant and
free from endotoxins. The surfaces may also be modified after
being given their form, so that the elevations may have a
surface energy above 20 mN/m.
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The novel structured surfaces may also have unstructured portions, in
particular unstructured portions with a surtace energy of from 10 to
20 mN/m.
On the unstructured portions it is possible for cells to adhere and/or
proliferate. It is thus possible, for example, to provide bioassays or cell-
culture test plates with the novel surfaces and using the
novel processes. The unstructured portions may be produced
mect~a~ically or lithographically. -
to
The unstructured portions may also have been treated or coated with
substances which promote cell adhesion, such as poly-L-amide, or with
substances which promote cell growth, such as fetal calf serum, bovine
serum, goat serum or horse serum.
The present invention also provides a process for producing structured
surfaces with properties which inhibit cell proliferation, by applying
elevations with an average height of from 50 nm to 10 ~m and with an
average separation of from 50 nm to 10 ~m to an unstructured material
2 o with a surface energy above 20 mN/m, using mechanical embossing,
lithographic etching processes or a molding process.
Mention may be made of other processes for altering the chemical
properties of surfaces by creating free-radical centers on the surface. The
structured or unstructured material may be treated using plasma, UV
2 5 radiation or gamma radiation, or other specific photoinitiators. After
activating the surface in this way, i.e. producing free radicals, it is also
possible to apply monomers by polymerization. A process of this type
generates a coating which is particularly chemically resistant. Examples of
possible monomers are acrylates, methacrylates, and other vinyl
3o derivatives, e.g. methyl methacrylate, ethylene oxide or vinyl chloride.
The surtace may be given its form or structure by embossinglrolling or
simultaneously with giving the article its macroscopic form, e.g. in casting,
injection molding or other shaping processes. These require the negative
35 molds corresponding to the structure desired.
Negative molds can be produced industrially, e.g. by the Liga technique
(R. Wechsung in Mikroelektronik, 9, 1995, pp. 34 et seq.). Here, one or
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more masks are first produced using electron-beam lithography, with the
dimensions of the elevations desired. These masks are used when
irradiating a photoresist layer using X-ray lithography, giving a positive
mold. The interstices in the photoresist are then filled by electrolytic
deposition of a metal. The resultant metal structure is a negative mold for
the structure desired.
In another embodiment of the present invention, the elevations may be
arranged on a somewhat coarser overstructure. The elevations have the
1 o dimensions listed above and can be applied to an overstructure with an
average height of from 10 ~m to 1 mm and with an average separation of
from 10 ~.m to 1 mm. The elevations of the overstructure may likewise be
applied by embossing, by lithographic processes or by molding processes.
The elevations and the overstructure may be applied simultaneously or
one after the other, i.e. first the overstructure, then the elevations, using
mechanical embossing, lithographic processes or a molding process.
In the case of surfaces with an overstructure, as with surfaces with
elevations only, it is useful to carry out the shaping and
2 o structuring in one operation. Subsequent chemical modification of a
previously produced double-structured surface is, of course, also possible.
Examples of mechanical processes for introducing structures onto
unstructured surfaces or for introducing unstructured portions on
2 5 structured surfaces are embossing or stamping processes with previously
produced molds or stamps (needling). Examples of lithographic processes
are the Liga technique, X-ray lithography and also ablative processes, e.g.
with laser beams.
3o Examples of molding processes are casting and injection molding, which
are conventional in plastics processing.
The invention also provides the use of the surfaces structured according to
the invention, or the structured surfaces produced according to the
35 invention.
The novel surfaces may, for example, be used as a cell culture vessel for
bioassays in cell screening, in the screening of new drugs in medicine, or
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in crop protection or in toxicology. The novel surfaces may also be used
for medical implants, e.g. cardiac valves or pacemakers.
The following examples are intended to describe the invention in greater
detail, but not to limit its scope.
Example:
The microstructured surfaces are produced using an embossing process.
The embossing tools (molding tools) are produced by the LIGA process, a
1 o process whose name derives from the German terms for the principles on
which it is based: X-ray lithography, electrodeposition and molding. The
process differs from micromechanics in that the structures are not
produced by a process of etching in the base material, but molded cost-
effectively using a mold, e.g. by injection molding. After the lithographic
process of resist exposure (radiation-sensitive polymer) and development,
the resultant structured coating is used as a template for an
electrodeposition process, in which a metal alloy is deposited into the
interstices which have been made available. The structured coating is then
removed, and the structured metal remaining behind is utilized as a mold
2 0 (G. Gerlach, W. Dotzel "Grundlagen der Mikrosystemtechnik" [Principles of
microsystem technology], Carl Hanser Verlag Munchen 1997, pp. 60 et
seq. ).
In the case of analyte 1, the resultant structures are merely fine structures
2 5 with an average height of 4 ~,m and an average separation of 3 ~,m. The
structures for analytes 2 to 4 also have an overstructure with an average
height of 20 ~,m and an average separation of 32 ~.m. The fine structure
corresponds to that of analyte 1.
3 o Fig. 4 shows a scanning electron micrograph with overstructure and fine
structure (analyte 4). Since the depth of focus in the scanning electron
micrograph is limited, the fine structure is detectable only on the "peaks" of
the overstructure.
35 Disks with a diameter of 9 mm are cut out from the polycarbonate surfaces
microstructured in this way and are converted into a cell culture plate
(Nunc, catalog No. 167008) with 96 welds, with the structured side facing
upward. Under sterile working conditions, the materials are inoculated with
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human dermal fibroblasts in passage 2 (3 x 10° cells/ml, 100 p.llwell).
Using various assays it is possible to assess the growth behavior of the
fibroblast cells. The control used is material with no surface structure but
likewise inoculated with human dermal fibroblasts. The non-adhering cell
fractions were counted on various days. It was clear that from 20 to 40%
fewer cells adhered to the microstructured surfaces at the floor. Fig. 1
shows the number of living cells which have adhered to the surface. Using
a MTT assay, the proliferation rate of the entire culture and the vitality
were determined. This assay is based on the absorption of a tetrazolium
1 o salt by cells, and reduction of this salt to formazan in a reaction
dependent
on mitochondria. After lysis of the cells this dye is liberated and can be
quantified photometrically. Since the MTT reduction involves only intact
mitochondria, the values measured allow conclusions to be drawn on the
vitality and the proliferation of the cells. An evaluation of the MTT
measurement for the cells which had adhered on various days showed
markedly reduced cell growth on the microstructured surfaces, compared
with the control surface. In some cases cell proliferation was less than
20% of that on the unstructured surface (see Fig. 2, MTT assay relative to
control).
Using a live/dead fluorescence assay, the number of living and dead cells
which had adhered was determined. Neither with the control nor with the
surface-structured specimens was there any significant difference in the
ratio of living to dead cells (Fig. 3), i.e. the adhesion and
proliferation of cells was successfully suppressed without killing the cells.
The possibility of toxic effects from the structured surfaces can thus be
excluded.
3 o Key to Figures 1 to 3
d1, d3, d5 and d8 indicate the 1 st, 3rd, 5th and 8th day after inoculation.
"Control° indicates a specimen of the same material without the
inventive
3 5 structuring of the surface.
Analyte 1, 2, 3 or 4 indicates in each case a differently structured surface
with the parameters:
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Overstructure Fine
in m structure
in
m
A B C A B C
Anal a 1 -- -- -- 3 4 1
Anal to 2 24 25 10 3 4 1
Anal a 3 15 15 7 3 4 1
Anal to 4 32 20 12 3 4 1
A
a i
Fig. 1 shows an adhesion assay, where the cells which have adhered per
analyte is plotted on the y axis. The corresponding absolute numbers are
found under each bar chart. In each case the value for d1 is indicated by
the left-hand column, the value for d2 by the middle column and the value
for d3 by the right-hand column.
1 o Fig. 2 shows an MTT assay, where the percentage of vital cells in relation
to the control assay is given on the y axis. In each case the corresponding
percentages are found under the bar chart. The columns indicate, from left
to right, the values for d1, d3, d5 and d8.
Fig. 3 shows a live/dead assay, where the percentage of living cells is
given on the y axis. The corresponding percentages are found under each
bar chart. The columns indicate, from left to right, the values for d1, d3, d5
and d8.
